The Pennsylvania State University

The Graduate School

Chemistry Department

ENHANCED PARTICLE DIFFUSION THROUGH ENZYME CATALYSIS

AND

SILVER TITANIA NANOMOTORS

A Thesis in

Chemistry

by

Shraddha Vishwas Surve

2010 Shraddha Vishwas Surve

Submitted in Partial Fulfillment

of the Requirements

for the Degree of

Master of Science

December 2010

ii

The thesis of Shraddha Vishwas Surve was reviewed and approved by the following

Ayusman Sen

Distinguished Professor of Chemistry

Thesis Advisor

Tom Mallouk

DuPont Professor of Materials Chemistry and Physics

Christine Keating

Associate Professor of Chemistry

Barbara Garrison

Shapiro Professor of Chemistry

Head of the Department of Chemistry

Signatures are on file in the Graduate School

iii

ABSTRACT

In the first part of this thesis I explore the use of enzymes as catalytic motors in order to

drive nanoparticles The two enzymes on which this thesis focuses are horseradish

peroxidase and glucose oxidase Chapter 2 describes the synthesis of carboxylated

polystyrene-silica Janus particles Horseradish peroxidase is immobilized on the

carboxylated polystyrene half of the Janus particle both with and without a linker

between the enzyme and the Janus particle Diffusion coefficients of these particles are

measured to check for enhanced diffusion In Chapter 3 an enzyme cascade system of

horseradish peroxidase and glucose oxidase has been investigated for predator-prey like

behavior Both the enzymes are immobilized on different polystyrene nanospheres and

motion of the spheres is observed in substrate solution of glucose and oxygen Optical

microscopy experiments and diffusion coefficient measurements are carried out in order

to study the predator-prey behavior of these nanoparticles Chapter 4 describes the second

part of the thesis which involves design and synthesis of Ag-TiO2 particles Autonomous

motion of Ag-TiO2 particles can be observed in the presence of hydrogen peroxide

solution Particle motion and inter-particle interactions can be altered by subjecting the

system to UV light trigger

iv

TABLE OF CONTENTS

LIST OF FIGURES vi

LIST OF TABLES ix

ACKNOWLEDGEMENTS x

Chapter 1 Introduction 1

11 Motion at the nanoscale 2

12 Existing nanomotors Mechanisms and uses 6

Chapter 2 Immobilized enzymes on Janus particles as catalytic motors 9

21 Introduction 9

211 Horseradish peroxidase 13

22 Experimental 14

221 Materials and methods 14

222 Synthesis of Janus particles 15

223 Immobilization of enzymes using EDCNHS coupling method 16

224 Enzyme assay 18

225 Diffusion coefficient measurements using Nanosight LM10 19

23 Results 21

231 Enzyme assay 21

232 Diffusion coefficient measurements 23

24 Discussion 25

Chapter 3 Predator-prey system of enzyme immobilized particles 28

31 Introduction 28

311 Glucose oxidase 31

32 Experimental 32

321 Materials and methods 32

v

322 Optical microscopy experiments 33

323 Diffusion coefficient measurements using Nanosight LM10 33

33 Results 34

331 Optical microscopy 34

332 Diffusion coefficient measurements 37

34 Discussion 39

Chapter 4 Silver-titania nanomotors 42

41 Introduction 42

42 Experimental 43

421 Materials and methods 43

422 Synthesis of silver colloidal particles 44

423 Preparation of silver-titania (Ag-TiO2) particles 45

424 Optical microscopy experiments 46

43 Results and Discussion 47

Chapter 5 Summary and Conclusion 52

References 54

vi

LIST OF FIGURES

Figure 1 1 Reciprocal and Nonreciprocal motion8 5

Figure 2 1 Enzyme functionalized Janus particle moves upon catalyzing

asymmetrical decomposition of substrate into product 10

Figure 2 2 Schematic showing idealized nonreciprocal conformation changes in

a theoretical enzyme 11

Figure 2 3 Horseradish peroxidase 13

Figure 2 4 Reaction catalyzed by horseradish peroxidase 14

Figure 2 5 Preparation of PS-SiO2 Janus particles by evaporation 16

Figure 2 6 EDCNHS coupling method for immobilization of enzyme on

carboxylated polystyrene particles 17

Figure 2 7 Immobilization of enzyme on Janus particles with and without 6-

aminohexanoic acid linker 17

Figure 2 8 Nanosight LM 10 instrumentation 19

Figure 2 9 NTA video analysis (a) Particles as they are seen on the screen

during experimentation in Nanosight LM10 (b) NTA screen as the particles

are being analyzed for their diffusion coefficients 20

Figure 2 10 Graph for the assay of solution of free enzyme containing 05mgL

of HRP ΔAmin = 01716 Unitsmg of free enzyme = 52158 22

Figure 2 11 Graph for the assay of of immobilized enzyme on 05 μm Janus

particles (~025 vv) ΔAmin = 01853 Unitsmg of free enzyme = 2133 23

ii

The thesis of Shraddha Vishwas Surve was reviewed and approved by the following

Ayusman Sen

Distinguished Professor of Chemistry

Thesis Advisor

Tom Mallouk

DuPont Professor of Materials Chemistry and Physics

Christine Keating

Associate Professor of Chemistry

Barbara Garrison

Shapiro Professor of Chemistry

Head of the Department of Chemistry

Signatures are on file in the Graduate School

iii

ABSTRACT

In the first part of this thesis I explore the use of enzymes as catalytic motors in order to

drive nanoparticles The two enzymes on which this thesis focuses are horseradish

peroxidase and glucose oxidase Chapter 2 describes the synthesis of carboxylated

polystyrene-silica Janus particles Horseradish peroxidase is immobilized on the

carboxylated polystyrene half of the Janus particle both with and without a linker

between the enzyme and the Janus particle Diffusion coefficients of these particles are

measured to check for enhanced diffusion In Chapter 3 an enzyme cascade system of

horseradish peroxidase and glucose oxidase has been investigated for predator-prey like

behavior Both the enzymes are immobilized on different polystyrene nanospheres and

motion of the spheres is observed in substrate solution of glucose and oxygen Optical

microscopy experiments and diffusion coefficient measurements are carried out in order

to study the predator-prey behavior of these nanoparticles Chapter 4 describes the second

part of the thesis which involves design and synthesis of Ag-TiO2 particles Autonomous

motion of Ag-TiO2 particles can be observed in the presence of hydrogen peroxide

solution Particle motion and inter-particle interactions can be altered by subjecting the

system to UV light trigger

iv

TABLE OF CONTENTS

LIST OF FIGURES vi

LIST OF TABLES ix

ACKNOWLEDGEMENTS x

Chapter 1 Introduction 1

11 Motion at the nanoscale 2

12 Existing nanomotors Mechanisms and uses 6

Chapter 2 Immobilized enzymes on Janus particles as catalytic motors 9

21 Introduction 9

211 Horseradish peroxidase 13

22 Experimental 14

221 Materials and methods 14

222 Synthesis of Janus particles 15

223 Immobilization of enzymes using EDCNHS coupling method 16

224 Enzyme assay 18

225 Diffusion coefficient measurements using Nanosight LM10 19

23 Results 21

231 Enzyme assay 21

232 Diffusion coefficient measurements 23

24 Discussion 25

Chapter 3 Predator-prey system of enzyme immobilized particles 28

31 Introduction 28

311 Glucose oxidase 31

32 Experimental 32

321 Materials and methods 32

v

322 Optical microscopy experiments 33

323 Diffusion coefficient measurements using Nanosight LM10 33

33 Results 34

331 Optical microscopy 34

332 Diffusion coefficient measurements 37

34 Discussion 39

Chapter 4 Silver-titania nanomotors 42

41 Introduction 42

42 Experimental 43

421 Materials and methods 43

422 Synthesis of silver colloidal particles 44

423 Preparation of silver-titania (Ag-TiO2) particles 45

424 Optical microscopy experiments 46

43 Results and Discussion 47

Chapter 5 Summary and Conclusion 52

References 54

vi

LIST OF FIGURES

Figure 1 1 Reciprocal and Nonreciprocal motion8 5

Figure 2 1 Enzyme functionalized Janus particle moves upon catalyzing

asymmetrical decomposition of substrate into product 10

Figure 2 2 Schematic showing idealized nonreciprocal conformation changes in

a theoretical enzyme 11

Figure 2 3 Horseradish peroxidase 13

Figure 2 4 Reaction catalyzed by horseradish peroxidase 14

Figure 2 5 Preparation of PS-SiO2 Janus particles by evaporation 16

Figure 2 6 EDCNHS coupling method for immobilization of enzyme on

carboxylated polystyrene particles 17

Figure 2 7 Immobilization of enzyme on Janus particles with and without 6-

aminohexanoic acid linker 17

Figure 2 8 Nanosight LM 10 instrumentation 19

Figure 2 9 NTA video analysis (a) Particles as they are seen on the screen

during experimentation in Nanosight LM10 (b) NTA screen as the particles

are being analyzed for their diffusion coefficients 20

Figure 2 10 Graph for the assay of solution of free enzyme containing 05mgL

of HRP ΔAmin = 01716 Unitsmg of free enzyme = 52158 22

Figure 2 11 Graph for the assay of of immobilized enzyme on 05 μm Janus

particles (~025 vv) ΔAmin = 01853 Unitsmg of free enzyme = 2133 23

iii

ABSTRACT

In the first part of this thesis I explore the use of enzymes as catalytic motors in order to

drive nanoparticles The two enzymes on which this thesis focuses are horseradish

peroxidase and glucose oxidase Chapter 2 describes the synthesis of carboxylated

polystyrene-silica Janus particles Horseradish peroxidase is immobilized on the

carboxylated polystyrene half of the Janus particle both with and without a linker

between the enzyme and the Janus particle Diffusion coefficients of these particles are

measured to check for enhanced diffusion In Chapter 3 an enzyme cascade system of

horseradish peroxidase and glucose oxidase has been investigated for predator-prey like

behavior Both the enzymes are immobilized on different polystyrene nanospheres and

motion of the spheres is observed in substrate solution of glucose and oxygen Optical

microscopy experiments and diffusion coefficient measurements are carried out in order

to study the predator-prey behavior of these nanoparticles Chapter 4 describes the second

part of the thesis which involves design and synthesis of Ag-TiO2 particles Autonomous

motion of Ag-TiO2 particles can be observed in the presence of hydrogen peroxide

solution Particle motion and inter-particle interactions can be altered by subjecting the

system to UV light trigger

iv

TABLE OF CONTENTS

LIST OF FIGURES vi

LIST OF TABLES ix

ACKNOWLEDGEMENTS x

Chapter 1 Introduction 1

11 Motion at the nanoscale 2

12 Existing nanomotors Mechanisms and uses 6

Chapter 2 Immobilized enzymes on Janus particles as catalytic motors 9

21 Introduction 9

211 Horseradish peroxidase 13

22 Experimental 14

221 Materials and methods 14

222 Synthesis of Janus particles 15

223 Immobilization of enzymes using EDCNHS coupling method 16

224 Enzyme assay 18

225 Diffusion coefficient measurements using Nanosight LM10 19

23 Results 21

231 Enzyme assay 21

232 Diffusion coefficient measurements 23

24 Discussion 25

Chapter 3 Predator-prey system of enzyme immobilized particles 28

31 Introduction 28

311 Glucose oxidase 31

32 Experimental 32

321 Materials and methods 32

v

322 Optical microscopy experiments 33

323 Diffusion coefficient measurements using Nanosight LM10 33

33 Results 34

331 Optical microscopy 34

332 Diffusion coefficient measurements 37

34 Discussion 39

Chapter 4 Silver-titania nanomotors 42

41 Introduction 42

42 Experimental 43

421 Materials and methods 43

422 Synthesis of silver colloidal particles 44

423 Preparation of silver-titania (Ag-TiO2) particles 45

424 Optical microscopy experiments 46

43 Results and Discussion 47

Chapter 5 Summary and Conclusion 52

References 54

vi

LIST OF FIGURES

Figure 1 1 Reciprocal and Nonreciprocal motion8 5

Figure 2 1 Enzyme functionalized Janus particle moves upon catalyzing

asymmetrical decomposition of substrate into product 10

Figure 2 2 Schematic showing idealized nonreciprocal conformation changes in

a theoretical enzyme 11

Figure 2 3 Horseradish peroxidase 13

Figure 2 4 Reaction catalyzed by horseradish peroxidase 14

Figure 2 5 Preparation of PS-SiO2 Janus particles by evaporation 16

Figure 2 6 EDCNHS coupling method for immobilization of enzyme on

carboxylated polystyrene particles 17

Figure 2 7 Immobilization of enzyme on Janus particles with and without 6-

aminohexanoic acid linker 17

Figure 2 8 Nanosight LM 10 instrumentation 19

Figure 2 9 NTA video analysis (a) Particles as they are seen on the screen

during experimentation in Nanosight LM10 (b) NTA screen as the particles

are being analyzed for their diffusion coefficients 20

Figure 2 10 Graph for the assay of solution of free enzyme containing 05mgL

of HRP ΔAmin = 01716 Unitsmg of free enzyme = 52158 22

Figure 2 11 Graph for the assay of of immobilized enzyme on 05 μm Janus

particles (~025 vv) ΔAmin = 01853 Unitsmg of free enzyme = 2133 23

iv

TABLE OF CONTENTS

LIST OF FIGURES vi

LIST OF TABLES ix

ACKNOWLEDGEMENTS x

Chapter 1 Introduction 1

11 Motion at the nanoscale 2

12 Existing nanomotors Mechanisms and uses 6

Chapter 2 Immobilized enzymes on Janus particles as catalytic motors 9

21 Introduction 9

211 Horseradish peroxidase 13

22 Experimental 14

221 Materials and methods 14

222 Synthesis of Janus particles 15

223 Immobilization of enzymes using EDCNHS coupling method 16

224 Enzyme assay 18

225 Diffusion coefficient measurements using Nanosight LM10 19

23 Results 21

231 Enzyme assay 21

232 Diffusion coefficient measurements 23

24 Discussion 25

Chapter 3 Predator-prey system of enzyme immobilized particles 28

31 Introduction 28

311 Glucose oxidase 31

32 Experimental 32

321 Materials and methods 32

v

322 Optical microscopy experiments 33

323 Diffusion coefficient measurements using Nanosight LM10 33

33 Results 34

331 Optical microscopy 34

332 Diffusion coefficient measurements 37

34 Discussion 39

Chapter 4 Silver-titania nanomotors 42

41 Introduction 42

42 Experimental 43

421 Materials and methods 43

422 Synthesis of silver colloidal particles 44

423 Preparation of silver-titania (Ag-TiO2) particles 45

424 Optical microscopy experiments 46

43 Results and Discussion 47

Chapter 5 Summary and Conclusion 52

References 54

vi

LIST OF FIGURES

Figure 1 1 Reciprocal and Nonreciprocal motion8 5

Figure 2 1 Enzyme functionalized Janus particle moves upon catalyzing

asymmetrical decomposition of substrate into product 10

Figure 2 2 Schematic showing idealized nonreciprocal conformation changes in

a theoretical enzyme 11

Figure 2 3 Horseradish peroxidase 13

Figure 2 4 Reaction catalyzed by horseradish peroxidase 14

Figure 2 5 Preparation of PS-SiO2 Janus particles by evaporation 16

Figure 2 6 EDCNHS coupling method for immobilization of enzyme on

carboxylated polystyrene particles 17

Figure 2 7 Immobilization of enzyme on Janus particles with and without 6-

aminohexanoic acid linker 17

Figure 2 8 Nanosight LM 10 instrumentation 19

Figure 2 9 NTA video analysis (a) Particles as they are seen on the screen

during experimentation in Nanosight LM10 (b) NTA screen as the particles

are being analyzed for their diffusion coefficients 20

Figure 2 10 Graph for the assay of solution of free enzyme containing 05mgL

of HRP ΔAmin = 01716 Unitsmg of free enzyme = 52158 22

Figure 2 11 Graph for the assay of of immobilized enzyme on 05 μm Janus

particles (~025 vv) ΔAmin = 01853 Unitsmg of free enzyme = 2133 23

v

322 Optical microscopy experiments 33

323 Diffusion coefficient measurements using Nanosight LM10 33

33 Results 34

331 Optical microscopy 34

332 Diffusion coefficient measurements 37

34 Discussion 39

Chapter 4 Silver-titania nanomotors 42

41 Introduction 42

42 Experimental 43

421 Materials and methods 43

422 Synthesis of silver colloidal particles 44

423 Preparation of silver-titania (Ag-TiO2) particles 45

424 Optical microscopy experiments 46

43 Results and Discussion 47

Chapter 5 Summary and Conclusion 52

References 54

vi

LIST OF FIGURES

Figure 1 1 Reciprocal and Nonreciprocal motion8 5

Figure 2 1 Enzyme functionalized Janus particle moves upon catalyzing

asymmetrical decomposition of substrate into product 10

Figure 2 2 Schematic showing idealized nonreciprocal conformation changes in

a theoretical enzyme 11

Figure 2 3 Horseradish peroxidase 13

Figure 2 4 Reaction catalyzed by horseradish peroxidase 14

Figure 2 5 Preparation of PS-SiO2 Janus particles by evaporation 16

Figure 2 6 EDCNHS coupling method for immobilization of enzyme on

carboxylated polystyrene particles 17

Figure 2 7 Immobilization of enzyme on Janus particles with and without 6-

aminohexanoic acid linker 17

Figure 2 8 Nanosight LM 10 instrumentation 19

Figure 2 9 NTA video analysis (a) Particles as they are seen on the screen

during experimentation in Nanosight LM10 (b) NTA screen as the particles

are being analyzed for their diffusion coefficients 20

Figure 2 10 Graph for the assay of solution of free enzyme containing 05mgL

of HRP ΔAmin = 01716 Unitsmg of free enzyme = 52158 22

Figure 2 11 Graph for the assay of of immobilized enzyme on 05 μm Janus

particles (~025 vv) ΔAmin = 01853 Unitsmg of free enzyme = 2133 23

vi

LIST OF FIGURES

Figure 1 1 Reciprocal and Nonreciprocal motion8 5

Figure 2 1 Enzyme functionalized Janus particle moves upon catalyzing

asymmetrical decomposition of substrate into product 10

Figure 2 2 Schematic showing idealized nonreciprocal conformation changes in

a theoretical enzyme 11

Figure 2 3 Horseradish peroxidase 13

Figure 2 4 Reaction catalyzed by horseradish peroxidase 14

Figure 2 5 Preparation of PS-SiO2 Janus particles by evaporation 16

Figure 2 6 EDCNHS coupling method for immobilization of enzyme on

carboxylated polystyrene particles 17

Figure 2 7 Immobilization of enzyme on Janus particles with and without 6-

aminohexanoic acid linker 17

Figure 2 8 Nanosight LM 10 instrumentation 19

Figure 2 9 NTA video analysis (a) Particles as they are seen on the screen

during experimentation in Nanosight LM10 (b) NTA screen as the particles

are being analyzed for their diffusion coefficients 20

Figure 2 10 Graph for the assay of solution of free enzyme containing 05mgL

of HRP ΔAmin = 01716 Unitsmg of free enzyme = 52158 22

Figure 2 11 Graph for the assay of of immobilized enzyme on 05 μm Janus

particles (~025 vv) ΔAmin = 01853 Unitsmg of free enzyme = 2133 23

vii

Figure 2 12 Diffusion coefficient measurements of PS-SiO2 Janus particles

without and with 6-aminohexanoic acid linker 25

Figure 3 1 Concept of particle aggregation due to cascade reactions of

immobilized enzymes 30

Figure 3 2 Glucose oxidase 31

Figure 3 3 Redox reaction of glucose catalyzed by glucose oxidase 31

Figure 3 4 Formation of fan-shaped structures when both HRP and GOx

immobilized particles were present in a substrate solution consisting of

glucose and TMB A In the initial stages single strands form on the surface

of liquid (5Ox) B Single strands branch out and intertwine to form fan-

shaped structures (50x) C Many such structures form and settle down after

20 minutes (20x) 35

Figure 3 5 Size dependence of fan-shaped structures on the size of HRP -

immobilized particles (20x) 36

Figure 3 6 Model for size dependence of fan-shaped structures on the particle

diameter of enzyme immobilized particles Larger the particle more surface

area is available for precipitation of TMB which eventually leads to bigger

fan-shaped structures 39

Figure 3 7 Hypothesis for formation of fan-shaped structures 40

Figure 4 1 Physvis particle tracking software 44

Figure 4 2 FESEM image of Ag-TiO2 particles 46

Figure 4 3 Mechanism of motion of Ag-TiO2 nanomotors 47

Figure 4 4 Silver-titania motors move faster when subjected to UV radiation 48

viii

Figure 4 5 Rotation due to particle aggregation of Ag-TiO2 particles 48

Figure 4 6 Mean square displacement curves of Ag-TiO2 Janus particles in

hydrogen peroxide and DI water 50

Figure 4 7 Higher concentrations of Ag-TiO2 particles showing aggregation

when UV is turned on 51

ix

LIST OF TABLES

Table 2 1 Diffusion coefficient measurements of HRP immobilized Janus

particles without linker 24

Table 2 2 Diffusion coefficient measurements of HRP immobilized Janus

particles in the presence of linker 24

Table 3 1 Observations for formation of fan-shaped structures in enzyme

cascade system in presence of different components 34

Table 3 2 Observations for particle aggregation in enzyme cascade system in

presence and absence of TMB 37

Table 3 3 Diffusion coefficients for 05 μm GOx immobilized particles in 01 M

glucose purged with O2 38

Table 3 4 Diffusion coefficients for GOx and HRP immobilized particles in 01

M glucose and purged with O2 38

Table 4 1 Translational diffusion coefficients of Ag-TiO2 particles in hydrogen

peroxide and water 49

x

ACKNOWLEDGEMENTS

I would like to thank my advisor Prof Ayusman Sen for his valuable guidance

throughout my research at Penn State He was not only inspiring and encouraging

regarding work but was also very supportive and understanding about life outside work

I would also like to thank my committee members Prof Tom Mallouk and Prof

Christine Keating for being available when I needed guidance and for providing their

valuable advice

I would like to thank my Mom Dad sister and other family members for being

supportive of my decision to come to United States to pursue my graduate studies and for

always being available to share my excitements and disappointments in graduate life I

cannot thank my friend Mahati Elluru enough for her encouraging talks constructive

criticism and praises genuine caring and the list can go on I would like to thank Ambuj

Sharma and Bhavana Achary for being a major part of my life during graduate school and

for making State College feel like home I would like to thank Samudra Sengupta for

being a good friend and mentor who was always around in lab and outside to provide his

valuable insights I would also like to thank Shakuntala Sundararajan Yiying Hong

Michale Ibele Natallia Kulyba and other Sen group members for their support during

graduate school and for creating an enjoyable working environment in the lab

1

Chapter 1

Introduction

Motion at the macroscale has been studied and applied for centuries Invention of the

wheel can be considered as the major step for the beginning of synthetic motors at

macroscale The potential applications of the wheel and other similar motors are still

being explored and seem to have endless possibilities Having said that motion at the

nanoscale is just beginning to get explored and can have similar if not greater

implications in the future This thesis is a part of an attempt to develop nanomotors made

from diverse materials operating in different environments to create a pool of

nanomotors which can be used in the future for specific applications

Some of the nanomotors which have already been developed include the pioneering

work by WFPaxton etal of platinum-gold nanomotors1 showing autonomous motion in

hydrogen peroxide motors made by Golestanian2 consisting of micron sized particles

half coated with Pt and others The mechanisms of motion differ in case of different

motors but all have some basic underlying principles which will be highlighted in the

following section An important point to notice in the above mentioned nanomotors is

that they involve metals or solutions which are not biocompatible This puts a restriction

on the application of such motors This thesis attempts to synthesize enzyme based

nanomotors which will be able to overcome these drawbacks and lead to applications in

biological systems like drug delivery and surgery in human body

2

Second part of this thesis consists of synthesis and motion of silver-titania nanomotors

which can move autonomously in hydrogen peroxide solution According to some recent

findings3 colloidal silver and hydrogen peroxide may prove to be an effective

combination for treatment of infections in human body Combination of silver particles

and hydrogen peroxide has been tested for disinfection of drinking water and may hold

promise as a secondary disinfecatant which can provide long lasting residual and biofilm

control that is required in water distribution systems4 Silver-titania nanomotors can open

interesting avenues in such applications

11 Motion at the nanoscale

Motion as we understand at macroscale is significantly different from motion at

micronnanoscale A definite discontinuity exists between the two This can be

emphasized by observing the biological systems surrounding us For example at the size

of a cell which is around 10 μm mixing of large molecules is instantaneous due to

diffusion and there is no need for additional convective mixing which is what we

generally think of when we picture macroscopic mixing For instance while convective

flow driven by the pumping of lungs or the use of gills is essential for transport of oxygen

inside an organism which is larger than 1mm in size such as a lizard or a man small

organisms receive nutrients and oxygen solely through diffusion through their tissues5

These differences in motion can be understood both physically and mathematically

Physically as the size of an object decreases the mass and weight decrease with the third

3

power of linear size A large body predominantly has gravitational pull of earth acting on

it due to its large mass and other forces like gravitational effect of surrounding objects are

relatively insignificant As the object gets smaller forces such as molecular attraction and

cohesion become more significant and the relative magnitude of the gravitational pull of

the earth on the object decreases due to reduction in mass5 Thus the forces that need to

be considered to cause motion at the macroscale are different than those at the

microscale

Mathematically the difference in motion at macro- and micro- scales can be explained by

means of a dimensionless number called Reynolds number Reynolds number is the ratio

of inertial forces of a system to its viscous forces As size decreases Reynolds number

decreases and the viscous forces become more dominant For a human swimming in

water Reynolds number is around 104 for goldfish it is around 10

2 whereas for an

organism of 1μm size it is around 10-4

to 10-5

[67]

What this means is that as we scale

down from the size of a goldfish to micron sized organisms the inertial forces become

insignificant and the familiar laws of macroscale motion lose their applicability Equation

11 gives the formula for calculating Reynolds number where ρ is density of the fluid

(kgmsup3) μ is the dynamic viscosity of the fluid (kg(mmiddots)) V is the mean fluid velocity

(ms) and D is diameter of particle (m)

Equation 1 1

To understand the implications of low-Reynolds-number systems we can consider the

motion of a macroscopic and an imaginary microscopic scallop Macroscopic scallop

4

moves by opening and closing of its shell Inertial thrust is created when a scallop closes

its shell and the scallop moves forward due to the clapping motion (Figure 1 1) This

however does not hold true at the nanoscale since the inertial forces are relatively

insignificant due to the large viscous forces If a microscopic scallop were to exhibit a

similar clapping motion it will just keep moving forward and backward in an oscillatory

kind of motion and will not experience a net motion This is an example of reciprocal

motion which can linearly move a body at macroscale however fails to cause motion at

the microscale

For motion at microscale nonreciprocal motion is necessary8 Nonreciprocal motion

occurs when the path chosen for going from Position A to Position B is different from the

path chosen to come back from Position B to Position A It can be better understood with

the help of Figure 1 1 which shows the change of shapes for a theoretical object going

from Position A to Position B and then returning to Position A by using a different set of

motions This kind of change can lead to motion at the nanomicron scale

5

Another factor which gains importance at nanoscale is the dominance and ubiquity of

Brownian motion13

The trajectory of a linearly propelled nanomotor would be

significantly affected by the continuous collision of randomly moving particles in the

environment These collisions deviate the nanomotor from its linear path and hence this

effect needs to be considered while projecting the path of a nanomotor

Motion at the nanoscale can be powered by different types of mechanisms such as

diffusiophoretic electrophoretic osmophoretic chemophoretic thermophoretic and

others Different types of mechanisms of motions may coexist or act individually to cause

Position A Position B

Figure 1 1 Reciprocal and Nonreciprocal motion8

Reciprocal motion

Nonreciprocal motion

6

motion Diffusiophoretic motion of particles is caused due to concentration gradients of

solutes in an electrolytenonelectrolyte solution9 Electrophoresis is the motion of a

particle relative to a fluid under the influence of an electric field10

This occurs as the

particle carries an electric surface charge which is acted upon by an electrostatic

coulombic force of the external electric field Osmophoresis is the motion of a

vesiclefluid particle encapsulated in a semi-permeable membrane towards lower

concentration gradient when it is placed in a solution containing solute concentration

gradient1112

Thus considering the numerous mechanisms of motion known to work at

the nanoscale several suitable options are available for the development of artificial

nanomotors

12 Existing nanomotors Mechanisms and uses

Several nanomotors have been developed in the past decade or so A general principle of

working of these nanomotors is that they function by catalytically decomposing the

dissolved fuel to generate asymmetry at their surface or at their interfacial region thereby

causing propulsion13

The self-electrophoretic bimetallic nanowires developed by

WPaxton and A Sen etal consisted of a 2 μm long rod with one tip made up of Au and

the other tip made up of Pt1 These motors were synthesized by electrodeposition in an

alumina template The rods moved autonomously in a solution of hydrogen peroxide

with platinum end forward Pt oxidizes H2O2 to produce H+ and O2 and Au utilizes H

+ to

reduce H2O2 to water This generates an electron flow through the rod and a proton flow

outside the rod resulting in an electric field around the rod in the solution This electric

7

field in solution acts on the unbalanced charges within the nanorodrsquos double layer This

moves the nanorod with respect to the surrounding fuel due to the ion flux that is

generated

Bubble propulsion has been predominantly used as mechanism of motion in the motors

developed by Feringa etal His group developed a micromotor by attaching catalase to a

microparticle and placing the particle in a solution containing hydrogen peroxide This

system allowed the microparticle to move by producing oxygen bubbles from the

decomposition of hydrogen peroxide14

A similar motor was later created by Feringarsquos

group which consisted of glucose oxidase and catalase immobilized on a carbon

nanotube in a substrate solution of hydrogen peroxide Glucose oxidase converted

glucose to H2O2 which was then utilized by catalase to produce water and oxygen

thereby propelling the carbon nanotube15

Another interesting example of a self-propelled motor is the polymerization driven motor

developed by Cameron et al16

It consists of polystyrene beads immobilized with ActA

protein which catalyzes actin polymerization leading to the formation of an actin

filament which resembles the flagella of bacteria This actin polymerization propels the

beads at the speed of 001-015 μms

A theoretical model has been proposed by Nafaji and Golestanian etal which consists

of three spheres linked by rigid rods in one dimension17

The length of the rods can

switch between two specific values Due to the nonreciprocal motion of the rod this

8

model can swim in low Reynolds number systems An actual model based on

nonreciprocal motion has been developed by Kim etal in which heart cells have been

linked to an asymmetric polydimethylsiloxane robot As the heart cells beat the

asymmetric structure of the device resembles a walking movement18

Most of the nanomicromotors mentioned above are either metallic or work in

environments not compatible with human body Enzymes being an integral part of the

body can solve this problem by operating in suitable substrates already present in the

body fluids The nonreciprocal conformation changes of the enzyme as well as

diffusiophoresis and electrophoresis can conceivably be used to generate motion in case

of enzyme driven nanomotors such motors will be discussed in detail in the following

chapter

9

Chapter 2

Immobilized enzymes on Janus particles as catalytic motors

21 Introduction

Enzymes are widely found in biological systems and act on a large variety of substrates

This can make enzymes an ideal choice for powering nanomotors which need to be

biocompatible Several fluids found in the body can act as substrates to the enzymes For

example dissolved glucose which is abundantly found in the body19

is a susbtrate for

enzyme glucose oxidase Urea which is found in the urinary tract20

is a substrate for

urease Hydrogen peroxide produced by phagocytic cells in the body21

is a substrate for

horseradish peroxidase Having motors driven by enzymes can open possibilities of

targeted drug delivery to these parts of the body containing such substrates

Enzymes have already been used to generate motion of carbon nanotubes by mechanism

of bubble propulsion by Feringa etal15

Two other possible mechanisms which may be

able to power motion of nanoparticles using enzymes are asymmetrical decomposition of

substrates into products leading to self-diffusiophoresis (Figure 2 1) and nonreciprocal

changes in enzyme conformation leading to propulsion (Figure 2 2) As seen in Figure 2

1 enzymes which are immobilized on a Janus particle can breakdown the available

substrate in the solution into products causing asymmetric distribution of solutes in the

surrounding fluid This can lead to movement of particle towards the region of lower

10

solute concentration by principle of self-diffusiophoresis Figure 2-2 shows the possible

nonreciprocal changes in the conformation of a theoretical protein which can lead to

motion of the protein22

These nonreciprocal changes in conformation are triggered by the

attachment of substrate molecule and release of product molecules from the enzymersquos

active site

Figure 2 1 Enzyme functionalized Janus particle moves upon catalyzing asymmetrical

decomposition of substrate into product

11

Figure 2 2 Schematic showing idealized nonreciprocal conformation changes in a

theoretical enzyme22

Enhanced diffusion of a single enzyme molecule by catalysis in a substrate solution has

been demonstrated23

The diffusion coefficient of a single urease molecule was measured

using fluorescence correlation spectroscopy and it was observed that the diffusion of the

urease molecule increases by 16-28 by varying urea concentrations from 0001 M to 1

M Addition of enzyme inhibitors to the system curbed the enhanced diffusion This

enhanced diffusion has been attributed to the force generated by self-electrophoresis by

the enzyme in substrate solution

12

Based on the above facts it might be possible to propel a nanoparticle by asymmetric

distribution of enzymes on its surface thereby leading to self-propulsion of the particles

in substrate solution (Figure 2 1) This propulsion may be due to self-

electrophoresisself-diffusiophoresis andor due to nonreciprocal changes in enzyme

conformations Here we aim to investigate a system consisting of polystyrene particles

half coated with enzyme introduced in a suitable substrate solution The enzyme can be

directly linked to the polystyrene particle or it can be linked through a long spacer

molecule (linker) to provide more degrees of freedom to the enzyme

Several factors need to be considered for choosing enzyme for powering motion at the

nanoscale Some of the important factors are the ratio of kcatKm which denotes the

catalytic efficiency of the enzyme possible substrates stability of enzymessubstrates

ease of immobilization relationship with other enzymes in cascade reactions and

commercial availability of the enzymes The enzyme being used in the present study is

horseradish peroxidase It acts on a molecule of hydrogen peroxide and reduces it while

simultaneously oxidizing another substrate molecule In the absence of alternate

oxidizable substrate HRP can oxidize water and oxygen back to hydrogen peroxide24

thus giving an equilibrium reaction

13

211 Horseradish peroxidase

Figure 2 3 Horseradish peroxidase

Horseradish peroxidase belongs to the group of peroxidases which are a group of heme-

containing oxidoreductases that act on peroxide as hydrogen donor25

It is a redox

enzyme which contains a protoheme a complex of trivalent iron with 1358-

tetramethyl-24-divinylporphin-67-dipropionic acid as a prosthetic group It is a 44 kDa

protein which includes 33900 Da polypeptide chain bound with eight oligosaccharide

chains The carbohydrate chains improve the stability of the enzyme by protecting it

against radicals formed in the course of the reaction it catalyzes26

Horseradish

peroxidase is found in horseradish and is used widely in bioanalytical applications such

as ELISA and Western blots It decomposes hydrogen peroxide into water and oxygen

while simultaneously oxidizing another substrate27

The oxidizable substrates can be

chromogenic substrates such as 22rsquo55rsquo-tetramethyl-benzidine (TMB) and 33-

diaminobenzidine (DMB) or chemoluminescent substrates like SuperSignal and ECL

luminal28

14

Figure 2 4 Reaction catalyzed by horseradish peroxidase

The functional parameters for horseradish peroxidase include Km of 015 mM and kcat of

790 s-1

at an optimum temperature of 20 ordmC and an optimum pH of 6429

Some of the

inhibitors of HRP are 110-phenanthroline aminotriazole and bromine

22 Experimental

221 Materials and methods

Uniform carboxyl modified polystyrene microspheres 05 microm in diameter were obtained

from Polysciences Inc The size of the purchased microspheres was verified by Field

Emission Scanning Electron Microscopy (FESEM) Peroxidase Type I from

Horseradish N-(3-dimethyl-aminopropyl)-Nrsquo-ethylcarbodiimide (EDC) and hydrogen

peroxide solution 50 wt in water were purchased from Sigma-Aldrich N-

hydroxysuccinimide (NHS) ge 97 was purchased from Fluka All chemicals were of

reagent grade and used without further purification Nanopure deionized water was used

for all experiments and preparation of solutions All measurements were made at room

15

temperature (25 oC) unless mentioned otherwise The UV-Visible spectroscopy

measurements were performed using Agilent 8453A diode-array UV-Visible

Spectrophotometer with ChemStation UV-Vis Software Diffusion coefficient

measurements were performed using Nanosight LM10

222 Synthesis of Janus particles

A monolayer of particles was prepared according to the procedure (Figure 2 5) by

Goldenberg L et al30

Glass slides were placed on the bottom of a petridish Water was

put into the petridish so that the entire slide was immersed in water A thin layer of

hexane was added on top of water in the petridish A suspension of microspheres in

ethanol (5 ww) was introduced to the water-hexane interface After the monolayer was

formed the glass slide was lifted up through the monolayer with tweezers The

microsphere-covered glass slides were left to dry overnight at room temperature

Following drying a thin film of silica was evaporated on the glass slides in Semicore

evaporator The spheres were partially coated with 30 nm of silica Particles were

removed from the glass slide by gentle sonication The polystyrene-silica (PS-SiO2)

Janus particles thus obtained were washed in deionized water several times before

further immobilization steps were performed FESEM was used to confirm synthesis of

Janus particles

16

Figure 2 5 Preparation of PS-SiO2 Janus particles by evaporation

223 Immobilization of enzymes using EDCNHS coupling method

1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide and N-hydroxysulfosuccinimide

(EDCNHS) coupling procedure was followed for immobilizing horseradish peroxidase

type I on 05 μm PS-SiO2 Janus particles (Figure 2 6) In this process EDC reacts with a

carboxyl groups on the Janus particles to form an amine-reactive O-acylisourea

intermediate If this intermediate does not encounter an amine it can hydrolyze and

regenerate the carboxyl group In the presence of NHS EDC can be used to convert

carboxyl groups to amine-reactive sulfo-NHS esters which are much more stable and do

not readily hydrolyze to regenerate carboxyl groups In the current experiment

immobilization was performed in the presence as well as absence of a long spacer

17

molecule (linker) between Janus particles and enzymes The chemical 6-aminohexanoic

acid was used as the linker (Figure 2 7) HRP immobilized PS-SiO2 Janus particles were

stored in deionized water until required for further experiments

Figure 2 6 EDCNHS coupling method for immobilization of enzyme on carboxylated

polystyrene particles

Figure 2 7 Immobilization of enzyme on Janus particles with and without 6-

aminohexanoic acid linker

18

224 Enzyme assay

Assay for activity of HRP was performed using a spectrophotometric method which

involved using 4-aminoantipyrene as hydrogen donor3132

The reaction rate was

determined by measuring increase in absorbance at 510 nm resulting from the

decomposition of hydrogen peroxide One unit represents the decomposition of 1 μM of

hydrogen peroxide per minute at 25 degC and pH 70 under the specified conditions

The spectrophotometer was set to the wavelength of 510 nm and temperature of 25 degC

Phenol4-aminoantipyrene solution (14 mL) and 00017 M H2O2 solution (15 mL) were

pipetted into each cuvette The cuvette was incubated for 3-4 mins at 25degC and blank was

established A 01 mL solution of diluted enzyme was added to the above mixture and

absorbance was recorded for 4-5 mins Standard graphs of absorbance versus time were

drawn for known concentration of the free enzyme and compared to that of the

immobilized enzyme on the Janus particles The estimate of the amount of enzyme

present on the Janus particles was deduced from the difference between the quantity of

the free enzyme previously added for immobilization and the amount that was measured

to be in the supernatant after some of the enzyme became attached to the Janus particles

The experiment was carried out in triplicate to assess reproducibility

19

The formula used to calculate the activity of enzyme is

where ∆Amin denotes the change in absorbance per minute

225 Diffusion coefficient measurements using Nanosight LM10

Diffusion coefficients for HRP immobilized particles were measured using Nanosight

LM 10 nanoparticle tracking instrument with Nanoparticle Tracking Analysis (NTA) 20

software in the presence of 003 H2O2 solution as the substrate and also in the presence

of only deionized water The videos were recorded for 30 seconds at the rate of 30 frames

per second Each experiment was carried out in triplicate to assess reproducibility

Particles

visualization

zone

Microscope with

LM 10 unit

LM 10 unit

Figure 2 8 Nanosight LM 10 instrumentation

20

Nanosight LM 10 (Figure 2 8) generates videos of population of moving nanoparticles in

a liquid when illuminated by laser light The particles which pass through the laser beam

path are seen by the instrument as small points of light undergoing Brownian motion

(Figure 2 9a)

The videos are further analyzed by NTA (Nanoparticle tracking analysis) software

(Figure 2 9b) which identifies and tracks each particle independently on a frame-by-

frame basis The software also calculates and subtracts any bulk flow in the system The

mean square displacement is calculated for each particle as long as it is visible If the

particle blinks that is if it disappears momentarily and then reappears on the screen the

software will consider the same particle as two separate ones All analysis data is

exported to the excel files in terms of diffusion coefficients of the tracked particles

B A

Figure 2 9 NTA video analysis (a) Particles as they are seen on the screen during

experimentation in Nanosight LM10 (b) NTA screen as the particles are being analyzed for

their diffusion coefficients

21

23 Results

231 Enzyme assay

The activity of free HRP enzyme reported by Sigma Aldrich is 150 unitsmg However

the activity of HRP upon analysis was found to be 52158 unitsmg which is much lower

than the reported value The activity of HRP was found to be further lowered to 2133

unitsmg when it was immobilized on the PS-SiO2 Janus particles in the presence of a

linker The following formula is used to calculate enzyme activity

where ΔAmin is the change in absorbance per minute

22

Figure 2 10 Graph for the assay of solution of free enzyme containing 05mgL of HRP

ΔAmin = 01716 Unitsmg of free enzyme = 52158

During EDCNHS coupling 04 mg enzyme was added to 1 ml of 025 vv suspension

of Janus particles Thus initial concentration of enzyme in the mixture was 04 mgml

Assuming retained activity of the enzyme in supernatant solution the amount of enzyme

in supernatant was calculated to be 03868 mgml from the assay Amount of enzyme

functionalized on the Janus particles was calculated as the difference between enzyme

previously added for immobilization (04 mgml) and the enzyme present in supernatant

(03868 mgml) and was found to be 00132 mgml Enzyme assay was performed on the

enzyme immobilized Janus particles to estimate the activity of immobilized enzyme

(Figure 2 11) and was found to be 2133 unitsmg

y = 0002x + 00516

0

01

02

03

04

05

06

07

08

0 50 100 150 200 250 300 350 400

Ab

sorb

an

ce

51

0 n

m

Time (sec)

Assay of free enzyme HRP

23

Figure 2 11 Graph for the assay of of immobilized enzyme on 05 μm Janus particles

(~025 vv) ΔAmin = 01853 Unitsmg of free enzyme = 2133

232 Diffusion coefficient measurements

No significant increase was observed in diffusion coefficient of HRP-immobilized Janus

particles in the presence of H2O2 as the substrate Similar results were obtained for

diffusion coefficient measurement with and without linker moieties bridging the enzymes

and particles(Table 2 1 Table 2 2) Thus the linker did not seem to assist in increasing

the particle diffusion by improving or retaining the activity of the enzyme

y = 00015x + 00953

0

01

02

03

04

05

06

07

0 50 100 150 200 250 300 350 400

Ab

sorb

an

ce

510 n

m

Time (sec)

Assay of HRP immobilized 05 μm Janus particles

24

Table 2 1 Diffusion coefficient measurements of HRP immobilized Janus particles

without linker

HRP immobilized

Janus particles in

003 H2O2

Control - Janus

particles in

003 H2O2

Control - HRP

immobilized Janus

particles in DI water

Diffusion coefficient

(10-10

cm2s)

1359416 1051777 1308675

Standard deviation 4534801 4122075 4918597

Table 2 2 Diffusion coefficient measurements of HRP immobilized Janus particles in

the presence of linker

HRP immobilized

Janus particles in

003 H2O2

Control - Janus

particles in

003 H2O2

Control - HRP

immobilized Janus

particles in DI water

Diffusion coefficient

(10-10

cm2s)

13838 1339829 1407891

Standard deviation 5644692 5649364 5797218

25

Figure 2 12 Diffusion coefficient measurements of PS-SiO2 Janus particles without and

with 6-aminohexanoic acid linker

24 Discussion

To compare the diffusion coefficients observed during the experiments theoretical

diffusion coefficient was calculated using following equation33

r

kTD altraslation

6 Equation 21

where r is the hydrodynamic radius of particle Dtranslational is translational diffusion

coefficient of the particle T is temperature of the system η is the viscosity of fluid

through which the particles are moving and k is the Boltzmann constant From FE-SEM

images the size of the particles used was determined to be 450 nm For 450 nm diameter

0

20

40

60

80

100

120

140

160

Without linker With linker

Dif

fusio

n c

oeff

icie

nt

(10

-10 c

m2s

)

HRP immobilized 05 μm Janus particles - Diffusion coefficient measurements

HRP immobilized Janus particles in 003 H2O2

Control - Janus particles in 003 H2O2

Control - HRP immobilized Janus particles in DI water

26

particle Dtranslational is equal to 109 μm2s which is comparable to the diffusion

coefficient measured for the enzyme immobilized particles

HRP was found to be active on the Janus particles however the activity of the enzyme

was reduced upon immobilization Lower enzyme activity can be due to the coupling

method used which in this case was EDCNHS covalent binding However EDCNHS

coupling method has been shown to significantly retain the enzyme activity when the

enzyme is immobilized on carboxylated multi-walled carbon nanotubes under appropriate

conditions34

Hence EDCNHS binding method by itself is not believed to be

responsible for reduction of enzyme activity upon immobilization

Direct binding of enzyme to the Janus particle may lead to reduction in the enzyme

activity because of space constraints for the enzyme and difficulty for the passage of

substrate to the active site of the enzyme due to stearic hindrance Hence a linker was

inserted between the enzyme and Janus particle during enzyme immobilization However

inserting a linker between the enzyme and the particle did not lead to enhanced diffusion

of Janus particles or increase in enzyme activity of immobilized enzyme

Lack of enhanced diffusion can be firstly attributed to the reduction in enzyme activity

upon immobilization Enzyme activity may be retained by modifying the conditions

during EDCNHS coupling or changing the binding method altogether The degree of

immobilization of HRP on carboxylated Janus particles can possibly be affected by

several factors such as temperature the concentration of the coupling reagent EDC

27

concentration HRP concentration in solution and incubation time34

Different

combinations of these factors can be tested to find optimum conditions for

immobilization of HRP Alternate binding method can involve thiolation of HRP by the

covalent conjugation of mercaptopropionic acid and then the thiolated HRP can then be

attached on the gold surface of a polystyrene-gold Janus particle35

Thus further

modifications can be made to the system in order to retain enzyme activity on the

immobilized particles and thereby power nanomotors using enzymes

28

Chapter 3

Predator-prey system of enzyme immobilized particles

31 Introduction

Predator-prey systems are widely found in nature and play a major role in biological

processes3637

Predator-prey systems include all the interactions in which one organism

pursues andor consumes another organism One familiar predator-prey system at the

macroscale is that of a tiger chasing and consuming a deer Another not so familiar one

includes Baleen whales which eat millions of microscopic planktons at once38

At the

microscale such kind of interactions can be observed in the case of phagocytes in the

body which are capable of engulfing foreign microorganisms and killing them with a

combination of microbicidal systems39

In the case of cancer the tumor is the prey and

the mature myeloid cells in the body are the predator However the tumor here has a

defense mechanism against the predator in which the tumor produces cytokines which

inhibits the maturation of the myeloid cells and thereby prevents predation40

One similarity that can be observed in all these systems is that the prey possesses some

attribute which is desired by the predator which makes the predator chase the prey A

similar relationship is seen in case of enzymes which are involved in cascade reactions41

In cascade reactions product synthesized by one enzyme is the reactant required by

another enzyme to carry out its own catalytic reaction As such there is a predator-prey

29

kind of dependence between the two enzymes which may cause one enzyme to chase the

other enzyme when placed in suitable substrate solutions However the mechanism

hypothesized for such a system is totally different than the mechanism involved in

biological systems In biological systems such as bacterial chemotaxis or phagocytes

chasing foreign particles in human body complex biochemical reactions are responsible

for the predator-prey behavior42

In the proposed enzyme cascade system there are no

complex biochemical reactions involved The principle hypothesized for predator-prey

behavior of enzyme cascade reactions can be explained as follows Motion of synthetic

motors at nanoscale involves two components translational diffusion and rotational

Brownian diffusion At very short time scales of less than 1Dr where Dr (s-1

) is

rotational Brownian diffusion coefficient the motion of particles is unidirectional For

longer time scales of greater than 1Dr the direction of motion is random giving

poweredactive diffusion43

If the chemical homogeneity is broken that is when there is

higher concentration of a substrate present in one direction of the enzyme the enzyme

might acquire a subtle bias which may lead to a spatial aggregation of the enzymes based

on the substrate concentration gradient The bias can be a result of the relationship

between the speed of enzyme diffusion and substrate concentration where higher

substrate concentration lead to higher enzyme diffusion As a result in an enzyme

cascade system one enzyme will migrate towards higher concentration of substrate

which is present near the other enzyme thus giving rise to a predator-prey behavior

Such enzyme cascades are widely found in signaling networks and metabolic pathways in

living cells and can be used to produce predator-prey like nanomotors systems44

The

30

enzymes being used in the present study are horseradish peroxidase and glucose oxidase

Both enzymes are extremely stable4546

Glucose is the substrate for glucose oxidase

which is widely available and highly stable47

Horseradish peroxidase acts on hydrogen

peroxide It is involved in a cascade reaction with glucose oxidase which produces

hydrogen peroxide by converting glucose into gluconic acid (Figure 3 1)48

Figure 3 1 Concept of particle aggregation due to cascade reactions of immobilized

enzymes

In this experiment glucose oxidase (GOx) and horseradish peroxidase (HRP) were

immobilized on polystyrene carboxylate beads of different sizes using EDCNHS

coupling method Glucose and 3355-Tetramethylbenzidine (TMB) were dissolved

together in water and this solution was used as the substrate solution All the components

were present in a single system in DI water Aggregation of particles and precipitation of

oxidized TMB from the solution was expected to be observed when all these components

were present in such a system

Properties of horseradish peroxidase have been discussed in the previous chapter (Section

211 Horseradish peroxidase)

GOxGOx HRPHRP

Glucose + O2Gluconic acid + H2O2

TMB TMB oxidizedBlue precipitate

H2O

Aggregation

31

311 Glucose oxidase

Figure 3 2 Glucose oxidase

Glucose oxidase (GOx) belongs to the group of oxidoreductases and is also called

glucose aerodehydrogenase49

It is obtained from fungi and has been identified as an

antimicrobial enzyme50

GOx binds to β-D-glucopyranose and catalyzes the oxidation

reaction to from gluconolactone while simultaneously reducing oxygen to hydrogen

peroxide51

GOx has high substrate affinity and specificity high stability and high

turnover number which makes it a suitable biocatalyst for industrial applications52

Figure 3 3 Redox reaction of glucose catalyzed by glucose oxidase53

32

GOx is a glycoprotein with molecular weight of 155 kDa and consists of two identical

polypeptide subunits linked together by a disulfide bond The optimum pH for GOx is

55 though it is stable in a broad pH range of 4 to 7 and can be stored for a long period of

time51

The functional parameters for glucose oxidase include Km of 151 mM and kcat of

337 s-1

at temperature of 20 ordmC and pH of 754

Some of the inhibitors of GOx are 2-

Deoxy-D-glucose adenine nucleotides and NaNO3

32 Experimental

321 Materials and methods

Uniform carboxyl modified polystyrene microspheres 05 microm in diameter were obtained

from Polysciences Inc The size of the purchased microspheres was verified by FESEM

Peroxidase Type I from horseradish glucose oxidase Type II-S from Aspergillus niger

N-(3-dimethyl-aminopropyl)-Nrsquo-ethylcarbodiimide (EDC) and Hydrogen peroxide

solution 50 wt in water were purchased from Sigma-Aldrich N-hydroxysuccinimide

(NHS) was purchased from Fluka D-Glucose (99) was purchased from VWR

International Secure-Seal imaging spacers (9mm diameter) for glass slides were obtained

from Sigma Aldrich All chemicals were of reagent grade and used without further

purification Nanopure deionized water was used for all experiments and preparation of

solutions All measurements were made at room temperature (25 oC) unless mentioned

otherwise The UV-Visible measurements were performed using Agilent 8453A diode-

array UV-Visible Spectrophotometer with ChemStation UV-Vis Software Optical

33

microscopy experiments were performed using Axiovert 200 MAT microscope by Carl

Zeiss Diffusion coefficient measurements were obtained using Nanosight LM10

322 Optical microscopy experiments

The enzyme substrate system was observed under optical microscope to check for

aggregation and precipitation due to cascade reactions Several such systems were

studied Each system in general consisted of GOx and HRP immobilized on different

polystyrene particles The substrate solutions consisted of 01 M glucose and different

concentrations of TMB solution The systems were studied in the presence of all the

constituents and in the absence of glucose GOxHRP and TMB Systems consisting of

different sized HRP immobilized particles in substrate solution of 003 hydrogen

peroxide and TMB were also studied Videos were recorded over a period of 24 hours to

observe changes in the system Each experiment was performed atleast thrice to assess

reproducibility of results

323 Diffusion coefficient measurements using Nanosight LM10

Diffusion coefficient of GOx and HRP immobilized particles were measured using

Nanosight LM10 nanoparticle tracking instrument with Nanoparticle Tracking Analysis

(NTA) 20 software Diffusion coefficients were measured for different systems namely

GOx particles in 01 M glucose and oxygen both GOx and HRP functionalized particles

in 01 M glucose and oxygen along with controls for the individual systems Videos were

34

recorded for 30 seconds at the rate of 30 frames per second Each experiment was carried

out in triplicate to assess reproducibility

33 Results

331 Optical microscopy

According to the enzyme cascade model precipitation of TMB was possible only upon

the interaction of GOx and HRP immobilized particles in presence of appropriate

substrate solution Hence in the first set of experiments precipitation of TMB was used

as an indication of the type of interaction between GOx and HRP immobilized particles

Fan-shaped structures were observed when both HRP and GOx immobilized particles

were present in a substrate solution consisting of glucose and TMB These structures

started forming as a single strand and then branched out and intertwined to form the fan-

shaped structures (Figure 3 4)

Table 3 1 Observations for formation of fan-shaped structures in enzyme cascade

system in presence of different components

GOx HRP Glucose TMB Observation

Yes Yes Yes Yes

Fan-shaped structures observed particles associated with

the fan-shaped structures

No Yes Yes Yes No fan-shaped structures

Yes No Yes Yes No fan-shaped structures

Yes Yes No Yes No fan-shaped structures

Yes Yes Yes No No fan-shaped structures

35

Fan-shaped structures can also be observed in the system consisting of only HRP

immobilized particles in substrate solution of hydrogen peroxide and TMB The size of

the fan-shaped structures depends on the size of the enzyme immobilized particles Free

enzymes show extremely tiny structures which are difficult to view under the optical

microscope

Figure 3 4 Formation of fan-shaped structures when both HRP and GOx

immobilized particles were present in a substrate solution consisting of glucose and

TMB A In the initial stages single strands form on the surface of liquid (5Ox) B

Single strands branch out and intertwine to form fan-shaped structures (50x) C Many

such structures form and settle down after 20 minutes (20x)

A

C

B

36

Experiments were performed in the absence of TMB that is the system consisted of GOx

and HRP immobilized particles in glucose substrate solution In the absence of TMB the

particles were expected to move closer to each other without any precipitation due to the

cascade reaction However there was no conclusive aggregation of particles observed in

the system

Free Enzyme 005 μm HRP - immobilized particle

10 μm HRP - immobilized particle 05 μm HRP - immobilized particle

Figure 3 5 Size dependence of fan-shaped structures on the size of HRP -

immobilized particles (20x)

37

Table 3 2 Observations for particle aggregation in enzyme cascade system in presence

and absence of TMB

Glucose GOx HRP TMB Observation

Present Present Present Present Aggregation and precipitate

Present Present Present Absent No conclusive aggregation

Present Absent Present Present No aggregation or ppt

Absent Present Present Present No aggregation or ppt

Present Present Absent Present No aggregation or ppt

332 Diffusion coefficient measurements

Diffusion coefficients were measured for GOx immobilized particles of 05 μm diameter

in substrate solution of 01 M glucose and oxygen Diffusion coefficients of particles

were also measured for system consisting of GOx and HRP functionalized particles in

glucose and oxygen Each experiment was done in triplicate to assess reproducibility

Nanosight visualization chamber being a closed chamber oxygen was purged into all the

solutions before starting the experiments to ensure sufficient supply of oxygen for

catalytic reaction of GOx

38

Table 3 3 Diffusion coefficients for 05 μm GOx immobilized particles in 01 M

glucose purged with O2

Mean

GOx

particles in

01 M

glucose

Control - GOx

particles in DI

water

Control - GOx

particles in 01 M

glucose without

oxygen purging

Control -

particles in 01

M Glucose

Diffusion

coefficient

(x10-10

cm2s)

13316 12888 13827 13702

Standard

deviation 6307 4926 4424 7468

Table 3 4 Diffusion coefficients for GOx and HRP immobilized particles in 01 M

glucose and purged with O2

Mean

GOx- HRP-

functionalized

particles in 01 M

glucose

Control ndash Gox-

HRP- functionalized

particles in DI water

Control - particles

in 01 M glucose

Diffusion coefficient

(x10-10

cm2s)

13005 14434 13702

Standard deviation 5263 6622 7468

No significant increase was observed in diffusion coefficient of enzyme immobilized

particles in any of the systems

39

34 Discussion

Formation of fan-shaped structures due to patterned deposition of oxidized TMB

indicates some kind of interaction between GOx and HRP immobilized particles Particle

diameter dependence of the size of structures can be explained with a simple model in

Figure 3 6 A larger particle will have a higher surface area as compared to a smaller

particle Hence more enzyme is immobilized on the larger particle As a result higher

quantity of TMB is oxidized and precipitated by the larger particle as compared to the

smaller particle This leads to thicker strands of fan-shaped structures eventually scaling

up the whole system to give bigger fan-shaped structures

The exact reason for the formation of such fan-shaped structures which are dependent on

particle size could not be determined It is hypothesized that hydrogen peroxide andor

Figure 3 6 Model for size dependence of fan-shaped structures on the particle

diameter of enzyme immobilized particles Larger the particle more surface area is

available for precipitation of TMB which eventually leads to bigger fan-shaped

structures

Larger particle Smaller particle

40

TMB gradient in the substrate solution might be responsible for such structures If there

is higher concentration of H2O2 on one side of the particle and assuming an even

concentration distribution of TMB there will be selective precipitation in the direction of

H2O2 When several particles come in the vicinity of higher concentration of H2O2 the

precipitation lines will converge to give fan-shaped structures (Figure 3 7)

Figure 3 7 Hypothesis for formation of fan-shaped structures

In the system consisting of GOx immobilized particles HRP immobilized particles

glucose and TMB it was observed that the fan-shaped structures formed at the liquid-air

interface and eventually settled down to the bottom of the glass slide This makes sense

as oxygen is necessary for the catalytic reaction of conversion of glucose to gluconic acid

and hydrogen peroxide which takes place on the surface of GOx immobilized particle As

a result H2O2 is primarily formed at the liquid-air interface and is then used by HRP for

precipitation of TMB

Following the observations that precipitation of TMB occurs at the surface GOx and

HRP immobilized particles are also expected to interact at the surface in the absence of

TMB Due the cascade reaction it was expected that HRP immobilized particles will be

41

attracted to GOx immobilized particles in appropriate substrate solution Experiments

were carried out to visualize inter-particle interactions at the liquid-air interface

However liquid-air interface not being a stable system to image the precise interaction

of GOx and HRP immobilized particles could not be determined Significant external

disturbances were involved at the interface To enable the cascade reaction in the bulk of

the liquid oxygen was purged into the reaction system before starting the experiment

However this did not lead to significant reaction in the bulk of the liquid and the reaction

prominently took place at the liquid-air interface where the oxygen concentration was

higher

In order to observe the interaction of GOx and HRP immobilized particles better imaging

techniques need to be used to observe diffusion of particles in absence of external

disturbances at the liquid-air interface Large quantity of oxygen needs to be purged into

the system and retained to make the reaction more pronounced in the bulk of the substrate

solution Higher concentration of oxygen in water may also be achieved by using oxygen

enriched water which is commercially available55

Alternatively a different enzyme cascade system may be used which does not require a

gaseous substrate56

For example glycogen synthase synthesizes glycogen from glucose-

1-phosphate and glycogen phosphorylase breaks down glycogen to glucose-1-

phosphate57

This system does not require a gaseous substrate and might be able to

overcome the deficiencies of GOx and HRP enzyme cascade system

42

Chapter 4

Silver-titania nanomotors

41 Introduction

Bimetallic nanowires made of platinum and gold have been developed by WPaxton

etal1 The rods move autonomously in a solution of hydrogen peroxide In the present

study silver-titania (Ag-TiO2) particles are being synthesized and investigated for motion

in presence of hydrogen peroxide and UV light With proper control of conditions and

UV light trigger it can be possible to control the speed and direction of motion of these

nanoparticles

Hydrogen peroxide is produced in the human body by phagocytes as an immune response

to foreign particles and pathogens5859

Also silver nanoparticles have been shown to be

biocompatible and capable of interacting with cells in human body without disrupting

cellular function60

Similarly it has been demonstrated that titania is compatible with

different cells DNA and proteins in human body and does not cause toxic effects Hence

silver-titania nanomotors which swim in hydrogen peroxide may open interesting

avenues in applications like targeted drug delivery in those parts of the body where

hydrogen peroxide is present

43

42 Experimental

421 Materials and methods

Silver nitrate (999995) was purchased from Alfa Aesar Gum arabic was purchased

from MP Biomedicals Hydrogen peroxide solution 50 wt in water and ascorbic acid

were purchased from Sigma-Aldrich Nanopure deionized water was used for all

experiments and preparation of all solutions Secure-Seal imaging spacers (9 mm

diameter) were obtained from Sigma Aldrich All experiments were carried out at room

temperature of 25ordmC unless otherwise mentioned Ag-TiO2 particles were synthesized by

evaporation of titania in Semicore electron beam evaporator Particles were characterized

using field-emission scanning electron microscopy Optical microscopy experiments

were performed using Axiovert 200 MAT microscope by Carl Zeiss Particles were

tracked using Physvis particle tracking software (Figure 4 1) in which the position

information of a particle on screen is stored on a frame by frame basis The data is stored

in a dat file as X and Y co-ordinates of particles tracked on the screen This information

can be converted to diffusion coefficients of the particles by further manipulation using

MathWorks-MATLABreg software

44

Figure 4 1 Physvis particle tracking software

422 Synthesis of silver colloidal particles

Silver particles were synthesized by a nucleationgrowth type reaction developed by

Goya and Matijevic61

and modified by Velikov62

Silver nitrate was used as precursor

metal salt for synthesis of silver particles Solution A was 75 ml of 50 mM AgNO3 and

035 by weight gum arabic Solution B was 75 mL of 100 mM 1-ascorbic acid and

035 by weight gum arabic Solution B was rapidly added to solution A in a 250 mL

reaction flask The reaction flask was covered and particles were stirred for 48 hrs After

48 hours particles were allowed to settle for 2-3 hours in refrigerator and supernatant

was separated from the particles Particles were then rinsed with DI water and centrifuged

thrice to obtain pure silver particles Zeta potential of these particles was reported to be

45

around -50 to -60 mV and the size was around 1μm The size of the particles can be

varied by varying the amounts of ascorbic acid and silver nitrate Using molar ratio of 31

of ascorbic acid silver nitrate can give particles of 200 nm diameter whereas ratio of

151 can give particles of upto 5 μm diameter

423 Preparation of silver-titania (Ag-TiO2) particles

A monolayer of silver particles was prepared according to the procedure by Goldenberg

et al63

Glass slides were placed on the bottom of a petridish Water was put into the

petridish so that the entire slide was immersed in water A thin layer of hexane was then

added on top of water in the petridish A suspension of silver particles in ethanol (5

ww) was slowly introduced to the water-hexane interface in the dish After the

monolayer was formed the glass slide was lifted up through the monolayer with tweezers

The silver particles-covered glass slides were left at room temperature to dry overnight

Following drying a thin film of titania was evaporated on the glass slides in Semicore

evaporator (Figure 2 7) The spheres were partially coated with a 10 nm film of titania

Ag-TiO2 particles were removed from the glass slide by gentle sonication FE-SEM was

used to characterize the particles (Figure 4 2) The thin titania layer was not easily

distinguishable due to roughness of the particles

46

Figure 4 2 FESEM image of Ag-TiO2 particles

424 Optical microscopy experiments

Imaging spacers were placed on a glass slide Suspension of Ag-TiO2 particles was

placed on the glass slide and the particles were allowed to settle Movement of particles

was observed in DI water and videos were recorded at 50x magnification of optical

microscope for 30 sec 05 H2O2 was added to the particle suspension on the slide

Videos were recorded at 50x ranging from 30 sec to 10 min to study the movement of

silver titania particles in hydrogen peroxide solution Particles in both DI water and

hydrogen peroxide were subjected to UV light and videos were recorded to study particle

dynamics in presence of UV radiation Particle tracking was performed using Physvis

particle tracking software Atleast 30 particles in every system were tracked for 10

seconds each Particle tracking data was obtained in terms of X and Y coordinates of

particles on screen This data was manipulated using MathWorks-Matlab software and

graphs of mean square displacement of particles versus time were plotted Diffusion

coefficients of particles were calculated from these graphs

47

43 Results and Discussion

Ag-TiO2 particles demonstrated autonomous motion in the presence of H2O2 No UV

light was required for this motion Motion of the particles was accelerated by addition of

fresh H2O2 to the system When subjected to UV radiation the particles displayed

momentary acceleration for 4-5 seconds

Figure 4 3 Mechanism of motion of Ag-TiO2 nanomotors

In the presence of H2O2 silver part of the Ag-TiO2 particle converts H2O2 to H2O and O2

This may lead to propulsion of the Ag-TiO2 particle due to bubble propulsion andor self-

diffusiophoresis In the presence of UV light additional reaction takes place on the

titania part of Ag-TiO2 particle which increases the velocity of the Ag-TiO2 particles

(Figure 4 4) This UV triggered photoactivity of titania is due to hole-electron separation

caused by photons having energy equal to or greater than the band gap of titania646566

Most of the holes-electrons recombine but a small fraction move to the surface and react

with the available redox species like water and oxygen to form protons (H+) superoxide

(O2-) and hydroxyl (OH) radicals

6566 This may lead to propulsion by diffusiophoresis

as well as electrophoresis

48

Figure 4 4 Silver-titania motors move faster when subjected to UV radiation

In addition to linear motion rotational motion is also observed in several particles and

particle aggregates Aggregation of particles can lead to a net circular propulsive force

which can rotate the aggregate as shown in Figure 4 5

Figure 4 5 Rotation due to particle aggregation of Ag-TiO2 particles

Particle tracking by Physvis software revealed a significant increase in diffusion

coefficient of Ag-TiO2 particles in hydrogen peroxide solution as compared to DI water

49

The Ag-TiO2 particles have a diffusion coefficient of 1491 μm2s in hydrogen peroxide

versus only 180 μm2s in DI water Thus the Ag-TiO2 particles move around more than

10 times their body length per second in hydrogen peroxide (Figure 4 6) When

subjected to UV light the particles experience a significant acceleration in hydrogen

peroxide which lasts only for a short duration of time (4-5 sec) after which the particles

return to their original velocity The diffusion coefficient of the particles during this UV

light trigger is found to be around 5093 μm2s UV light trigger does not have any visible

effect on the Ag-TiO2 particles in DI water

Table 4 1 Translational diffusion coefficients of Ag-TiO2 particles in hydrogen

peroxide and water

Mean 05 H2O2 solution DI water

Diffusion coefficient of Ag-TiO2 (μm2s)

without UV light

1491 180

Diffusion coefficient of Ag-TiO2 (μm2s)

in the presence of UV light

5093 113

50

Figure 4 6 Mean square displacement curves of Ag-TiO2 Janus particles in hydrogen

peroxide and DI water

When higher concentration (~30 vv) of Ag-TiO2 particles in hydrogen peroxide is

subjected to UV radiation the particles exhibit pattern formation (Figure 4 7)

Aggregation of Ag-TiO2 Janus particles is observed when UV light is turned on and the

particles disaggregated when UV light is switched off This can be due to predator-prey

kind of interaction between silver and titania half of the Janus particles in the presence of

UV light Oxygen and water that are produced at the silver end by decomposition of

hydrogen peroxide are used up by titania when subjected to UV radiation This can lead

to attraction between titania and silver parts of neighboring Janus particles causing

aggregation

y = 20373x

y = 59994x

y = 71848x

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10 12

Mean

sq

uare

dis

pla

cem

en

t (μ

m2s

)

Time (sec)

Mean square displacement vs Time for Ag-TiO2

particles

05 Hydrogen peroxide and UV05 Hydrogen peroxide

DI Water

51

A UV OFF B UV ON

C UV OFF

Figure 4 7 Higher concentrations of Ag-TiO2 particles showing aggregation when UV

is turned on

52

Chapter 5

Summary and Conclusion

In summary no significant enhancement in diffusion was observed in case of enzyme

immobilized Janus particles in substrate solution Presence of a linker between enzyme

and particle did not increase the diffusion coefficient of particles Alternative enzyme

immobilization methods need to be tested like thiolation of enzyme and subsequent

binding to gold surface of a Janus particle35

or expressing the enzyme with polyhistidine

or avidin tag67

This can result in higher enzyme activity upon immobilization and may

lead to enhanced diffusion of Janus particles

Predator-prey system of GOx and HRP immobilized particles showed some promising

results Aggregation of particles and formation of fan-shaped structures by TMB

oxidation were observed under optical microscope when GOx and HRP immobilized

particles glucose and TMB were present in the system The size of the fan-shaped

structures was dependent on the size of enzyme immobilized particles This can be

indicative of some kind of interaction between the GOx and HRP immobilized particles

No conclusive aggregation of GOx and HRP immobilized particles was observed in

absence of TMB Better imaging techniques are required to study the interaction of GOx

and HRP immobilized particles at the unstable liquid-air interface where majority of the

reaction takes place Other enzyme cascade systems not requiring gaseous reactants like

glycogen synthase and glycogen phosphorylase should be tested for similar activity

53

Ag-TiO2 particles were found to exhibit autonomous motion in the presence of hydrogen

peroxide solution The motion was momentarily accelerated upon exposure to UV

radiation The particles showed a diffusion coefficient of 1491 μm2s in hydrogen

peroxide versus only 180 μm2s in DI water Upon subjecting particles to UV radiation in

presence of H2O2 diffusion coefficient increased to 5093 μm2s for a period of 4-5

seconds In addition to linear motion rotating motion could also be observed in some

particle aggregates Further development of these aggregate structures can lead to

formation of rotors at nanoscale Higher concentration of silver-titania particles in

hydrogen peroxide displayed aggregation in presence of UV light and disaggregated

when UV light was switched off This may be attributed to the predator-prey kind of

interaction between silver and titania where oxygen and water that are produced by

silver by decomposition of hydrogen peroxide are used up by titania when subjected to

UV radiation This promises to be an interesting system and needs to be further

investigated to explore its properties

54

References

1 Paxton WF Sundararajan S Mallouk TE Sen A Angew Chem Int Ed Engl

2006 45 5420-5429

2 Golestanian R Liverpool TB Adjari A Phys Rev Lett 2005 94 220801

3 httpwwwsilvermedicineorgh2o2html

4 Rami Pedahzur Ovadia Lev Badri Fattal Hillel Shuval Water Science and Technology

1995 31(5-6) 123-129

5 W Went F American Scientist 1968 56

6 Adriano Cavalcanti Tad Hogg Bijan Shirinzadeh IEEE MHS 2006 International

Symposium on Micro-NanoMechatronics and Human Science 2006 226-232

7 EM Purcell American Journal of Physics 1977 45 3-11

8 Golestanian R Phys Rev Lett 2010 105 018103(1)-018103(4)

9 S S Dukhin Z R Ulberg G L Dvornichenko B V Deryagin Russian Chemical

Bulletin 1983 31(8) 1535-1544

10 Dukhin AS Goetz PJ Ultrasound for characterizing colloids Particle sizing zeta

potential rheology Elsevier 2002 59

11 HJKeh HJTu Trends in Colloid and Interface Science XIV Progress in Colloid and

Polymer Science 2000 115 174-180

12 D Zinemanas A Nir International Journal of Multiphase Flow 1995 21(5) 787-800

55

13 Stephen Ebbens Jonathan Howse Soft matter 2010 6 726-739

14 J Vicario R Eelkema W R Browne A Meetsma R M La Crois B L Feringa

Chem Commun 2005 31 3936

15 Davide Pantarotto Wesley R Browne Ben L Feringa Chem Commun 2008 1533 -

1535

16 Lisa A Cameron Matthew J Footer Alexander van Oudenaarden Julie A Theriot

PNAS 1999 96(9) 4908-4913

17 Ali Najafi Ramin Golestanian Phys Rev E 2004 69 062901

18 Jinseok Kim Jungyul Park Sungwook Yang Jeongeun Baek Byungkyu Kim Sang

Ho Lee Eui-Sung Yoon Kukjin Chun Sukho Park Lab Chip 2007 7 1504 - 1508

19 Kurt M Dubowski Clinical Chemistry 2008 5411 1919ndash1920

20 Spector DA Yang Q Wade JB Am J Physiol Renal Physiol 2007 292(1) 467-474

21 Rhee SG Chang TS Bae YS Lee SR Kang SW J Am Soc Nephrol 2003 14(8-3)

211-215

22 Sakaue T Kapral R Mikhailov A S Eur Phys J B 2010 75 381ndash387

23 Muddana H S Sengupta S Mallouk T E Sen A Butler P J J Am Chem Soc

2010 132 (7) 2110ndash2111

24 Baker CJ Deahl K Domek J Orlandi EW Arch Biochem Biophys 2000 382(2) 232-

237

25 V Vojinovic RH Carvalho F Lemos JMS Cabral LP Fonseca BS Ferreira

Biochemical Engineering Journal 2007 35 126-135

56

26

OV Lebedeva NN Ugarova Russian Chemical Bulletin 1996 45(1) 18-25

27 Nigel C Veitch Phytochemistry 2004 65(3) 249-259

28 Kenneth H Petersen Journal of Immunological Methods 2009 340(1) 86-89

29 httpwwwbrenda-enzymesorgphpresult_flatphp4ecno=11117

30 Goldenberg L M Wagner J Stumpe J Paulke B-R Goumlrnitz E Langmuir 2002 18

5627-5629

31 httpwwwgoldbiocompdf3761-Protocol201pdf

32 Av Rovisco Pais Lisboa Portugal T amp D Technology West Yorkshire UK Journal

of Molecular Catalysis B Enzymatic 2004 28(2-3) 129-135

33 httpwwwphysicsnyuedugrierlabmethodsnode11html

34 Yoon-Mee Lee1 O-Yul Kwon Yeo-Joon Yoon Keungarp Ryu Biotechnology Letters

2006 28 39ndash43

35 JC Pyun SD Kim JW Chung Analytical Biochemistry 2005 347(2) 227-233

36 George H Pimbley Jr Mathematical Biosciences 1974 20(1-2) 27-51

37 Andy Fenton Sarah E Perkins Parasitology 2010 137 1027-1038

38 Mead James G Brownell Robert L Mammal Species of the World A Taxonomic and

Geographic Reference (3rd ed) Johns Hopkins University Press 2005 2 2142

57

39

Krause KH Schweiz Med Wochenschr 2000 130(4) 97-100

40 Irina Kareva Faina Berezovskaya Carlos Castillo-Chavez Journal of Biological

Dynamics 2010 4(4)315 - 327

41 Delaittre G Reynhout I Cornelissen J Nolte R Chemistry ndash A European Journal

2009 15 12600ndash12603

42 C Buller DH Johnson RC Tschumper Investigative Ophthalmology amp Visual Science

1990 31 2156-2163

43 Robert M Mazo Brownian Motion Fluctuations Dynamics and Applications Oxford

Science Publications 2009 57

44 Vivek K Mutalik KV Venkatesh Theoretical Biology and Medical Modelling 2005 2

19

45 Satoshi Nakamura Kunimasa Koga Biochemical and Biophysical Research

Communications 1977 78(2) 806-810

46 Krishnananda Chattopadhyay Shyamalava Mazumdar Biochemistry 2000 39 (1)

263ndash270

47 Satoshi Nakamura Yasuyuki Ogura J Biochem 1968 63 (3) 308-316

48 van Dongen SF Nallani M Cornelissen JJ Nolte RJ van Hest JC Chemistry 2009

15(5) 1107-1114

49 Ronald Bentley Methods in Enzymology 1955 1 340-345

50 Vartiainen J Raumlttouml M Paulussen S Packaging Technology and Science 2005

18 243ndash251

58

51 GA Sherbeny AA Shindia YMMM Sheriff International Journal of Agriculture

and Biology 2005 7

52 D Rando G-W Kohring F Giffhorn Appl Microbiol Biotechnol 1997 48

53 Worthington enzyme manual Worthington Biochemical Corporation

httpworthington-biochemcomgopdefaulthtml

54 httpwwwbrenda-enzymesorgphpresult_flatphp4ecno=11117

55 httpwwwoxygenphwatercom

56 P Boon Chock Sue Goo Rhee and Earl R Stadtman Annu Rev Biochem 1980 49

813-841

57 Sybil Golden P A Wals Joseph Katz Analytical Biochemistry 1977 77(2) 436-445

58 R L Baehner S K Murrmann J Davis R B Johnston Jr J Clin Invest 1975 56(3)

571ndash576

59 I Bejarano M P Terroacuten S D Paredes C Barriga A B Rodriacuteguez J A Pariente

Molecular and Cellular Biochemistry 2007 296(1-2) 77-84

60 Michael C Moulton Laura K Braydich-Stolle Mallikarjuna N Nadagouda Samantha

Kunzelman Saber M Hussain Rajender S Varma Nanoscale 2010 2 763-770

61 Goya DV Matijevic E Colloids and Surfaces A 1999 146 139

62 Velikov KP Zegers GE van Blaanderen A Langmuir 2003 19 1384

63 Goldenberg L M Wagner J Stumpe J Paulke B-R Goumlrnitz E Langmuir 2002 18

5627-5629

59

64

Banerjee S Gopal J Muraleedharan P Tyagi A K Raj B Curr Sci 2006

901378ndash1383

65 Bogomolov V N Kudinov E K Firsov Y A Sov Phys Solid State 1968 35 555

66 Kudo A Miseki Y Chem Soc Rev 2009 38 253ndash278

67 Gaj T Meyer SC Ghosh I Protein expression and Purification 2007 56 54-61

x

ACKNOWLEDGEMENTS

I would like to thank my advisor Prof Ayusman Sen for his valuable guidance

throughout my research at Penn State He was not only inspiring and encouraging

regarding work but was also very supportive and understanding about life outside work

I would also like to thank my committee members Prof Tom Mallouk and Prof

Christine Keating for being available when I needed guidance and for providing their

valuable advice

I would like to thank my Mom Dad sister and other family members for being

supportive of my decision to come to United States to pursue my graduate studies and for

always being available to share my excitements and disappointments in graduate life I

cannot thank my friend Mahati Elluru enough for her encouraging talks constructive

criticism and praises genuine caring and the list can go on I would like to thank Ambuj

Sharma and Bhavana Achary for being a major part of my life during graduate school and

for making State College feel like home I would like to thank Samudra Sengupta for

being a good friend and mentor who was always around in lab and outside to provide his

valuable insights I would also like to thank Shakuntala Sundararajan Yiying Hong

Michale Ibele Natallia Kulyba and other Sen group members for their support during

graduate school and for creating an enjoyable working environment in the lab

1

Chapter 1

Introduction

Motion at the macroscale has been studied and applied for centuries Invention of the

wheel can be considered as the major step for the beginning of synthetic motors at

macroscale The potential applications of the wheel and other similar motors are still

being explored and seem to have endless possibilities Having said that motion at the

nanoscale is just beginning to get explored and can have similar if not greater

implications in the future This thesis is a part of an attempt to develop nanomotors made

from diverse materials operating in different environments to create a pool of

nanomotors which can be used in the future for specific applications

Some of the nanomotors which have already been developed include the pioneering

work by WFPaxton etal of platinum-gold nanomotors1 showing autonomous motion in

hydrogen peroxide motors made by Golestanian2 consisting of micron sized particles

half coated with Pt and others The mechanisms of motion differ in case of different

motors but all have some basic underlying principles which will be highlighted in the

following section An important point to notice in the above mentioned nanomotors is

that they involve metals or solutions which are not biocompatible This puts a restriction

on the application of such motors This thesis attempts to synthesize enzyme based

nanomotors which will be able to overcome these drawbacks and lead to applications in

biological systems like drug delivery and surgery in human body

2

Second part of this thesis consists of synthesis and motion of silver-titania nanomotors

which can move autonomously in hydrogen peroxide solution According to some recent

findings3 colloidal silver and hydrogen peroxide may prove to be an effective

combination for treatment of infections in human body Combination of silver particles

and hydrogen peroxide has been tested for disinfection of drinking water and may hold

promise as a secondary disinfecatant which can provide long lasting residual and biofilm

control that is required in water distribution systems4 Silver-titania nanomotors can open

interesting avenues in such applications

11 Motion at the nanoscale

Motion as we understand at macroscale is significantly different from motion at

micronnanoscale A definite discontinuity exists between the two This can be

emphasized by observing the biological systems surrounding us For example at the size

of a cell which is around 10 μm mixing of large molecules is instantaneous due to

diffusion and there is no need for additional convective mixing which is what we

generally think of when we picture macroscopic mixing For instance while convective

flow driven by the pumping of lungs or the use of gills is essential for transport of oxygen

inside an organism which is larger than 1mm in size such as a lizard or a man small

organisms receive nutrients and oxygen solely through diffusion through their tissues5

These differences in motion can be understood both physically and mathematically

Physically as the size of an object decreases the mass and weight decrease with the third

3

power of linear size A large body predominantly has gravitational pull of earth acting on

it due to its large mass and other forces like gravitational effect of surrounding objects are

relatively insignificant As the object gets smaller forces such as molecular attraction and

cohesion become more significant and the relative magnitude of the gravitational pull of

the earth on the object decreases due to reduction in mass5 Thus the forces that need to

be considered to cause motion at the macroscale are different than those at the

microscale

Mathematically the difference in motion at macro- and micro- scales can be explained by

means of a dimensionless number called Reynolds number Reynolds number is the ratio

of inertial forces of a system to its viscous forces As size decreases Reynolds number

decreases and the viscous forces become more dominant For a human swimming in

water Reynolds number is around 104 for goldfish it is around 10

2 whereas for an

organism of 1μm size it is around 10-4

to 10-5

[67]

What this means is that as we scale

down from the size of a goldfish to micron sized organisms the inertial forces become

insignificant and the familiar laws of macroscale motion lose their applicability Equation

11 gives the formula for calculating Reynolds number where ρ is density of the fluid

(kgmsup3) μ is the dynamic viscosity of the fluid (kg(mmiddots)) V is the mean fluid velocity

(ms) and D is diameter of particle (m)

Equation 1 1

To understand the implications of low-Reynolds-number systems we can consider the

motion of a macroscopic and an imaginary microscopic scallop Macroscopic scallop

4

moves by opening and closing of its shell Inertial thrust is created when a scallop closes

its shell and the scallop moves forward due to the clapping motion (Figure 1 1) This

however does not hold true at the nanoscale since the inertial forces are relatively

insignificant due to the large viscous forces If a microscopic scallop were to exhibit a

similar clapping motion it will just keep moving forward and backward in an oscillatory

kind of motion and will not experience a net motion This is an example of reciprocal

motion which can linearly move a body at macroscale however fails to cause motion at

the microscale

For motion at microscale nonreciprocal motion is necessary8 Nonreciprocal motion

occurs when the path chosen for going from Position A to Position B is different from the

path chosen to come back from Position B to Position A It can be better understood with

the help of Figure 1 1 which shows the change of shapes for a theoretical object going

from Position A to Position B and then returning to Position A by using a different set of

motions This kind of change can lead to motion at the nanomicron scale

5

Another factor which gains importance at nanoscale is the dominance and ubiquity of

Brownian motion13

The trajectory of a linearly propelled nanomotor would be

significantly affected by the continuous collision of randomly moving particles in the

environment These collisions deviate the nanomotor from its linear path and hence this

effect needs to be considered while projecting the path of a nanomotor

Motion at the nanoscale can be powered by different types of mechanisms such as

diffusiophoretic electrophoretic osmophoretic chemophoretic thermophoretic and

others Different types of mechanisms of motions may coexist or act individually to cause

Position A Position B

Figure 1 1 Reciprocal and Nonreciprocal motion8

Reciprocal motion

Nonreciprocal motion

6

motion Diffusiophoretic motion of particles is caused due to concentration gradients of

solutes in an electrolytenonelectrolyte solution9 Electrophoresis is the motion of a

particle relative to a fluid under the influence of an electric field10

This occurs as the

particle carries an electric surface charge which is acted upon by an electrostatic

coulombic force of the external electric field Osmophoresis is the motion of a

vesiclefluid particle encapsulated in a semi-permeable membrane towards lower

concentration gradient when it is placed in a solution containing solute concentration

gradient1112

Thus considering the numerous mechanisms of motion known to work at

the nanoscale several suitable options are available for the development of artificial

nanomotors

12 Existing nanomotors Mechanisms and uses

Several nanomotors have been developed in the past decade or so A general principle of

working of these nanomotors is that they function by catalytically decomposing the

dissolved fuel to generate asymmetry at their surface or at their interfacial region thereby

causing propulsion13

The self-electrophoretic bimetallic nanowires developed by

WPaxton and A Sen etal consisted of a 2 μm long rod with one tip made up of Au and

the other tip made up of Pt1 These motors were synthesized by electrodeposition in an

alumina template The rods moved autonomously in a solution of hydrogen peroxide

with platinum end forward Pt oxidizes H2O2 to produce H+ and O2 and Au utilizes H

+ to

reduce H2O2 to water This generates an electron flow through the rod and a proton flow

outside the rod resulting in an electric field around the rod in the solution This electric

7

field in solution acts on the unbalanced charges within the nanorodrsquos double layer This

moves the nanorod with respect to the surrounding fuel due to the ion flux that is

generated

Bubble propulsion has been predominantly used as mechanism of motion in the motors

developed by Feringa etal His group developed a micromotor by attaching catalase to a

microparticle and placing the particle in a solution containing hydrogen peroxide This

system allowed the microparticle to move by producing oxygen bubbles from the

decomposition of hydrogen peroxide14

A similar motor was later created by Feringarsquos

group which consisted of glucose oxidase and catalase immobilized on a carbon

nanotube in a substrate solution of hydrogen peroxide Glucose oxidase converted

glucose to H2O2 which was then utilized by catalase to produce water and oxygen

thereby propelling the carbon nanotube15

Another interesting example of a self-propelled motor is the polymerization driven motor

developed by Cameron et al16

It consists of polystyrene beads immobilized with ActA

protein which catalyzes actin polymerization leading to the formation of an actin

filament which resembles the flagella of bacteria This actin polymerization propels the

beads at the speed of 001-015 μms

A theoretical model has been proposed by Nafaji and Golestanian etal which consists

of three spheres linked by rigid rods in one dimension17

The length of the rods can

switch between two specific values Due to the nonreciprocal motion of the rod this

8

model can swim in low Reynolds number systems An actual model based on

nonreciprocal motion has been developed by Kim etal in which heart cells have been

linked to an asymmetric polydimethylsiloxane robot As the heart cells beat the

asymmetric structure of the device resembles a walking movement18

Most of the nanomicromotors mentioned above are either metallic or work in

environments not compatible with human body Enzymes being an integral part of the

body can solve this problem by operating in suitable substrates already present in the

body fluids The nonreciprocal conformation changes of the enzyme as well as

diffusiophoresis and electrophoresis can conceivably be used to generate motion in case

of enzyme driven nanomotors such motors will be discussed in detail in the following

chapter

9

Chapter 2

Immobilized enzymes on Janus particles as catalytic motors

21 Introduction

Enzymes are widely found in biological systems and act on a large variety of substrates

This can make enzymes an ideal choice for powering nanomotors which need to be

biocompatible Several fluids found in the body can act as substrates to the enzymes For

example dissolved glucose which is abundantly found in the body19

is a susbtrate for

enzyme glucose oxidase Urea which is found in the urinary tract20

is a substrate for

urease Hydrogen peroxide produced by phagocytic cells in the body21

is a substrate for

horseradish peroxidase Having motors driven by enzymes can open possibilities of

targeted drug delivery to these parts of the body containing such substrates

Enzymes have already been used to generate motion of carbon nanotubes by mechanism

of bubble propulsion by Feringa etal15

Two other possible mechanisms which may be

able to power motion of nanoparticles using enzymes are asymmetrical decomposition of

substrates into products leading to self-diffusiophoresis (Figure 2 1) and nonreciprocal

changes in enzyme conformation leading to propulsion (Figure 2 2) As seen in Figure 2

1 enzymes which are immobilized on a Janus particle can breakdown the available

substrate in the solution into products causing asymmetric distribution of solutes in the

surrounding fluid This can lead to movement of particle towards the region of lower

10

solute concentration by principle of self-diffusiophoresis Figure 2-2 shows the possible

nonreciprocal changes in the conformation of a theoretical protein which can lead to

motion of the protein22

These nonreciprocal changes in conformation are triggered by the

attachment of substrate molecule and release of product molecules from the enzymersquos

active site

Figure 2 1 Enzyme functionalized Janus particle moves upon catalyzing asymmetrical

decomposition of substrate into product

11

Figure 2 2 Schematic showing idealized nonreciprocal conformation changes in a

theoretical enzyme22

Enhanced diffusion of a single enzyme molecule by catalysis in a substrate solution has

been demonstrated23

The diffusion coefficient of a single urease molecule was measured

using fluorescence correlation spectroscopy and it was observed that the diffusion of the

urease molecule increases by 16-28 by varying urea concentrations from 0001 M to 1

M Addition of enzyme inhibitors to the system curbed the enhanced diffusion This

enhanced diffusion has been attributed to the force generated by self-electrophoresis by

the enzyme in substrate solution

12

Based on the above facts it might be possible to propel a nanoparticle by asymmetric

distribution of enzymes on its surface thereby leading to self-propulsion of the particles

in substrate solution (Figure 2 1) This propulsion may be due to self-

electrophoresisself-diffusiophoresis andor due to nonreciprocal changes in enzyme

conformations Here we aim to investigate a system consisting of polystyrene particles

half coated with enzyme introduced in a suitable substrate solution The enzyme can be

directly linked to the polystyrene particle or it can be linked through a long spacer

molecule (linker) to provide more degrees of freedom to the enzyme

Several factors need to be considered for choosing enzyme for powering motion at the

nanoscale Some of the important factors are the ratio of kcatKm which denotes the

catalytic efficiency of the enzyme possible substrates stability of enzymessubstrates

ease of immobilization relationship with other enzymes in cascade reactions and

commercial availability of the enzymes The enzyme being used in the present study is

horseradish peroxidase It acts on a molecule of hydrogen peroxide and reduces it while

simultaneously oxidizing another substrate molecule In the absence of alternate

oxidizable substrate HRP can oxidize water and oxygen back to hydrogen peroxide24

thus giving an equilibrium reaction

13

211 Horseradish peroxidase

Figure 2 3 Horseradish peroxidase

Horseradish peroxidase belongs to the group of peroxidases which are a group of heme-

containing oxidoreductases that act on peroxide as hydrogen donor25

It is a redox

enzyme which contains a protoheme a complex of trivalent iron with 1358-

tetramethyl-24-divinylporphin-67-dipropionic acid as a prosthetic group It is a 44 kDa

protein which includes 33900 Da polypeptide chain bound with eight oligosaccharide

chains The carbohydrate chains improve the stability of the enzyme by protecting it

against radicals formed in the course of the reaction it catalyzes26

Horseradish

peroxidase is found in horseradish and is used widely in bioanalytical applications such

as ELISA and Western blots It decomposes hydrogen peroxide into water and oxygen

while simultaneously oxidizing another substrate27

The oxidizable substrates can be

chromogenic substrates such as 22rsquo55rsquo-tetramethyl-benzidine (TMB) and 33-

diaminobenzidine (DMB) or chemoluminescent substrates like SuperSignal and ECL

luminal28

14

Figure 2 4 Reaction catalyzed by horseradish peroxidase

The functional parameters for horseradish peroxidase include Km of 015 mM and kcat of

790 s-1

at an optimum temperature of 20 ordmC and an optimum pH of 6429

Some of the

inhibitors of HRP are 110-phenanthroline aminotriazole and bromine

22 Experimental

221 Materials and methods

Uniform carboxyl modified polystyrene microspheres 05 microm in diameter were obtained

from Polysciences Inc The size of the purchased microspheres was verified by Field

Emission Scanning Electron Microscopy (FESEM) Peroxidase Type I from

Horseradish N-(3-dimethyl-aminopropyl)-Nrsquo-ethylcarbodiimide (EDC) and hydrogen

peroxide solution 50 wt in water were purchased from Sigma-Aldrich N-

hydroxysuccinimide (NHS) ge 97 was purchased from Fluka All chemicals were of

reagent grade and used without further purification Nanopure deionized water was used

for all experiments and preparation of solutions All measurements were made at room

15

temperature (25 oC) unless mentioned otherwise The UV-Visible spectroscopy

measurements were performed using Agilent 8453A diode-array UV-Visible

Spectrophotometer with ChemStation UV-Vis Software Diffusion coefficient

measurements were performed using Nanosight LM10

222 Synthesis of Janus particles

A monolayer of particles was prepared according to the procedure (Figure 2 5) by

Goldenberg L et al30

Glass slides were placed on the bottom of a petridish Water was

put into the petridish so that the entire slide was immersed in water A thin layer of

hexane was added on top of water in the petridish A suspension of microspheres in

ethanol (5 ww) was introduced to the water-hexane interface After the monolayer was

formed the glass slide was lifted up through the monolayer with tweezers The

microsphere-covered glass slides were left to dry overnight at room temperature

Following drying a thin film of silica was evaporated on the glass slides in Semicore

evaporator The spheres were partially coated with 30 nm of silica Particles were

removed from the glass slide by gentle sonication The polystyrene-silica (PS-SiO2)

Janus particles thus obtained were washed in deionized water several times before

further immobilization steps were performed FESEM was used to confirm synthesis of

Janus particles

16

Figure 2 5 Preparation of PS-SiO2 Janus particles by evaporation

223 Immobilization of enzymes using EDCNHS coupling method

1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide and N-hydroxysulfosuccinimide

(EDCNHS) coupling procedure was followed for immobilizing horseradish peroxidase

type I on 05 μm PS-SiO2 Janus particles (Figure 2 6) In this process EDC reacts with a

carboxyl groups on the Janus particles to form an amine-reactive O-acylisourea

intermediate If this intermediate does not encounter an amine it can hydrolyze and

regenerate the carboxyl group In the presence of NHS EDC can be used to convert

carboxyl groups to amine-reactive sulfo-NHS esters which are much more stable and do

not readily hydrolyze to regenerate carboxyl groups In the current experiment

immobilization was performed in the presence as well as absence of a long spacer

17

molecule (linker) between Janus particles and enzymes The chemical 6-aminohexanoic

acid was used as the linker (Figure 2 7) HRP immobilized PS-SiO2 Janus particles were

stored in deionized water until required for further experiments

Figure 2 6 EDCNHS coupling method for immobilization of enzyme on carboxylated

polystyrene particles

Figure 2 7 Immobilization of enzyme on Janus particles with and without 6-

aminohexanoic acid linker

18

224 Enzyme assay

Assay for activity of HRP was performed using a spectrophotometric method which

involved using 4-aminoantipyrene as hydrogen donor3132

The reaction rate was

determined by measuring increase in absorbance at 510 nm resulting from the

decomposition of hydrogen peroxide One unit represents the decomposition of 1 μM of

hydrogen peroxide per minute at 25 degC and pH 70 under the specified conditions

The spectrophotometer was set to the wavelength of 510 nm and temperature of 25 degC

Phenol4-aminoantipyrene solution (14 mL) and 00017 M H2O2 solution (15 mL) were

pipetted into each cuvette The cuvette was incubated for 3-4 mins at 25degC and blank was

established A 01 mL solution of diluted enzyme was added to the above mixture and

absorbance was recorded for 4-5 mins Standard graphs of absorbance versus time were

drawn for known concentration of the free enzyme and compared to that of the

immobilized enzyme on the Janus particles The estimate of the amount of enzyme

present on the Janus particles was deduced from the difference between the quantity of

the free enzyme previously added for immobilization and the amount that was measured

to be in the supernatant after some of the enzyme became attached to the Janus particles

The experiment was carried out in triplicate to assess reproducibility

19

The formula used to calculate the activity of enzyme is

where ∆Amin denotes the change in absorbance per minute

225 Diffusion coefficient measurements using Nanosight LM10

Diffusion coefficients for HRP immobilized particles were measured using Nanosight

LM 10 nanoparticle tracking instrument with Nanoparticle Tracking Analysis (NTA) 20

software in the presence of 003 H2O2 solution as the substrate and also in the presence

of only deionized water The videos were recorded for 30 seconds at the rate of 30 frames

per second Each experiment was carried out in triplicate to assess reproducibility

Particles

visualization

zone

Microscope with

LM 10 unit

LM 10 unit

Figure 2 8 Nanosight LM 10 instrumentation

20

Nanosight LM 10 (Figure 2 8) generates videos of population of moving nanoparticles in

a liquid when illuminated by laser light The particles which pass through the laser beam

path are seen by the instrument as small points of light undergoing Brownian motion

(Figure 2 9a)

The videos are further analyzed by NTA (Nanoparticle tracking analysis) software

(Figure 2 9b) which identifies and tracks each particle independently on a frame-by-

frame basis The software also calculates and subtracts any bulk flow in the system The

mean square displacement is calculated for each particle as long as it is visible If the

particle blinks that is if it disappears momentarily and then reappears on the screen the

software will consider the same particle as two separate ones All analysis data is

exported to the excel files in terms of diffusion coefficients of the tracked particles

B A

Figure 2 9 NTA video analysis (a) Particles as they are seen on the screen during

experimentation in Nanosight LM10 (b) NTA screen as the particles are being analyzed for

their diffusion coefficients

21

23 Results

231 Enzyme assay

The activity of free HRP enzyme reported by Sigma Aldrich is 150 unitsmg However

the activity of HRP upon analysis was found to be 52158 unitsmg which is much lower

than the reported value The activity of HRP was found to be further lowered to 2133

unitsmg when it was immobilized on the PS-SiO2 Janus particles in the presence of a

linker The following formula is used to calculate enzyme activity

where ΔAmin is the change in absorbance per minute

22

Figure 2 10 Graph for the assay of solution of free enzyme containing 05mgL of HRP

ΔAmin = 01716 Unitsmg of free enzyme = 52158

During EDCNHS coupling 04 mg enzyme was added to 1 ml of 025 vv suspension

of Janus particles Thus initial concentration of enzyme in the mixture was 04 mgml

Assuming retained activity of the enzyme in supernatant solution the amount of enzyme

in supernatant was calculated to be 03868 mgml from the assay Amount of enzyme

functionalized on the Janus particles was calculated as the difference between enzyme

previously added for immobilization (04 mgml) and the enzyme present in supernatant

(03868 mgml) and was found to be 00132 mgml Enzyme assay was performed on the

enzyme immobilized Janus particles to estimate the activity of immobilized enzyme

(Figure 2 11) and was found to be 2133 unitsmg

y = 0002x + 00516

0

01

02

03

04

05

06

07

08

0 50 100 150 200 250 300 350 400

Ab

sorb

an

ce

51

0 n

m

Time (sec)

Assay of free enzyme HRP

23

Figure 2 11 Graph for the assay of of immobilized enzyme on 05 μm Janus particles

(~025 vv) ΔAmin = 01853 Unitsmg of free enzyme = 2133

232 Diffusion coefficient measurements

No significant increase was observed in diffusion coefficient of HRP-immobilized Janus

particles in the presence of H2O2 as the substrate Similar results were obtained for

diffusion coefficient measurement with and without linker moieties bridging the enzymes

and particles(Table 2 1 Table 2 2) Thus the linker did not seem to assist in increasing

the particle diffusion by improving or retaining the activity of the enzyme

y = 00015x + 00953

0

01

02

03

04

05

06

07

0 50 100 150 200 250 300 350 400

Ab

sorb

an

ce

510 n

m

Time (sec)

Assay of HRP immobilized 05 μm Janus particles

24

Table 2 1 Diffusion coefficient measurements of HRP immobilized Janus particles

without linker

HRP immobilized

Janus particles in

003 H2O2

Control - Janus

particles in

003 H2O2

Control - HRP

immobilized Janus

particles in DI water

Diffusion coefficient

(10-10

cm2s)

1359416 1051777 1308675

Standard deviation 4534801 4122075 4918597

Table 2 2 Diffusion coefficient measurements of HRP immobilized Janus particles in

the presence of linker

HRP immobilized

Janus particles in

003 H2O2

Control - Janus

particles in

003 H2O2

Control - HRP

immobilized Janus

particles in DI water

Diffusion coefficient

(10-10

cm2s)

13838 1339829 1407891

Standard deviation 5644692 5649364 5797218

25

Figure 2 12 Diffusion coefficient measurements of PS-SiO2 Janus particles without and

with 6-aminohexanoic acid linker

24 Discussion

To compare the diffusion coefficients observed during the experiments theoretical

diffusion coefficient was calculated using following equation33

r

kTD altraslation

6 Equation 21

where r is the hydrodynamic radius of particle Dtranslational is translational diffusion

coefficient of the particle T is temperature of the system η is the viscosity of fluid

through which the particles are moving and k is the Boltzmann constant From FE-SEM

images the size of the particles used was determined to be 450 nm For 450 nm diameter

0

20

40

60

80

100

120

140

160

Without linker With linker

Dif

fusio

n c

oeff

icie

nt

(10

-10 c

m2s

)

HRP immobilized 05 μm Janus particles - Diffusion coefficient measurements

HRP immobilized Janus particles in 003 H2O2

Control - Janus particles in 003 H2O2

Control - HRP immobilized Janus particles in DI water

26

particle Dtranslational is equal to 109 μm2s which is comparable to the diffusion

coefficient measured for the enzyme immobilized particles

HRP was found to be active on the Janus particles however the activity of the enzyme

was reduced upon immobilization Lower enzyme activity can be due to the coupling

method used which in this case was EDCNHS covalent binding However EDCNHS

coupling method has been shown to significantly retain the enzyme activity when the

enzyme is immobilized on carboxylated multi-walled carbon nanotubes under appropriate

conditions34

Hence EDCNHS binding method by itself is not believed to be

responsible for reduction of enzyme activity upon immobilization

Direct binding of enzyme to the Janus particle may lead to reduction in the enzyme

activity because of space constraints for the enzyme and difficulty for the passage of

substrate to the active site of the enzyme due to stearic hindrance Hence a linker was

inserted between the enzyme and Janus particle during enzyme immobilization However

inserting a linker between the enzyme and the particle did not lead to enhanced diffusion

of Janus particles or increase in enzyme activity of immobilized enzyme

Lack of enhanced diffusion can be firstly attributed to the reduction in enzyme activity

upon immobilization Enzyme activity may be retained by modifying the conditions

during EDCNHS coupling or changing the binding method altogether The degree of

immobilization of HRP on carboxylated Janus particles can possibly be affected by

several factors such as temperature the concentration of the coupling reagent EDC

27

concentration HRP concentration in solution and incubation time34

Different

combinations of these factors can be tested to find optimum conditions for

immobilization of HRP Alternate binding method can involve thiolation of HRP by the

covalent conjugation of mercaptopropionic acid and then the thiolated HRP can then be

attached on the gold surface of a polystyrene-gold Janus particle35

Thus further

modifications can be made to the system in order to retain enzyme activity on the

immobilized particles and thereby power nanomotors using enzymes

28

Chapter 3

Predator-prey system of enzyme immobilized particles

31 Introduction

Predator-prey systems are widely found in nature and play a major role in biological

processes3637

Predator-prey systems include all the interactions in which one organism

pursues andor consumes another organism One familiar predator-prey system at the

macroscale is that of a tiger chasing and consuming a deer Another not so familiar one

includes Baleen whales which eat millions of microscopic planktons at once38

At the

microscale such kind of interactions can be observed in the case of phagocytes in the

body which are capable of engulfing foreign microorganisms and killing them with a

combination of microbicidal systems39

In the case of cancer the tumor is the prey and

the mature myeloid cells in the body are the predator However the tumor here has a

defense mechanism against the predator in which the tumor produces cytokines which

inhibits the maturation of the myeloid cells and thereby prevents predation40

One similarity that can be observed in all these systems is that the prey possesses some

attribute which is desired by the predator which makes the predator chase the prey A

similar relationship is seen in case of enzymes which are involved in cascade reactions41

In cascade reactions product synthesized by one enzyme is the reactant required by

another enzyme to carry out its own catalytic reaction As such there is a predator-prey

29

kind of dependence between the two enzymes which may cause one enzyme to chase the

other enzyme when placed in suitable substrate solutions However the mechanism

hypothesized for such a system is totally different than the mechanism involved in

biological systems In biological systems such as bacterial chemotaxis or phagocytes

chasing foreign particles in human body complex biochemical reactions are responsible

for the predator-prey behavior42

In the proposed enzyme cascade system there are no

complex biochemical reactions involved The principle hypothesized for predator-prey

behavior of enzyme cascade reactions can be explained as follows Motion of synthetic

motors at nanoscale involves two components translational diffusion and rotational

Brownian diffusion At very short time scales of less than 1Dr where Dr (s-1

) is

rotational Brownian diffusion coefficient the motion of particles is unidirectional For

longer time scales of greater than 1Dr the direction of motion is random giving

poweredactive diffusion43

If the chemical homogeneity is broken that is when there is

higher concentration of a substrate present in one direction of the enzyme the enzyme

might acquire a subtle bias which may lead to a spatial aggregation of the enzymes based

on the substrate concentration gradient The bias can be a result of the relationship

between the speed of enzyme diffusion and substrate concentration where higher

substrate concentration lead to higher enzyme diffusion As a result in an enzyme

cascade system one enzyme will migrate towards higher concentration of substrate

which is present near the other enzyme thus giving rise to a predator-prey behavior

Such enzyme cascades are widely found in signaling networks and metabolic pathways in

living cells and can be used to produce predator-prey like nanomotors systems44

The

30

enzymes being used in the present study are horseradish peroxidase and glucose oxidase

Both enzymes are extremely stable4546

Glucose is the substrate for glucose oxidase

which is widely available and highly stable47

Horseradish peroxidase acts on hydrogen

peroxide It is involved in a cascade reaction with glucose oxidase which produces

hydrogen peroxide by converting glucose into gluconic acid (Figure 3 1)48

Figure 3 1 Concept of particle aggregation due to cascade reactions of immobilized

enzymes

In this experiment glucose oxidase (GOx) and horseradish peroxidase (HRP) were

immobilized on polystyrene carboxylate beads of different sizes using EDCNHS

coupling method Glucose and 3355-Tetramethylbenzidine (TMB) were dissolved

together in water and this solution was used as the substrate solution All the components

were present in a single system in DI water Aggregation of particles and precipitation of

oxidized TMB from the solution was expected to be observed when all these components

were present in such a system

Properties of horseradish peroxidase have been discussed in the previous chapter (Section

211 Horseradish peroxidase)

GOxGOx HRPHRP

Glucose + O2Gluconic acid + H2O2

TMB TMB oxidizedBlue precipitate

H2O

Aggregation

31

311 Glucose oxidase

Figure 3 2 Glucose oxidase

Glucose oxidase (GOx) belongs to the group of oxidoreductases and is also called

glucose aerodehydrogenase49

It is obtained from fungi and has been identified as an

antimicrobial enzyme50

GOx binds to β-D-glucopyranose and catalyzes the oxidation

reaction to from gluconolactone while simultaneously reducing oxygen to hydrogen

peroxide51

GOx has high substrate affinity and specificity high stability and high

turnover number which makes it a suitable biocatalyst for industrial applications52

Figure 3 3 Redox reaction of glucose catalyzed by glucose oxidase53

32

GOx is a glycoprotein with molecular weight of 155 kDa and consists of two identical

polypeptide subunits linked together by a disulfide bond The optimum pH for GOx is

55 though it is stable in a broad pH range of 4 to 7 and can be stored for a long period of

time51

The functional parameters for glucose oxidase include Km of 151 mM and kcat of

337 s-1

at temperature of 20 ordmC and pH of 754

Some of the inhibitors of GOx are 2-

Deoxy-D-glucose adenine nucleotides and NaNO3

32 Experimental

321 Materials and methods

Uniform carboxyl modified polystyrene microspheres 05 microm in diameter were obtained

from Polysciences Inc The size of the purchased microspheres was verified by FESEM

Peroxidase Type I from horseradish glucose oxidase Type II-S from Aspergillus niger

N-(3-dimethyl-aminopropyl)-Nrsquo-ethylcarbodiimide (EDC) and Hydrogen peroxide

solution 50 wt in water were purchased from Sigma-Aldrich N-hydroxysuccinimide

(NHS) was purchased from Fluka D-Glucose (99) was purchased from VWR

International Secure-Seal imaging spacers (9mm diameter) for glass slides were obtained

from Sigma Aldrich All chemicals were of reagent grade and used without further

purification Nanopure deionized water was used for all experiments and preparation of

solutions All measurements were made at room temperature (25 oC) unless mentioned

otherwise The UV-Visible measurements were performed using Agilent 8453A diode-

array UV-Visible Spectrophotometer with ChemStation UV-Vis Software Optical

33

microscopy experiments were performed using Axiovert 200 MAT microscope by Carl

Zeiss Diffusion coefficient measurements were obtained using Nanosight LM10

322 Optical microscopy experiments

The enzyme substrate system was observed under optical microscope to check for

aggregation and precipitation due to cascade reactions Several such systems were

studied Each system in general consisted of GOx and HRP immobilized on different

polystyrene particles The substrate solutions consisted of 01 M glucose and different

concentrations of TMB solution The systems were studied in the presence of all the

constituents and in the absence of glucose GOxHRP and TMB Systems consisting of

different sized HRP immobilized particles in substrate solution of 003 hydrogen

peroxide and TMB were also studied Videos were recorded over a period of 24 hours to

observe changes in the system Each experiment was performed atleast thrice to assess

reproducibility of results

323 Diffusion coefficient measurements using Nanosight LM10

Diffusion coefficient of GOx and HRP immobilized particles were measured using

Nanosight LM10 nanoparticle tracking instrument with Nanoparticle Tracking Analysis

(NTA) 20 software Diffusion coefficients were measured for different systems namely

GOx particles in 01 M glucose and oxygen both GOx and HRP functionalized particles

in 01 M glucose and oxygen along with controls for the individual systems Videos were

34

recorded for 30 seconds at the rate of 30 frames per second Each experiment was carried

out in triplicate to assess reproducibility

33 Results

331 Optical microscopy

According to the enzyme cascade model precipitation of TMB was possible only upon

the interaction of GOx and HRP immobilized particles in presence of appropriate

substrate solution Hence in the first set of experiments precipitation of TMB was used

as an indication of the type of interaction between GOx and HRP immobilized particles

Fan-shaped structures were observed when both HRP and GOx immobilized particles

were present in a substrate solution consisting of glucose and TMB These structures

started forming as a single strand and then branched out and intertwined to form the fan-

shaped structures (Figure 3 4)

Table 3 1 Observations for formation of fan-shaped structures in enzyme cascade

system in presence of different components

GOx HRP Glucose TMB Observation

Yes Yes Yes Yes

Fan-shaped structures observed particles associated with

the fan-shaped structures

No Yes Yes Yes No fan-shaped structures

Yes No Yes Yes No fan-shaped structures

Yes Yes No Yes No fan-shaped structures

Yes Yes Yes No No fan-shaped structures

35

Fan-shaped structures can also be observed in the system consisting of only HRP

immobilized particles in substrate solution of hydrogen peroxide and TMB The size of

the fan-shaped structures depends on the size of the enzyme immobilized particles Free

enzymes show extremely tiny structures which are difficult to view under the optical

microscope

Figure 3 4 Formation of fan-shaped structures when both HRP and GOx

immobilized particles were present in a substrate solution consisting of glucose and

TMB A In the initial stages single strands form on the surface of liquid (5Ox) B

Single strands branch out and intertwine to form fan-shaped structures (50x) C Many

such structures form and settle down after 20 minutes (20x)

A

C

B

36

Experiments were performed in the absence of TMB that is the system consisted of GOx

and HRP immobilized particles in glucose substrate solution In the absence of TMB the

particles were expected to move closer to each other without any precipitation due to the

cascade reaction However there was no conclusive aggregation of particles observed in

the system

Free Enzyme 005 μm HRP - immobilized particle

10 μm HRP - immobilized particle 05 μm HRP - immobilized particle

Figure 3 5 Size dependence of fan-shaped structures on the size of HRP -

immobilized particles (20x)

37

Table 3 2 Observations for particle aggregation in enzyme cascade system in presence

and absence of TMB

Glucose GOx HRP TMB Observation

Present Present Present Present Aggregation and precipitate

Present Present Present Absent No conclusive aggregation

Present Absent Present Present No aggregation or ppt

Absent Present Present Present No aggregation or ppt

Present Present Absent Present No aggregation or ppt

332 Diffusion coefficient measurements

Diffusion coefficients were measured for GOx immobilized particles of 05 μm diameter

in substrate solution of 01 M glucose and oxygen Diffusion coefficients of particles

were also measured for system consisting of GOx and HRP functionalized particles in

glucose and oxygen Each experiment was done in triplicate to assess reproducibility

Nanosight visualization chamber being a closed chamber oxygen was purged into all the

solutions before starting the experiments to ensure sufficient supply of oxygen for

catalytic reaction of GOx

38

Table 3 3 Diffusion coefficients for 05 μm GOx immobilized particles in 01 M

glucose purged with O2

Mean

GOx

particles in

01 M

glucose

Control - GOx

particles in DI

water

Control - GOx

particles in 01 M

glucose without

oxygen purging

Control -

particles in 01

M Glucose

Diffusion

coefficient

(x10-10

cm2s)

13316 12888 13827 13702

Standard

deviation 6307 4926 4424 7468

Table 3 4 Diffusion coefficients for GOx and HRP immobilized particles in 01 M

glucose and purged with O2

Mean

GOx- HRP-

functionalized

particles in 01 M

glucose

Control ndash Gox-

HRP- functionalized

particles in DI water

Control - particles

in 01 M glucose

Diffusion coefficient

(x10-10

cm2s)

13005 14434 13702

Standard deviation 5263 6622 7468

No significant increase was observed in diffusion coefficient of enzyme immobilized

particles in any of the systems

39

34 Discussion

Formation of fan-shaped structures due to patterned deposition of oxidized TMB

indicates some kind of interaction between GOx and HRP immobilized particles Particle

diameter dependence of the size of structures can be explained with a simple model in

Figure 3 6 A larger particle will have a higher surface area as compared to a smaller

particle Hence more enzyme is immobilized on the larger particle As a result higher

quantity of TMB is oxidized and precipitated by the larger particle as compared to the

smaller particle This leads to thicker strands of fan-shaped structures eventually scaling

up the whole system to give bigger fan-shaped structures

The exact reason for the formation of such fan-shaped structures which are dependent on

particle size could not be determined It is hypothesized that hydrogen peroxide andor

Figure 3 6 Model for size dependence of fan-shaped structures on the particle

diameter of enzyme immobilized particles Larger the particle more surface area is

available for precipitation of TMB which eventually leads to bigger fan-shaped

structures

Larger particle Smaller particle

40

TMB gradient in the substrate solution might be responsible for such structures If there

is higher concentration of H2O2 on one side of the particle and assuming an even

concentration distribution of TMB there will be selective precipitation in the direction of

H2O2 When several particles come in the vicinity of higher concentration of H2O2 the

precipitation lines will converge to give fan-shaped structures (Figure 3 7)

Figure 3 7 Hypothesis for formation of fan-shaped structures

In the system consisting of GOx immobilized particles HRP immobilized particles

glucose and TMB it was observed that the fan-shaped structures formed at the liquid-air

interface and eventually settled down to the bottom of the glass slide This makes sense

as oxygen is necessary for the catalytic reaction of conversion of glucose to gluconic acid

and hydrogen peroxide which takes place on the surface of GOx immobilized particle As

a result H2O2 is primarily formed at the liquid-air interface and is then used by HRP for

precipitation of TMB

Following the observations that precipitation of TMB occurs at the surface GOx and

HRP immobilized particles are also expected to interact at the surface in the absence of

TMB Due the cascade reaction it was expected that HRP immobilized particles will be

41

attracted to GOx immobilized particles in appropriate substrate solution Experiments

were carried out to visualize inter-particle interactions at the liquid-air interface

However liquid-air interface not being a stable system to image the precise interaction

of GOx and HRP immobilized particles could not be determined Significant external

disturbances were involved at the interface To enable the cascade reaction in the bulk of

the liquid oxygen was purged into the reaction system before starting the experiment

However this did not lead to significant reaction in the bulk of the liquid and the reaction

prominently took place at the liquid-air interface where the oxygen concentration was

higher

In order to observe the interaction of GOx and HRP immobilized particles better imaging

techniques need to be used to observe diffusion of particles in absence of external

disturbances at the liquid-air interface Large quantity of oxygen needs to be purged into

the system and retained to make the reaction more pronounced in the bulk of the substrate

solution Higher concentration of oxygen in water may also be achieved by using oxygen

enriched water which is commercially available55

Alternatively a different enzyme cascade system may be used which does not require a

gaseous substrate56

For example glycogen synthase synthesizes glycogen from glucose-

1-phosphate and glycogen phosphorylase breaks down glycogen to glucose-1-

phosphate57

This system does not require a gaseous substrate and might be able to

overcome the deficiencies of GOx and HRP enzyme cascade system

42

Chapter 4

Silver-titania nanomotors

41 Introduction

Bimetallic nanowires made of platinum and gold have been developed by WPaxton

etal1 The rods move autonomously in a solution of hydrogen peroxide In the present

study silver-titania (Ag-TiO2) particles are being synthesized and investigated for motion

in presence of hydrogen peroxide and UV light With proper control of conditions and

UV light trigger it can be possible to control the speed and direction of motion of these

nanoparticles

Hydrogen peroxide is produced in the human body by phagocytes as an immune response

to foreign particles and pathogens5859

Also silver nanoparticles have been shown to be

biocompatible and capable of interacting with cells in human body without disrupting

cellular function60

Similarly it has been demonstrated that titania is compatible with

different cells DNA and proteins in human body and does not cause toxic effects Hence

silver-titania nanomotors which swim in hydrogen peroxide may open interesting

avenues in applications like targeted drug delivery in those parts of the body where

hydrogen peroxide is present

43

42 Experimental

421 Materials and methods

Silver nitrate (999995) was purchased from Alfa Aesar Gum arabic was purchased

from MP Biomedicals Hydrogen peroxide solution 50 wt in water and ascorbic acid

were purchased from Sigma-Aldrich Nanopure deionized water was used for all

experiments and preparation of all solutions Secure-Seal imaging spacers (9 mm

diameter) were obtained from Sigma Aldrich All experiments were carried out at room

temperature of 25ordmC unless otherwise mentioned Ag-TiO2 particles were synthesized by

evaporation of titania in Semicore electron beam evaporator Particles were characterized

using field-emission scanning electron microscopy Optical microscopy experiments

were performed using Axiovert 200 MAT microscope by Carl Zeiss Particles were

tracked using Physvis particle tracking software (Figure 4 1) in which the position

information of a particle on screen is stored on a frame by frame basis The data is stored

in a dat file as X and Y co-ordinates of particles tracked on the screen This information

can be converted to diffusion coefficients of the particles by further manipulation using

MathWorks-MATLABreg software

44

Figure 4 1 Physvis particle tracking software

422 Synthesis of silver colloidal particles

Silver particles were synthesized by a nucleationgrowth type reaction developed by

Goya and Matijevic61

and modified by Velikov62

Silver nitrate was used as precursor

metal salt for synthesis of silver particles Solution A was 75 ml of 50 mM AgNO3 and

035 by weight gum arabic Solution B was 75 mL of 100 mM 1-ascorbic acid and

035 by weight gum arabic Solution B was rapidly added to solution A in a 250 mL

reaction flask The reaction flask was covered and particles were stirred for 48 hrs After

48 hours particles were allowed to settle for 2-3 hours in refrigerator and supernatant

was separated from the particles Particles were then rinsed with DI water and centrifuged

thrice to obtain pure silver particles Zeta potential of these particles was reported to be

45

around -50 to -60 mV and the size was around 1μm The size of the particles can be

varied by varying the amounts of ascorbic acid and silver nitrate Using molar ratio of 31

of ascorbic acid silver nitrate can give particles of 200 nm diameter whereas ratio of

151 can give particles of upto 5 μm diameter

423 Preparation of silver-titania (Ag-TiO2) particles

A monolayer of silver particles was prepared according to the procedure by Goldenberg

et al63

Glass slides were placed on the bottom of a petridish Water was put into the

petridish so that the entire slide was immersed in water A thin layer of hexane was then

added on top of water in the petridish A suspension of silver particles in ethanol (5

ww) was slowly introduced to the water-hexane interface in the dish After the

monolayer was formed the glass slide was lifted up through the monolayer with tweezers

The silver particles-covered glass slides were left at room temperature to dry overnight

Following drying a thin film of titania was evaporated on the glass slides in Semicore

evaporator (Figure 2 7) The spheres were partially coated with a 10 nm film of titania

Ag-TiO2 particles were removed from the glass slide by gentle sonication FE-SEM was

used to characterize the particles (Figure 4 2) The thin titania layer was not easily

distinguishable due to roughness of the particles

46

Figure 4 2 FESEM image of Ag-TiO2 particles

424 Optical microscopy experiments

Imaging spacers were placed on a glass slide Suspension of Ag-TiO2 particles was

placed on the glass slide and the particles were allowed to settle Movement of particles

was observed in DI water and videos were recorded at 50x magnification of optical

microscope for 30 sec 05 H2O2 was added to the particle suspension on the slide

Videos were recorded at 50x ranging from 30 sec to 10 min to study the movement of

silver titania particles in hydrogen peroxide solution Particles in both DI water and

hydrogen peroxide were subjected to UV light and videos were recorded to study particle

dynamics in presence of UV radiation Particle tracking was performed using Physvis

particle tracking software Atleast 30 particles in every system were tracked for 10

seconds each Particle tracking data was obtained in terms of X and Y coordinates of

particles on screen This data was manipulated using MathWorks-Matlab software and

graphs of mean square displacement of particles versus time were plotted Diffusion

coefficients of particles were calculated from these graphs

47

43 Results and Discussion

Ag-TiO2 particles demonstrated autonomous motion in the presence of H2O2 No UV

light was required for this motion Motion of the particles was accelerated by addition of

fresh H2O2 to the system When subjected to UV radiation the particles displayed

momentary acceleration for 4-5 seconds

Figure 4 3 Mechanism of motion of Ag-TiO2 nanomotors

In the presence of H2O2 silver part of the Ag-TiO2 particle converts H2O2 to H2O and O2

This may lead to propulsion of the Ag-TiO2 particle due to bubble propulsion andor self-

diffusiophoresis In the presence of UV light additional reaction takes place on the

titania part of Ag-TiO2 particle which increases the velocity of the Ag-TiO2 particles

(Figure 4 4) This UV triggered photoactivity of titania is due to hole-electron separation

caused by photons having energy equal to or greater than the band gap of titania646566

Most of the holes-electrons recombine but a small fraction move to the surface and react

with the available redox species like water and oxygen to form protons (H+) superoxide

(O2-) and hydroxyl (OH) radicals

6566 This may lead to propulsion by diffusiophoresis

as well as electrophoresis

48

Figure 4 4 Silver-titania motors move faster when subjected to UV radiation

In addition to linear motion rotational motion is also observed in several particles and

particle aggregates Aggregation of particles can lead to a net circular propulsive force

which can rotate the aggregate as shown in Figure 4 5

Figure 4 5 Rotation due to particle aggregation of Ag-TiO2 particles

Particle tracking by Physvis software revealed a significant increase in diffusion

coefficient of Ag-TiO2 particles in hydrogen peroxide solution as compared to DI water

49

The Ag-TiO2 particles have a diffusion coefficient of 1491 μm2s in hydrogen peroxide

versus only 180 μm2s in DI water Thus the Ag-TiO2 particles move around more than

10 times their body length per second in hydrogen peroxide (Figure 4 6) When

subjected to UV light the particles experience a significant acceleration in hydrogen

peroxide which lasts only for a short duration of time (4-5 sec) after which the particles

return to their original velocity The diffusion coefficient of the particles during this UV

light trigger is found to be around 5093 μm2s UV light trigger does not have any visible

effect on the Ag-TiO2 particles in DI water

Table 4 1 Translational diffusion coefficients of Ag-TiO2 particles in hydrogen

peroxide and water

Mean 05 H2O2 solution DI water

Diffusion coefficient of Ag-TiO2 (μm2s)

without UV light

1491 180

Diffusion coefficient of Ag-TiO2 (μm2s)

in the presence of UV light

5093 113

50

Figure 4 6 Mean square displacement curves of Ag-TiO2 Janus particles in hydrogen

peroxide and DI water

When higher concentration (~30 vv) of Ag-TiO2 particles in hydrogen peroxide is

subjected to UV radiation the particles exhibit pattern formation (Figure 4 7)

Aggregation of Ag-TiO2 Janus particles is observed when UV light is turned on and the

particles disaggregated when UV light is switched off This can be due to predator-prey

kind of interaction between silver and titania half of the Janus particles in the presence of

UV light Oxygen and water that are produced at the silver end by decomposition of

hydrogen peroxide are used up by titania when subjected to UV radiation This can lead

to attraction between titania and silver parts of neighboring Janus particles causing

aggregation

y = 20373x

y = 59994x

y = 71848x

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10 12

Mean

sq

uare

dis

pla

cem

en

t (μ

m2s

)

Time (sec)

Mean square displacement vs Time for Ag-TiO2

particles

05 Hydrogen peroxide and UV05 Hydrogen peroxide

DI Water

51

A UV OFF B UV ON

C UV OFF

Figure 4 7 Higher concentrations of Ag-TiO2 particles showing aggregation when UV

is turned on

52

Chapter 5

Summary and Conclusion

In summary no significant enhancement in diffusion was observed in case of enzyme

immobilized Janus particles in substrate solution Presence of a linker between enzyme

and particle did not increase the diffusion coefficient of particles Alternative enzyme

immobilization methods need to be tested like thiolation of enzyme and subsequent

binding to gold surface of a Janus particle35

or expressing the enzyme with polyhistidine

or avidin tag67

This can result in higher enzyme activity upon immobilization and may

lead to enhanced diffusion of Janus particles

Predator-prey system of GOx and HRP immobilized particles showed some promising

results Aggregation of particles and formation of fan-shaped structures by TMB

oxidation were observed under optical microscope when GOx and HRP immobilized

particles glucose and TMB were present in the system The size of the fan-shaped

structures was dependent on the size of enzyme immobilized particles This can be

indicative of some kind of interaction between the GOx and HRP immobilized particles

No conclusive aggregation of GOx and HRP immobilized particles was observed in

absence of TMB Better imaging techniques are required to study the interaction of GOx

and HRP immobilized particles at the unstable liquid-air interface where majority of the

reaction takes place Other enzyme cascade systems not requiring gaseous reactants like

glycogen synthase and glycogen phosphorylase should be tested for similar activity

53

Ag-TiO2 particles were found to exhibit autonomous motion in the presence of hydrogen

peroxide solution The motion was momentarily accelerated upon exposure to UV

radiation The particles showed a diffusion coefficient of 1491 μm2s in hydrogen

peroxide versus only 180 μm2s in DI water Upon subjecting particles to UV radiation in

presence of H2O2 diffusion coefficient increased to 5093 μm2s for a period of 4-5

seconds In addition to linear motion rotating motion could also be observed in some

particle aggregates Further development of these aggregate structures can lead to

formation of rotors at nanoscale Higher concentration of silver-titania particles in

hydrogen peroxide displayed aggregation in presence of UV light and disaggregated

when UV light was switched off This may be attributed to the predator-prey kind of

interaction between silver and titania where oxygen and water that are produced by

silver by decomposition of hydrogen peroxide are used up by titania when subjected to

UV radiation This promises to be an interesting system and needs to be further

investigated to explore its properties

54

References

1 Paxton WF Sundararajan S Mallouk TE Sen A Angew Chem Int Ed Engl

2006 45 5420-5429

2 Golestanian R Liverpool TB Adjari A Phys Rev Lett 2005 94 220801

3 httpwwwsilvermedicineorgh2o2html

4 Rami Pedahzur Ovadia Lev Badri Fattal Hillel Shuval Water Science and Technology

1995 31(5-6) 123-129

5 W Went F American Scientist 1968 56

6 Adriano Cavalcanti Tad Hogg Bijan Shirinzadeh IEEE MHS 2006 International

Symposium on Micro-NanoMechatronics and Human Science 2006 226-232

7 EM Purcell American Journal of Physics 1977 45 3-11

8 Golestanian R Phys Rev Lett 2010 105 018103(1)-018103(4)

9 S S Dukhin Z R Ulberg G L Dvornichenko B V Deryagin Russian Chemical

Bulletin 1983 31(8) 1535-1544

10 Dukhin AS Goetz PJ Ultrasound for characterizing colloids Particle sizing zeta

potential rheology Elsevier 2002 59

11 HJKeh HJTu Trends in Colloid and Interface Science XIV Progress in Colloid and

Polymer Science 2000 115 174-180

12 D Zinemanas A Nir International Journal of Multiphase Flow 1995 21(5) 787-800

55

13 Stephen Ebbens Jonathan Howse Soft matter 2010 6 726-739

14 J Vicario R Eelkema W R Browne A Meetsma R M La Crois B L Feringa

Chem Commun 2005 31 3936

15 Davide Pantarotto Wesley R Browne Ben L Feringa Chem Commun 2008 1533 -

1535

16 Lisa A Cameron Matthew J Footer Alexander van Oudenaarden Julie A Theriot

PNAS 1999 96(9) 4908-4913

17 Ali Najafi Ramin Golestanian Phys Rev E 2004 69 062901

18 Jinseok Kim Jungyul Park Sungwook Yang Jeongeun Baek Byungkyu Kim Sang

Ho Lee Eui-Sung Yoon Kukjin Chun Sukho Park Lab Chip 2007 7 1504 - 1508

19 Kurt M Dubowski Clinical Chemistry 2008 5411 1919ndash1920

20 Spector DA Yang Q Wade JB Am J Physiol Renal Physiol 2007 292(1) 467-474

21 Rhee SG Chang TS Bae YS Lee SR Kang SW J Am Soc Nephrol 2003 14(8-3)

211-215

22 Sakaue T Kapral R Mikhailov A S Eur Phys J B 2010 75 381ndash387

23 Muddana H S Sengupta S Mallouk T E Sen A Butler P J J Am Chem Soc

2010 132 (7) 2110ndash2111

24 Baker CJ Deahl K Domek J Orlandi EW Arch Biochem Biophys 2000 382(2) 232-

237

25 V Vojinovic RH Carvalho F Lemos JMS Cabral LP Fonseca BS Ferreira

Biochemical Engineering Journal 2007 35 126-135

56

26

OV Lebedeva NN Ugarova Russian Chemical Bulletin 1996 45(1) 18-25

27 Nigel C Veitch Phytochemistry 2004 65(3) 249-259

28 Kenneth H Petersen Journal of Immunological Methods 2009 340(1) 86-89

29 httpwwwbrenda-enzymesorgphpresult_flatphp4ecno=11117

30 Goldenberg L M Wagner J Stumpe J Paulke B-R Goumlrnitz E Langmuir 2002 18

5627-5629

31 httpwwwgoldbiocompdf3761-Protocol201pdf

32 Av Rovisco Pais Lisboa Portugal T amp D Technology West Yorkshire UK Journal

of Molecular Catalysis B Enzymatic 2004 28(2-3) 129-135

33 httpwwwphysicsnyuedugrierlabmethodsnode11html

34 Yoon-Mee Lee1 O-Yul Kwon Yeo-Joon Yoon Keungarp Ryu Biotechnology Letters

2006 28 39ndash43

35 JC Pyun SD Kim JW Chung Analytical Biochemistry 2005 347(2) 227-233

36 George H Pimbley Jr Mathematical Biosciences 1974 20(1-2) 27-51

37 Andy Fenton Sarah E Perkins Parasitology 2010 137 1027-1038

38 Mead James G Brownell Robert L Mammal Species of the World A Taxonomic and

Geographic Reference (3rd ed) Johns Hopkins University Press 2005 2 2142

57

39

Krause KH Schweiz Med Wochenschr 2000 130(4) 97-100

40 Irina Kareva Faina Berezovskaya Carlos Castillo-Chavez Journal of Biological

Dynamics 2010 4(4)315 - 327

41 Delaittre G Reynhout I Cornelissen J Nolte R Chemistry ndash A European Journal

2009 15 12600ndash12603

42 C Buller DH Johnson RC Tschumper Investigative Ophthalmology amp Visual Science

1990 31 2156-2163

43 Robert M Mazo Brownian Motion Fluctuations Dynamics and Applications Oxford

Science Publications 2009 57

44 Vivek K Mutalik KV Venkatesh Theoretical Biology and Medical Modelling 2005 2

19

45 Satoshi Nakamura Kunimasa Koga Biochemical and Biophysical Research

Communications 1977 78(2) 806-810

46 Krishnananda Chattopadhyay Shyamalava Mazumdar Biochemistry 2000 39 (1)

263ndash270

47 Satoshi Nakamura Yasuyuki Ogura J Biochem 1968 63 (3) 308-316

48 van Dongen SF Nallani M Cornelissen JJ Nolte RJ van Hest JC Chemistry 2009

15(5) 1107-1114

49 Ronald Bentley Methods in Enzymology 1955 1 340-345

50 Vartiainen J Raumlttouml M Paulussen S Packaging Technology and Science 2005

18 243ndash251

58

51 GA Sherbeny AA Shindia YMMM Sheriff International Journal of Agriculture

and Biology 2005 7

52 D Rando G-W Kohring F Giffhorn Appl Microbiol Biotechnol 1997 48

53 Worthington enzyme manual Worthington Biochemical Corporation

httpworthington-biochemcomgopdefaulthtml

54 httpwwwbrenda-enzymesorgphpresult_flatphp4ecno=11117

55 httpwwwoxygenphwatercom

56 P Boon Chock Sue Goo Rhee and Earl R Stadtman Annu Rev Biochem 1980 49

813-841

57 Sybil Golden P A Wals Joseph Katz Analytical Biochemistry 1977 77(2) 436-445

58 R L Baehner S K Murrmann J Davis R B Johnston Jr J Clin Invest 1975 56(3)

571ndash576

59 I Bejarano M P Terroacuten S D Paredes C Barriga A B Rodriacuteguez J A Pariente

Molecular and Cellular Biochemistry 2007 296(1-2) 77-84

60 Michael C Moulton Laura K Braydich-Stolle Mallikarjuna N Nadagouda Samantha

Kunzelman Saber M Hussain Rajender S Varma Nanoscale 2010 2 763-770

61 Goya DV Matijevic E Colloids and Surfaces A 1999 146 139

62 Velikov KP Zegers GE van Blaanderen A Langmuir 2003 19 1384

63 Goldenberg L M Wagner J Stumpe J Paulke B-R Goumlrnitz E Langmuir 2002 18

5627-5629

59

64

Banerjee S Gopal J Muraleedharan P Tyagi A K Raj B Curr Sci 2006

901378ndash1383

65 Bogomolov V N Kudinov E K Firsov Y A Sov Phys Solid State 1968 35 555

66 Kudo A Miseki Y Chem Soc Rev 2009 38 253ndash278

67 Gaj T Meyer SC Ghosh I Protein expression and Purification 2007 56 54-61

1

Chapter 1

Introduction

Motion at the macroscale has been studied and applied for centuries Invention of the

wheel can be considered as the major step for the beginning of synthetic motors at

macroscale The potential applications of the wheel and other similar motors are still

being explored and seem to have endless possibilities Having said that motion at the

nanoscale is just beginning to get explored and can have similar if not greater

implications in the future This thesis is a part of an attempt to develop nanomotors made

from diverse materials operating in different environments to create a pool of

nanomotors which can be used in the future for specific applications

Some of the nanomotors which have already been developed include the pioneering

work by WFPaxton etal of platinum-gold nanomotors1 showing autonomous motion in

hydrogen peroxide motors made by Golestanian2 consisting of micron sized particles

half coated with Pt and others The mechanisms of motion differ in case of different

motors but all have some basic underlying principles which will be highlighted in the

following section An important point to notice in the above mentioned nanomotors is

that they involve metals or solutions which are not biocompatible This puts a restriction

on the application of such motors This thesis attempts to synthesize enzyme based

nanomotors which will be able to overcome these drawbacks and lead to applications in

biological systems like drug delivery and surgery in human body

2

Second part of this thesis consists of synthesis and motion of silver-titania nanomotors

which can move autonomously in hydrogen peroxide solution According to some recent

findings3 colloidal silver and hydrogen peroxide may prove to be an effective

combination for treatment of infections in human body Combination of silver particles

and hydrogen peroxide has been tested for disinfection of drinking water and may hold

promise as a secondary disinfecatant which can provide long lasting residual and biofilm

control that is required in water distribution systems4 Silver-titania nanomotors can open

interesting avenues in such applications

11 Motion at the nanoscale

Motion as we understand at macroscale is significantly different from motion at

micronnanoscale A definite discontinuity exists between the two This can be

emphasized by observing the biological systems surrounding us For example at the size

of a cell which is around 10 μm mixing of large molecules is instantaneous due to

diffusion and there is no need for additional convective mixing which is what we

generally think of when we picture macroscopic mixing For instance while convective

flow driven by the pumping of lungs or the use of gills is essential for transport of oxygen

inside an organism which is larger than 1mm in size such as a lizard or a man small

organisms receive nutrients and oxygen solely through diffusion through their tissues5

These differences in motion can be understood both physically and mathematically

Physically as the size of an object decreases the mass and weight decrease with the third

3

power of linear size A large body predominantly has gravitational pull of earth acting on

it due to its large mass and other forces like gravitational effect of surrounding objects are

relatively insignificant As the object gets smaller forces such as molecular attraction and

cohesion become more significant and the relative magnitude of the gravitational pull of

the earth on the object decreases due to reduction in mass5 Thus the forces that need to

be considered to cause motion at the macroscale are different than those at the

microscale

Mathematically the difference in motion at macro- and micro- scales can be explained by

means of a dimensionless number called Reynolds number Reynolds number is the ratio

of inertial forces of a system to its viscous forces As size decreases Reynolds number

decreases and the viscous forces become more dominant For a human swimming in

water Reynolds number is around 104 for goldfish it is around 10

2 whereas for an

organism of 1μm size it is around 10-4

to 10-5

[67]

What this means is that as we scale

down from the size of a goldfish to micron sized organisms the inertial forces become

insignificant and the familiar laws of macroscale motion lose their applicability Equation

11 gives the formula for calculating Reynolds number where ρ is density of the fluid

(kgmsup3) μ is the dynamic viscosity of the fluid (kg(mmiddots)) V is the mean fluid velocity

(ms) and D is diameter of particle (m)

Equation 1 1

To understand the implications of low-Reynolds-number systems we can consider the

motion of a macroscopic and an imaginary microscopic scallop Macroscopic scallop

4

moves by opening and closing of its shell Inertial thrust is created when a scallop closes

its shell and the scallop moves forward due to the clapping motion (Figure 1 1) This

however does not hold true at the nanoscale since the inertial forces are relatively

insignificant due to the large viscous forces If a microscopic scallop were to exhibit a

similar clapping motion it will just keep moving forward and backward in an oscillatory

kind of motion and will not experience a net motion This is an example of reciprocal

motion which can linearly move a body at macroscale however fails to cause motion at

the microscale

For motion at microscale nonreciprocal motion is necessary8 Nonreciprocal motion

occurs when the path chosen for going from Position A to Position B is different from the

path chosen to come back from Position B to Position A It can be better understood with

the help of Figure 1 1 which shows the change of shapes for a theoretical object going

from Position A to Position B and then returning to Position A by using a different set of

motions This kind of change can lead to motion at the nanomicron scale

5

Another factor which gains importance at nanoscale is the dominance and ubiquity of

Brownian motion13

The trajectory of a linearly propelled nanomotor would be

significantly affected by the continuous collision of randomly moving particles in the

environment These collisions deviate the nanomotor from its linear path and hence this

effect needs to be considered while projecting the path of a nanomotor

Motion at the nanoscale can be powered by different types of mechanisms such as

diffusiophoretic electrophoretic osmophoretic chemophoretic thermophoretic and

others Different types of mechanisms of motions may coexist or act individually to cause

Position A Position B

Figure 1 1 Reciprocal and Nonreciprocal motion8

Reciprocal motion

Nonreciprocal motion

6

motion Diffusiophoretic motion of particles is caused due to concentration gradients of

solutes in an electrolytenonelectrolyte solution9 Electrophoresis is the motion of a

particle relative to a fluid under the influence of an electric field10

This occurs as the

particle carries an electric surface charge which is acted upon by an electrostatic

coulombic force of the external electric field Osmophoresis is the motion of a

vesiclefluid particle encapsulated in a semi-permeable membrane towards lower

concentration gradient when it is placed in a solution containing solute concentration

gradient1112

Thus considering the numerous mechanisms of motion known to work at

the nanoscale several suitable options are available for the development of artificial

nanomotors

12 Existing nanomotors Mechanisms and uses

Several nanomotors have been developed in the past decade or so A general principle of

working of these nanomotors is that they function by catalytically decomposing the

dissolved fuel to generate asymmetry at their surface or at their interfacial region thereby

causing propulsion13

The self-electrophoretic bimetallic nanowires developed by

WPaxton and A Sen etal consisted of a 2 μm long rod with one tip made up of Au and

the other tip made up of Pt1 These motors were synthesized by electrodeposition in an

alumina template The rods moved autonomously in a solution of hydrogen peroxide

with platinum end forward Pt oxidizes H2O2 to produce H+ and O2 and Au utilizes H

+ to

reduce H2O2 to water This generates an electron flow through the rod and a proton flow

outside the rod resulting in an electric field around the rod in the solution This electric

7

field in solution acts on the unbalanced charges within the nanorodrsquos double layer This

moves the nanorod with respect to the surrounding fuel due to the ion flux that is

generated

Bubble propulsion has been predominantly used as mechanism of motion in the motors

developed by Feringa etal His group developed a micromotor by attaching catalase to a

microparticle and placing the particle in a solution containing hydrogen peroxide This

system allowed the microparticle to move by producing oxygen bubbles from the

decomposition of hydrogen peroxide14

A similar motor was later created by Feringarsquos

group which consisted of glucose oxidase and catalase immobilized on a carbon

nanotube in a substrate solution of hydrogen peroxide Glucose oxidase converted

glucose to H2O2 which was then utilized by catalase to produce water and oxygen

thereby propelling the carbon nanotube15

Another interesting example of a self-propelled motor is the polymerization driven motor

developed by Cameron et al16

It consists of polystyrene beads immobilized with ActA

protein which catalyzes actin polymerization leading to the formation of an actin

filament which resembles the flagella of bacteria This actin polymerization propels the

beads at the speed of 001-015 μms

A theoretical model has been proposed by Nafaji and Golestanian etal which consists

of three spheres linked by rigid rods in one dimension17

The length of the rods can

switch between two specific values Due to the nonreciprocal motion of the rod this

8

model can swim in low Reynolds number systems An actual model based on

nonreciprocal motion has been developed by Kim etal in which heart cells have been

linked to an asymmetric polydimethylsiloxane robot As the heart cells beat the

asymmetric structure of the device resembles a walking movement18

Most of the nanomicromotors mentioned above are either metallic or work in

environments not compatible with human body Enzymes being an integral part of the

body can solve this problem by operating in suitable substrates already present in the

body fluids The nonreciprocal conformation changes of the enzyme as well as

diffusiophoresis and electrophoresis can conceivably be used to generate motion in case

of enzyme driven nanomotors such motors will be discussed in detail in the following

chapter

9

Chapter 2

Immobilized enzymes on Janus particles as catalytic motors

21 Introduction

Enzymes are widely found in biological systems and act on a large variety of substrates

This can make enzymes an ideal choice for powering nanomotors which need to be

biocompatible Several fluids found in the body can act as substrates to the enzymes For

example dissolved glucose which is abundantly found in the body19

is a susbtrate for

enzyme glucose oxidase Urea which is found in the urinary tract20

is a substrate for

urease Hydrogen peroxide produced by phagocytic cells in the body21

is a substrate for

horseradish peroxidase Having motors driven by enzymes can open possibilities of

targeted drug delivery to these parts of the body containing such substrates

Enzymes have already been used to generate motion of carbon nanotubes by mechanism

of bubble propulsion by Feringa etal15

Two other possible mechanisms which may be

able to power motion of nanoparticles using enzymes are asymmetrical decomposition of

substrates into products leading to self-diffusiophoresis (Figure 2 1) and nonreciprocal

changes in enzyme conformation leading to propulsion (Figure 2 2) As seen in Figure 2

1 enzymes which are immobilized on a Janus particle can breakdown the available

substrate in the solution into products causing asymmetric distribution of solutes in the

surrounding fluid This can lead to movement of particle towards the region of lower

10

solute concentration by principle of self-diffusiophoresis Figure 2-2 shows the possible

nonreciprocal changes in the conformation of a theoretical protein which can lead to

motion of the protein22

These nonreciprocal changes in conformation are triggered by the

attachment of substrate molecule and release of product molecules from the enzymersquos

active site

Figure 2 1 Enzyme functionalized Janus particle moves upon catalyzing asymmetrical

decomposition of substrate into product

11

Figure 2 2 Schematic showing idealized nonreciprocal conformation changes in a

theoretical enzyme22

Enhanced diffusion of a single enzyme molecule by catalysis in a substrate solution has

been demonstrated23

The diffusion coefficient of a single urease molecule was measured

using fluorescence correlation spectroscopy and it was observed that the diffusion of the

urease molecule increases by 16-28 by varying urea concentrations from 0001 M to 1

M Addition of enzyme inhibitors to the system curbed the enhanced diffusion This

enhanced diffusion has been attributed to the force generated by self-electrophoresis by

the enzyme in substrate solution

12

Based on the above facts it might be possible to propel a nanoparticle by asymmetric

distribution of enzymes on its surface thereby leading to self-propulsion of the particles

in substrate solution (Figure 2 1) This propulsion may be due to self-

electrophoresisself-diffusiophoresis andor due to nonreciprocal changes in enzyme

conformations Here we aim to investigate a system consisting of polystyrene particles

half coated with enzyme introduced in a suitable substrate solution The enzyme can be

directly linked to the polystyrene particle or it can be linked through a long spacer

molecule (linker) to provide more degrees of freedom to the enzyme

Several factors need to be considered for choosing enzyme for powering motion at the

nanoscale Some of the important factors are the ratio of kcatKm which denotes the

catalytic efficiency of the enzyme possible substrates stability of enzymessubstrates

ease of immobilization relationship with other enzymes in cascade reactions and

commercial availability of the enzymes The enzyme being used in the present study is

horseradish peroxidase It acts on a molecule of hydrogen peroxide and reduces it while

simultaneously oxidizing another substrate molecule In the absence of alternate

oxidizable substrate HRP can oxidize water and oxygen back to hydrogen peroxide24

thus giving an equilibrium reaction

13

211 Horseradish peroxidase

Figure 2 3 Horseradish peroxidase

Horseradish peroxidase belongs to the group of peroxidases which are a group of heme-

containing oxidoreductases that act on peroxide as hydrogen donor25

It is a redox

enzyme which contains a protoheme a complex of trivalent iron with 1358-

tetramethyl-24-divinylporphin-67-dipropionic acid as a prosthetic group It is a 44 kDa

protein which includes 33900 Da polypeptide chain bound with eight oligosaccharide

chains The carbohydrate chains improve the stability of the enzyme by protecting it

against radicals formed in the course of the reaction it catalyzes26

Horseradish

peroxidase is found in horseradish and is used widely in bioanalytical applications such

as ELISA and Western blots It decomposes hydrogen peroxide into water and oxygen

while simultaneously oxidizing another substrate27

The oxidizable substrates can be

chromogenic substrates such as 22rsquo55rsquo-tetramethyl-benzidine (TMB) and 33-

diaminobenzidine (DMB) or chemoluminescent substrates like SuperSignal and ECL

luminal28

14

Figure 2 4 Reaction catalyzed by horseradish peroxidase

The functional parameters for horseradish peroxidase include Km of 015 mM and kcat of

790 s-1

at an optimum temperature of 20 ordmC and an optimum pH of 6429

Some of the

inhibitors of HRP are 110-phenanthroline aminotriazole and bromine

22 Experimental

221 Materials and methods

Uniform carboxyl modified polystyrene microspheres 05 microm in diameter were obtained

from Polysciences Inc The size of the purchased microspheres was verified by Field

Emission Scanning Electron Microscopy (FESEM) Peroxidase Type I from

Horseradish N-(3-dimethyl-aminopropyl)-Nrsquo-ethylcarbodiimide (EDC) and hydrogen

peroxide solution 50 wt in water were purchased from Sigma-Aldrich N-

hydroxysuccinimide (NHS) ge 97 was purchased from Fluka All chemicals were of

reagent grade and used without further purification Nanopure deionized water was used

for all experiments and preparation of solutions All measurements were made at room

15

temperature (25 oC) unless mentioned otherwise The UV-Visible spectroscopy

measurements were performed using Agilent 8453A diode-array UV-Visible

Spectrophotometer with ChemStation UV-Vis Software Diffusion coefficient

measurements were performed using Nanosight LM10

222 Synthesis of Janus particles

A monolayer of particles was prepared according to the procedure (Figure 2 5) by

Goldenberg L et al30

Glass slides were placed on the bottom of a petridish Water was

put into the petridish so that the entire slide was immersed in water A thin layer of

hexane was added on top of water in the petridish A suspension of microspheres in

ethanol (5 ww) was introduced to the water-hexane interface After the monolayer was

formed the glass slide was lifted up through the monolayer with tweezers The

microsphere-covered glass slides were left to dry overnight at room temperature

Following drying a thin film of silica was evaporated on the glass slides in Semicore

evaporator The spheres were partially coated with 30 nm of silica Particles were

removed from the glass slide by gentle sonication The polystyrene-silica (PS-SiO2)

Janus particles thus obtained were washed in deionized water several times before

further immobilization steps were performed FESEM was used to confirm synthesis of

Janus particles

16

Figure 2 5 Preparation of PS-SiO2 Janus particles by evaporation

223 Immobilization of enzymes using EDCNHS coupling method

1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide and N-hydroxysulfosuccinimide

(EDCNHS) coupling procedure was followed for immobilizing horseradish peroxidase

type I on 05 μm PS-SiO2 Janus particles (Figure 2 6) In this process EDC reacts with a

carboxyl groups on the Janus particles to form an amine-reactive O-acylisourea

intermediate If this intermediate does not encounter an amine it can hydrolyze and

regenerate the carboxyl group In the presence of NHS EDC can be used to convert

carboxyl groups to amine-reactive sulfo-NHS esters which are much more stable and do

not readily hydrolyze to regenerate carboxyl groups In the current experiment

immobilization was performed in the presence as well as absence of a long spacer

17

molecule (linker) between Janus particles and enzymes The chemical 6-aminohexanoic

acid was used as the linker (Figure 2 7) HRP immobilized PS-SiO2 Janus particles were

stored in deionized water until required for further experiments

Figure 2 6 EDCNHS coupling method for immobilization of enzyme on carboxylated

polystyrene particles

Figure 2 7 Immobilization of enzyme on Janus particles with and without 6-

aminohexanoic acid linker

18

224 Enzyme assay

Assay for activity of HRP was performed using a spectrophotometric method which

involved using 4-aminoantipyrene as hydrogen donor3132

The reaction rate was

determined by measuring increase in absorbance at 510 nm resulting from the

decomposition of hydrogen peroxide One unit represents the decomposition of 1 μM of

hydrogen peroxide per minute at 25 degC and pH 70 under the specified conditions

The spectrophotometer was set to the wavelength of 510 nm and temperature of 25 degC

Phenol4-aminoantipyrene solution (14 mL) and 00017 M H2O2 solution (15 mL) were

pipetted into each cuvette The cuvette was incubated for 3-4 mins at 25degC and blank was

established A 01 mL solution of diluted enzyme was added to the above mixture and

absorbance was recorded for 4-5 mins Standard graphs of absorbance versus time were

drawn for known concentration of the free enzyme and compared to that of the

immobilized enzyme on the Janus particles The estimate of the amount of enzyme

present on the Janus particles was deduced from the difference between the quantity of

the free enzyme previously added for immobilization and the amount that was measured

to be in the supernatant after some of the enzyme became attached to the Janus particles

The experiment was carried out in triplicate to assess reproducibility

19

The formula used to calculate the activity of enzyme is

where ∆Amin denotes the change in absorbance per minute

225 Diffusion coefficient measurements using Nanosight LM10

Diffusion coefficients for HRP immobilized particles were measured using Nanosight

LM 10 nanoparticle tracking instrument with Nanoparticle Tracking Analysis (NTA) 20

software in the presence of 003 H2O2 solution as the substrate and also in the presence

of only deionized water The videos were recorded for 30 seconds at the rate of 30 frames

per second Each experiment was carried out in triplicate to assess reproducibility

Particles

visualization

zone

Microscope with

LM 10 unit

LM 10 unit

Figure 2 8 Nanosight LM 10 instrumentation

20

Nanosight LM 10 (Figure 2 8) generates videos of population of moving nanoparticles in

a liquid when illuminated by laser light The particles which pass through the laser beam

path are seen by the instrument as small points of light undergoing Brownian motion

(Figure 2 9a)

The videos are further analyzed by NTA (Nanoparticle tracking analysis) software

(Figure 2 9b) which identifies and tracks each particle independently on a frame-by-

frame basis The software also calculates and subtracts any bulk flow in the system The

mean square displacement is calculated for each particle as long as it is visible If the

particle blinks that is if it disappears momentarily and then reappears on the screen the

software will consider the same particle as two separate ones All analysis data is

exported to the excel files in terms of diffusion coefficients of the tracked particles

B A

Figure 2 9 NTA video analysis (a) Particles as they are seen on the screen during

experimentation in Nanosight LM10 (b) NTA screen as the particles are being analyzed for

their diffusion coefficients

21

23 Results

231 Enzyme assay

The activity of free HRP enzyme reported by Sigma Aldrich is 150 unitsmg However

the activity of HRP upon analysis was found to be 52158 unitsmg which is much lower

than the reported value The activity of HRP was found to be further lowered to 2133

unitsmg when it was immobilized on the PS-SiO2 Janus particles in the presence of a

linker The following formula is used to calculate enzyme activity

where ΔAmin is the change in absorbance per minute

22

Figure 2 10 Graph for the assay of solution of free enzyme containing 05mgL of HRP

ΔAmin = 01716 Unitsmg of free enzyme = 52158

During EDCNHS coupling 04 mg enzyme was added to 1 ml of 025 vv suspension

of Janus particles Thus initial concentration of enzyme in the mixture was 04 mgml

Assuming retained activity of the enzyme in supernatant solution the amount of enzyme

in supernatant was calculated to be 03868 mgml from the assay Amount of enzyme

functionalized on the Janus particles was calculated as the difference between enzyme

previously added for immobilization (04 mgml) and the enzyme present in supernatant

(03868 mgml) and was found to be 00132 mgml Enzyme assay was performed on the

enzyme immobilized Janus particles to estimate the activity of immobilized enzyme

(Figure 2 11) and was found to be 2133 unitsmg

y = 0002x + 00516

0

01

02

03

04

05

06

07

08

0 50 100 150 200 250 300 350 400

Ab

sorb

an

ce

51

0 n

m

Time (sec)

Assay of free enzyme HRP

23

Figure 2 11 Graph for the assay of of immobilized enzyme on 05 μm Janus particles

(~025 vv) ΔAmin = 01853 Unitsmg of free enzyme = 2133

232 Diffusion coefficient measurements

No significant increase was observed in diffusion coefficient of HRP-immobilized Janus

particles in the presence of H2O2 as the substrate Similar results were obtained for

diffusion coefficient measurement with and without linker moieties bridging the enzymes

and particles(Table 2 1 Table 2 2) Thus the linker did not seem to assist in increasing

the particle diffusion by improving or retaining the activity of the enzyme

y = 00015x + 00953

0

01

02

03

04

05

06

07

0 50 100 150 200 250 300 350 400

Ab

sorb

an

ce

510 n

m

Time (sec)

Assay of HRP immobilized 05 μm Janus particles

24

Table 2 1 Diffusion coefficient measurements of HRP immobilized Janus particles

without linker

HRP immobilized

Janus particles in

003 H2O2

Control - Janus

particles in

003 H2O2

Control - HRP

immobilized Janus

particles in DI water

Diffusion coefficient

(10-10

cm2s)

1359416 1051777 1308675

Standard deviation 4534801 4122075 4918597

Table 2 2 Diffusion coefficient measurements of HRP immobilized Janus particles in

the presence of linker

HRP immobilized

Janus particles in

003 H2O2

Control - Janus

particles in

003 H2O2

Control - HRP

immobilized Janus

particles in DI water

Diffusion coefficient

(10-10

cm2s)

13838 1339829 1407891

Standard deviation 5644692 5649364 5797218

25

Figure 2 12 Diffusion coefficient measurements of PS-SiO2 Janus particles without and

with 6-aminohexanoic acid linker

24 Discussion

To compare the diffusion coefficients observed during the experiments theoretical

diffusion coefficient was calculated using following equation33

r

kTD altraslation

6 Equation 21

where r is the hydrodynamic radius of particle Dtranslational is translational diffusion

coefficient of the particle T is temperature of the system η is the viscosity of fluid

through which the particles are moving and k is the Boltzmann constant From FE-SEM

images the size of the particles used was determined to be 450 nm For 450 nm diameter

0

20

40

60

80

100

120

140

160

Without linker With linker

Dif

fusio

n c

oeff

icie

nt

(10

-10 c

m2s

)

HRP immobilized 05 μm Janus particles - Diffusion coefficient measurements

HRP immobilized Janus particles in 003 H2O2

Control - Janus particles in 003 H2O2

Control - HRP immobilized Janus particles in DI water

26

particle Dtranslational is equal to 109 μm2s which is comparable to the diffusion

coefficient measured for the enzyme immobilized particles

HRP was found to be active on the Janus particles however the activity of the enzyme

was reduced upon immobilization Lower enzyme activity can be due to the coupling

method used which in this case was EDCNHS covalent binding However EDCNHS

coupling method has been shown to significantly retain the enzyme activity when the

enzyme is immobilized on carboxylated multi-walled carbon nanotubes under appropriate

conditions34

Hence EDCNHS binding method by itself is not believed to be

responsible for reduction of enzyme activity upon immobilization

Direct binding of enzyme to the Janus particle may lead to reduction in the enzyme

activity because of space constraints for the enzyme and difficulty for the passage of

substrate to the active site of the enzyme due to stearic hindrance Hence a linker was

inserted between the enzyme and Janus particle during enzyme immobilization However

inserting a linker between the enzyme and the particle did not lead to enhanced diffusion

of Janus particles or increase in enzyme activity of immobilized enzyme

Lack of enhanced diffusion can be firstly attributed to the reduction in enzyme activity

upon immobilization Enzyme activity may be retained by modifying the conditions

during EDCNHS coupling or changing the binding method altogether The degree of

immobilization of HRP on carboxylated Janus particles can possibly be affected by

several factors such as temperature the concentration of the coupling reagent EDC

27

concentration HRP concentration in solution and incubation time34

Different

combinations of these factors can be tested to find optimum conditions for

immobilization of HRP Alternate binding method can involve thiolation of HRP by the

covalent conjugation of mercaptopropionic acid and then the thiolated HRP can then be

attached on the gold surface of a polystyrene-gold Janus particle35

Thus further

modifications can be made to the system in order to retain enzyme activity on the

immobilized particles and thereby power nanomotors using enzymes

28

Chapter 3

Predator-prey system of enzyme immobilized particles

31 Introduction

Predator-prey systems are widely found in nature and play a major role in biological

processes3637

Predator-prey systems include all the interactions in which one organism

pursues andor consumes another organism One familiar predator-prey system at the

macroscale is that of a tiger chasing and consuming a deer Another not so familiar one

includes Baleen whales which eat millions of microscopic planktons at once38

At the

microscale such kind of interactions can be observed in the case of phagocytes in the

body which are capable of engulfing foreign microorganisms and killing them with a

combination of microbicidal systems39

In the case of cancer the tumor is the prey and

the mature myeloid cells in the body are the predator However the tumor here has a

defense mechanism against the predator in which the tumor produces cytokines which

inhibits the maturation of the myeloid cells and thereby prevents predation40

One similarity that can be observed in all these systems is that the prey possesses some

attribute which is desired by the predator which makes the predator chase the prey A

similar relationship is seen in case of enzymes which are involved in cascade reactions41

In cascade reactions product synthesized by one enzyme is the reactant required by

another enzyme to carry out its own catalytic reaction As such there is a predator-prey

29

kind of dependence between the two enzymes which may cause one enzyme to chase the

other enzyme when placed in suitable substrate solutions However the mechanism

hypothesized for such a system is totally different than the mechanism involved in

biological systems In biological systems such as bacterial chemotaxis or phagocytes

chasing foreign particles in human body complex biochemical reactions are responsible

for the predator-prey behavior42

In the proposed enzyme cascade system there are no

complex biochemical reactions involved The principle hypothesized for predator-prey

behavior of enzyme cascade reactions can be explained as follows Motion of synthetic

motors at nanoscale involves two components translational diffusion and rotational

Brownian diffusion At very short time scales of less than 1Dr where Dr (s-1

) is

rotational Brownian diffusion coefficient the motion of particles is unidirectional For

longer time scales of greater than 1Dr the direction of motion is random giving

poweredactive diffusion43

If the chemical homogeneity is broken that is when there is

higher concentration of a substrate present in one direction of the enzyme the enzyme

might acquire a subtle bias which may lead to a spatial aggregation of the enzymes based

on the substrate concentration gradient The bias can be a result of the relationship

between the speed of enzyme diffusion and substrate concentration where higher

substrate concentration lead to higher enzyme diffusion As a result in an enzyme

cascade system one enzyme will migrate towards higher concentration of substrate

which is present near the other enzyme thus giving rise to a predator-prey behavior

Such enzyme cascades are widely found in signaling networks and metabolic pathways in

living cells and can be used to produce predator-prey like nanomotors systems44

The

30

enzymes being used in the present study are horseradish peroxidase and glucose oxidase

Both enzymes are extremely stable4546

Glucose is the substrate for glucose oxidase

which is widely available and highly stable47

Horseradish peroxidase acts on hydrogen

peroxide It is involved in a cascade reaction with glucose oxidase which produces

hydrogen peroxide by converting glucose into gluconic acid (Figure 3 1)48

Figure 3 1 Concept of particle aggregation due to cascade reactions of immobilized

enzymes

In this experiment glucose oxidase (GOx) and horseradish peroxidase (HRP) were

immobilized on polystyrene carboxylate beads of different sizes using EDCNHS

coupling method Glucose and 3355-Tetramethylbenzidine (TMB) were dissolved

together in water and this solution was used as the substrate solution All the components

were present in a single system in DI water Aggregation of particles and precipitation of

oxidized TMB from the solution was expected to be observed when all these components

were present in such a system

Properties of horseradish peroxidase have been discussed in the previous chapter (Section

211 Horseradish peroxidase)

GOxGOx HRPHRP

Glucose + O2Gluconic acid + H2O2

TMB TMB oxidizedBlue precipitate

H2O

Aggregation

31

311 Glucose oxidase

Figure 3 2 Glucose oxidase

Glucose oxidase (GOx) belongs to the group of oxidoreductases and is also called

glucose aerodehydrogenase49

It is obtained from fungi and has been identified as an

antimicrobial enzyme50

GOx binds to β-D-glucopyranose and catalyzes the oxidation

reaction to from gluconolactone while simultaneously reducing oxygen to hydrogen

peroxide51

GOx has high substrate affinity and specificity high stability and high

turnover number which makes it a suitable biocatalyst for industrial applications52

Figure 3 3 Redox reaction of glucose catalyzed by glucose oxidase53

32

GOx is a glycoprotein with molecular weight of 155 kDa and consists of two identical

polypeptide subunits linked together by a disulfide bond The optimum pH for GOx is

55 though it is stable in a broad pH range of 4 to 7 and can be stored for a long period of

time51

The functional parameters for glucose oxidase include Km of 151 mM and kcat of

337 s-1

at temperature of 20 ordmC and pH of 754

Some of the inhibitors of GOx are 2-

Deoxy-D-glucose adenine nucleotides and NaNO3

32 Experimental

321 Materials and methods

Uniform carboxyl modified polystyrene microspheres 05 microm in diameter were obtained

from Polysciences Inc The size of the purchased microspheres was verified by FESEM

Peroxidase Type I from horseradish glucose oxidase Type II-S from Aspergillus niger

N-(3-dimethyl-aminopropyl)-Nrsquo-ethylcarbodiimide (EDC) and Hydrogen peroxide

solution 50 wt in water were purchased from Sigma-Aldrich N-hydroxysuccinimide

(NHS) was purchased from Fluka D-Glucose (99) was purchased from VWR

International Secure-Seal imaging spacers (9mm diameter) for glass slides were obtained

from Sigma Aldrich All chemicals were of reagent grade and used without further

purification Nanopure deionized water was used for all experiments and preparation of

solutions All measurements were made at room temperature (25 oC) unless mentioned

otherwise The UV-Visible measurements were performed using Agilent 8453A diode-

array UV-Visible Spectrophotometer with ChemStation UV-Vis Software Optical

33

microscopy experiments were performed using Axiovert 200 MAT microscope by Carl

Zeiss Diffusion coefficient measurements were obtained using Nanosight LM10

322 Optical microscopy experiments

The enzyme substrate system was observed under optical microscope to check for

aggregation and precipitation due to cascade reactions Several such systems were

studied Each system in general consisted of GOx and HRP immobilized on different

polystyrene particles The substrate solutions consisted of 01 M glucose and different

concentrations of TMB solution The systems were studied in the presence of all the

constituents and in the absence of glucose GOxHRP and TMB Systems consisting of

different sized HRP immobilized particles in substrate solution of 003 hydrogen

peroxide and TMB were also studied Videos were recorded over a period of 24 hours to

observe changes in the system Each experiment was performed atleast thrice to assess

reproducibility of results

323 Diffusion coefficient measurements using Nanosight LM10

Diffusion coefficient of GOx and HRP immobilized particles were measured using

Nanosight LM10 nanoparticle tracking instrument with Nanoparticle Tracking Analysis

(NTA) 20 software Diffusion coefficients were measured for different systems namely

GOx particles in 01 M glucose and oxygen both GOx and HRP functionalized particles

in 01 M glucose and oxygen along with controls for the individual systems Videos were

34

recorded for 30 seconds at the rate of 30 frames per second Each experiment was carried

out in triplicate to assess reproducibility

33 Results

331 Optical microscopy

According to the enzyme cascade model precipitation of TMB was possible only upon

the interaction of GOx and HRP immobilized particles in presence of appropriate

substrate solution Hence in the first set of experiments precipitation of TMB was used

as an indication of the type of interaction between GOx and HRP immobilized particles

Fan-shaped structures were observed when both HRP and GOx immobilized particles

were present in a substrate solution consisting of glucose and TMB These structures

started forming as a single strand and then branched out and intertwined to form the fan-

shaped structures (Figure 3 4)

Table 3 1 Observations for formation of fan-shaped structures in enzyme cascade

system in presence of different components

GOx HRP Glucose TMB Observation

Yes Yes Yes Yes

Fan-shaped structures observed particles associated with

the fan-shaped structures

No Yes Yes Yes No fan-shaped structures

Yes No Yes Yes No fan-shaped structures

Yes Yes No Yes No fan-shaped structures

Yes Yes Yes No No fan-shaped structures

35

Fan-shaped structures can also be observed in the system consisting of only HRP

immobilized particles in substrate solution of hydrogen peroxide and TMB The size of

the fan-shaped structures depends on the size of the enzyme immobilized particles Free

enzymes show extremely tiny structures which are difficult to view under the optical

microscope

Figure 3 4 Formation of fan-shaped structures when both HRP and GOx

immobilized particles were present in a substrate solution consisting of glucose and

TMB A In the initial stages single strands form on the surface of liquid (5Ox) B

Single strands branch out and intertwine to form fan-shaped structures (50x) C Many

such structures form and settle down after 20 minutes (20x)

A

C

B

36

Experiments were performed in the absence of TMB that is the system consisted of GOx

and HRP immobilized particles in glucose substrate solution In the absence of TMB the

particles were expected to move closer to each other without any precipitation due to the

cascade reaction However there was no conclusive aggregation of particles observed in

the system

Free Enzyme 005 μm HRP - immobilized particle

10 μm HRP - immobilized particle 05 μm HRP - immobilized particle

Figure 3 5 Size dependence of fan-shaped structures on the size of HRP -

immobilized particles (20x)

37

Table 3 2 Observations for particle aggregation in enzyme cascade system in presence

and absence of TMB

Glucose GOx HRP TMB Observation

Present Present Present Present Aggregation and precipitate

Present Present Present Absent No conclusive aggregation

Present Absent Present Present No aggregation or ppt

Absent Present Present Present No aggregation or ppt

Present Present Absent Present No aggregation or ppt

332 Diffusion coefficient measurements

Diffusion coefficients were measured for GOx immobilized particles of 05 μm diameter

in substrate solution of 01 M glucose and oxygen Diffusion coefficients of particles

were also measured for system consisting of GOx and HRP functionalized particles in

glucose and oxygen Each experiment was done in triplicate to assess reproducibility

Nanosight visualization chamber being a closed chamber oxygen was purged into all the

solutions before starting the experiments to ensure sufficient supply of oxygen for

catalytic reaction of GOx

38

Table 3 3 Diffusion coefficients for 05 μm GOx immobilized particles in 01 M

glucose purged with O2

Mean

GOx

particles in

01 M

glucose

Control - GOx

particles in DI

water

Control - GOx

particles in 01 M

glucose without

oxygen purging

Control -

particles in 01

M Glucose

Diffusion

coefficient

(x10-10

cm2s)

13316 12888 13827 13702

Standard

deviation 6307 4926 4424 7468

Table 3 4 Diffusion coefficients for GOx and HRP immobilized particles in 01 M

glucose and purged with O2

Mean

GOx- HRP-

functionalized

particles in 01 M

glucose

Control ndash Gox-

HRP- functionalized

particles in DI water

Control - particles

in 01 M glucose

Diffusion coefficient

(x10-10

cm2s)

13005 14434 13702

Standard deviation 5263 6622 7468

No significant increase was observed in diffusion coefficient of enzyme immobilized

particles in any of the systems

39

34 Discussion

Formation of fan-shaped structures due to patterned deposition of oxidized TMB

indicates some kind of interaction between GOx and HRP immobilized particles Particle

diameter dependence of the size of structures can be explained with a simple model in

Figure 3 6 A larger particle will have a higher surface area as compared to a smaller

particle Hence more enzyme is immobilized on the larger particle As a result higher

quantity of TMB is oxidized and precipitated by the larger particle as compared to the

smaller particle This leads to thicker strands of fan-shaped structures eventually scaling

up the whole system to give bigger fan-shaped structures

The exact reason for the formation of such fan-shaped structures which are dependent on

particle size could not be determined It is hypothesized that hydrogen peroxide andor

Figure 3 6 Model for size dependence of fan-shaped structures on the particle

diameter of enzyme immobilized particles Larger the particle more surface area is

available for precipitation of TMB which eventually leads to bigger fan-shaped

structures

Larger particle Smaller particle

40

TMB gradient in the substrate solution might be responsible for such structures If there

is higher concentration of H2O2 on one side of the particle and assuming an even

concentration distribution of TMB there will be selective precipitation in the direction of

H2O2 When several particles come in the vicinity of higher concentration of H2O2 the

precipitation lines will converge to give fan-shaped structures (Figure 3 7)

Figure 3 7 Hypothesis for formation of fan-shaped structures

In the system consisting of GOx immobilized particles HRP immobilized particles

glucose and TMB it was observed that the fan-shaped structures formed at the liquid-air

interface and eventually settled down to the bottom of the glass slide This makes sense

as oxygen is necessary for the catalytic reaction of conversion of glucose to gluconic acid

and hydrogen peroxide which takes place on the surface of GOx immobilized particle As

a result H2O2 is primarily formed at the liquid-air interface and is then used by HRP for

precipitation of TMB

Following the observations that precipitation of TMB occurs at the surface GOx and

HRP immobilized particles are also expected to interact at the surface in the absence of

TMB Due the cascade reaction it was expected that HRP immobilized particles will be

41

attracted to GOx immobilized particles in appropriate substrate solution Experiments

were carried out to visualize inter-particle interactions at the liquid-air interface

However liquid-air interface not being a stable system to image the precise interaction

of GOx and HRP immobilized particles could not be determined Significant external

disturbances were involved at the interface To enable the cascade reaction in the bulk of

the liquid oxygen was purged into the reaction system before starting the experiment

However this did not lead to significant reaction in the bulk of the liquid and the reaction

prominently took place at the liquid-air interface where the oxygen concentration was

higher

In order to observe the interaction of GOx and HRP immobilized particles better imaging

techniques need to be used to observe diffusion of particles in absence of external

disturbances at the liquid-air interface Large quantity of oxygen needs to be purged into

the system and retained to make the reaction more pronounced in the bulk of the substrate

solution Higher concentration of oxygen in water may also be achieved by using oxygen

enriched water which is commercially available55

Alternatively a different enzyme cascade system may be used which does not require a

gaseous substrate56

For example glycogen synthase synthesizes glycogen from glucose-

1-phosphate and glycogen phosphorylase breaks down glycogen to glucose-1-

phosphate57

This system does not require a gaseous substrate and might be able to

overcome the deficiencies of GOx and HRP enzyme cascade system

42

Chapter 4

Silver-titania nanomotors

41 Introduction

Bimetallic nanowires made of platinum and gold have been developed by WPaxton

etal1 The rods move autonomously in a solution of hydrogen peroxide In the present

study silver-titania (Ag-TiO2) particles are being synthesized and investigated for motion

in presence of hydrogen peroxide and UV light With proper control of conditions and

UV light trigger it can be possible to control the speed and direction of motion of these

nanoparticles

Hydrogen peroxide is produced in the human body by phagocytes as an immune response

to foreign particles and pathogens5859

Also silver nanoparticles have been shown to be

biocompatible and capable of interacting with cells in human body without disrupting

cellular function60

Similarly it has been demonstrated that titania is compatible with

different cells DNA and proteins in human body and does not cause toxic effects Hence

silver-titania nanomotors which swim in hydrogen peroxide may open interesting

avenues in applications like targeted drug delivery in those parts of the body where

hydrogen peroxide is present

43

42 Experimental

421 Materials and methods

Silver nitrate (999995) was purchased from Alfa Aesar Gum arabic was purchased

from MP Biomedicals Hydrogen peroxide solution 50 wt in water and ascorbic acid

were purchased from Sigma-Aldrich Nanopure deionized water was used for all

experiments and preparation of all solutions Secure-Seal imaging spacers (9 mm

diameter) were obtained from Sigma Aldrich All experiments were carried out at room

temperature of 25ordmC unless otherwise mentioned Ag-TiO2 particles were synthesized by

evaporation of titania in Semicore electron beam evaporator Particles were characterized

using field-emission scanning electron microscopy Optical microscopy experiments

were performed using Axiovert 200 MAT microscope by Carl Zeiss Particles were

tracked using Physvis particle tracking software (Figure 4 1) in which the position

information of a particle on screen is stored on a frame by frame basis The data is stored

in a dat file as X and Y co-ordinates of particles tracked on the screen This information

can be converted to diffusion coefficients of the particles by further manipulation using

MathWorks-MATLABreg software

44

Figure 4 1 Physvis particle tracking software

422 Synthesis of silver colloidal particles

Silver particles were synthesized by a nucleationgrowth type reaction developed by

Goya and Matijevic61

and modified by Velikov62

Silver nitrate was used as precursor

metal salt for synthesis of silver particles Solution A was 75 ml of 50 mM AgNO3 and

035 by weight gum arabic Solution B was 75 mL of 100 mM 1-ascorbic acid and

035 by weight gum arabic Solution B was rapidly added to solution A in a 250 mL

reaction flask The reaction flask was covered and particles were stirred for 48 hrs After

48 hours particles were allowed to settle for 2-3 hours in refrigerator and supernatant

was separated from the particles Particles were then rinsed with DI water and centrifuged

thrice to obtain pure silver particles Zeta potential of these particles was reported to be

45

around -50 to -60 mV and the size was around 1μm The size of the particles can be

varied by varying the amounts of ascorbic acid and silver nitrate Using molar ratio of 31

of ascorbic acid silver nitrate can give particles of 200 nm diameter whereas ratio of

151 can give particles of upto 5 μm diameter

423 Preparation of silver-titania (Ag-TiO2) particles

A monolayer of silver particles was prepared according to the procedure by Goldenberg

et al63

Glass slides were placed on the bottom of a petridish Water was put into the

petridish so that the entire slide was immersed in water A thin layer of hexane was then

added on top of water in the petridish A suspension of silver particles in ethanol (5

ww) was slowly introduced to the water-hexane interface in the dish After the

monolayer was formed the glass slide was lifted up through the monolayer with tweezers

The silver particles-covered glass slides were left at room temperature to dry overnight

Following drying a thin film of titania was evaporated on the glass slides in Semicore

evaporator (Figure 2 7) The spheres were partially coated with a 10 nm film of titania

Ag-TiO2 particles were removed from the glass slide by gentle sonication FE-SEM was

used to characterize the particles (Figure 4 2) The thin titania layer was not easily

distinguishable due to roughness of the particles

46

Figure 4 2 FESEM image of Ag-TiO2 particles

424 Optical microscopy experiments

Imaging spacers were placed on a glass slide Suspension of Ag-TiO2 particles was

placed on the glass slide and the particles were allowed to settle Movement of particles

was observed in DI water and videos were recorded at 50x magnification of optical

microscope for 30 sec 05 H2O2 was added to the particle suspension on the slide

Videos were recorded at 50x ranging from 30 sec to 10 min to study the movement of

silver titania particles in hydrogen peroxide solution Particles in both DI water and

hydrogen peroxide were subjected to UV light and videos were recorded to study particle

dynamics in presence of UV radiation Particle tracking was performed using Physvis

particle tracking software Atleast 30 particles in every system were tracked for 10

seconds each Particle tracking data was obtained in terms of X and Y coordinates of

particles on screen This data was manipulated using MathWorks-Matlab software and

graphs of mean square displacement of particles versus time were plotted Diffusion

coefficients of particles were calculated from these graphs

47

43 Results and Discussion

Ag-TiO2 particles demonstrated autonomous motion in the presence of H2O2 No UV

light was required for this motion Motion of the particles was accelerated by addition of

fresh H2O2 to the system When subjected to UV radiation the particles displayed

momentary acceleration for 4-5 seconds

Figure 4 3 Mechanism of motion of Ag-TiO2 nanomotors

In the presence of H2O2 silver part of the Ag-TiO2 particle converts H2O2 to H2O and O2

This may lead to propulsion of the Ag-TiO2 particle due to bubble propulsion andor self-

diffusiophoresis In the presence of UV light additional reaction takes place on the

titania part of Ag-TiO2 particle which increases the velocity of the Ag-TiO2 particles

(Figure 4 4) This UV triggered photoactivity of titania is due to hole-electron separation

caused by photons having energy equal to or greater than the band gap of titania646566

Most of the holes-electrons recombine but a small fraction move to the surface and react

with the available redox species like water and oxygen to form protons (H+) superoxide

(O2-) and hydroxyl (OH) radicals

6566 This may lead to propulsion by diffusiophoresis

as well as electrophoresis

48

Figure 4 4 Silver-titania motors move faster when subjected to UV radiation

In addition to linear motion rotational motion is also observed in several particles and

particle aggregates Aggregation of particles can lead to a net circular propulsive force

which can rotate the aggregate as shown in Figure 4 5

Figure 4 5 Rotation due to particle aggregation of Ag-TiO2 particles

Particle tracking by Physvis software revealed a significant increase in diffusion

coefficient of Ag-TiO2 particles in hydrogen peroxide solution as compared to DI water

49

The Ag-TiO2 particles have a diffusion coefficient of 1491 μm2s in hydrogen peroxide

versus only 180 μm2s in DI water Thus the Ag-TiO2 particles move around more than

10 times their body length per second in hydrogen peroxide (Figure 4 6) When

subjected to UV light the particles experience a significant acceleration in hydrogen

peroxide which lasts only for a short duration of time (4-5 sec) after which the particles

return to their original velocity The diffusion coefficient of the particles during this UV

light trigger is found to be around 5093 μm2s UV light trigger does not have any visible

effect on the Ag-TiO2 particles in DI water

Table 4 1 Translational diffusion coefficients of Ag-TiO2 particles in hydrogen

peroxide and water

Mean 05 H2O2 solution DI water

Diffusion coefficient of Ag-TiO2 (μm2s)

without UV light

1491 180

Diffusion coefficient of Ag-TiO2 (μm2s)

in the presence of UV light

5093 113

50

Figure 4 6 Mean square displacement curves of Ag-TiO2 Janus particles in hydrogen

peroxide and DI water

When higher concentration (~30 vv) of Ag-TiO2 particles in hydrogen peroxide is

subjected to UV radiation the particles exhibit pattern formation (Figure 4 7)

Aggregation of Ag-TiO2 Janus particles is observed when UV light is turned on and the

particles disaggregated when UV light is switched off This can be due to predator-prey

kind of interaction between silver and titania half of the Janus particles in the presence of

UV light Oxygen and water that are produced at the silver end by decomposition of

hydrogen peroxide are used up by titania when subjected to UV radiation This can lead

to attraction between titania and silver parts of neighboring Janus particles causing

aggregation

y = 20373x

y = 59994x

y = 71848x

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10 12

Mean

sq

uare

dis

pla

cem

en

t (μ

m2s

)

Time (sec)

Mean square displacement vs Time for Ag-TiO2

particles

05 Hydrogen peroxide and UV05 Hydrogen peroxide

DI Water

51

A UV OFF B UV ON

C UV OFF

Figure 4 7 Higher concentrations of Ag-TiO2 particles showing aggregation when UV

is turned on

52

Chapter 5

Summary and Conclusion

In summary no significant enhancement in diffusion was observed in case of enzyme

immobilized Janus particles in substrate solution Presence of a linker between enzyme

and particle did not increase the diffusion coefficient of particles Alternative enzyme

immobilization methods need to be tested like thiolation of enzyme and subsequent

binding to gold surface of a Janus particle35

or expressing the enzyme with polyhistidine

or avidin tag67

This can result in higher enzyme activity upon immobilization and may

lead to enhanced diffusion of Janus particles

Predator-prey system of GOx and HRP immobilized particles showed some promising

results Aggregation of particles and formation of fan-shaped structures by TMB

oxidation were observed under optical microscope when GOx and HRP immobilized

particles glucose and TMB were present in the system The size of the fan-shaped

structures was dependent on the size of enzyme immobilized particles This can be

indicative of some kind of interaction between the GOx and HRP immobilized particles

No conclusive aggregation of GOx and HRP immobilized particles was observed in

absence of TMB Better imaging techniques are required to study the interaction of GOx

and HRP immobilized particles at the unstable liquid-air interface where majority of the

reaction takes place Other enzyme cascade systems not requiring gaseous reactants like

glycogen synthase and glycogen phosphorylase should be tested for similar activity

53

Ag-TiO2 particles were found to exhibit autonomous motion in the presence of hydrogen

peroxide solution The motion was momentarily accelerated upon exposure to UV

radiation The particles showed a diffusion coefficient of 1491 μm2s in hydrogen

peroxide versus only 180 μm2s in DI water Upon subjecting particles to UV radiation in

presence of H2O2 diffusion coefficient increased to 5093 μm2s for a period of 4-5

seconds In addition to linear motion rotating motion could also be observed in some

particle aggregates Further development of these aggregate structures can lead to

formation of rotors at nanoscale Higher concentration of silver-titania particles in

hydrogen peroxide displayed aggregation in presence of UV light and disaggregated

when UV light was switched off This may be attributed to the predator-prey kind of

interaction between silver and titania where oxygen and water that are produced by

silver by decomposition of hydrogen peroxide are used up by titania when subjected to

UV radiation This promises to be an interesting system and needs to be further

investigated to explore its properties

54

References

1 Paxton WF Sundararajan S Mallouk TE Sen A Angew Chem Int Ed Engl

2006 45 5420-5429

2 Golestanian R Liverpool TB Adjari A Phys Rev Lett 2005 94 220801

3 httpwwwsilvermedicineorgh2o2html

4 Rami Pedahzur Ovadia Lev Badri Fattal Hillel Shuval Water Science and Technology

1995 31(5-6) 123-129

5 W Went F American Scientist 1968 56

6 Adriano Cavalcanti Tad Hogg Bijan Shirinzadeh IEEE MHS 2006 International

Symposium on Micro-NanoMechatronics and Human Science 2006 226-232

7 EM Purcell American Journal of Physics 1977 45 3-11

8 Golestanian R Phys Rev Lett 2010 105 018103(1)-018103(4)

9 S S Dukhin Z R Ulberg G L Dvornichenko B V Deryagin Russian Chemical

Bulletin 1983 31(8) 1535-1544

10 Dukhin AS Goetz PJ Ultrasound for characterizing colloids Particle sizing zeta

potential rheology Elsevier 2002 59

11 HJKeh HJTu Trends in Colloid and Interface Science XIV Progress in Colloid and

Polymer Science 2000 115 174-180

12 D Zinemanas A Nir International Journal of Multiphase Flow 1995 21(5) 787-800

55

13 Stephen Ebbens Jonathan Howse Soft matter 2010 6 726-739

14 J Vicario R Eelkema W R Browne A Meetsma R M La Crois B L Feringa

Chem Commun 2005 31 3936

15 Davide Pantarotto Wesley R Browne Ben L Feringa Chem Commun 2008 1533 -

1535

16 Lisa A Cameron Matthew J Footer Alexander van Oudenaarden Julie A Theriot

PNAS 1999 96(9) 4908-4913

17 Ali Najafi Ramin Golestanian Phys Rev E 2004 69 062901

18 Jinseok Kim Jungyul Park Sungwook Yang Jeongeun Baek Byungkyu Kim Sang

Ho Lee Eui-Sung Yoon Kukjin Chun Sukho Park Lab Chip 2007 7 1504 - 1508

19 Kurt M Dubowski Clinical Chemistry 2008 5411 1919ndash1920

20 Spector DA Yang Q Wade JB Am J Physiol Renal Physiol 2007 292(1) 467-474

21 Rhee SG Chang TS Bae YS Lee SR Kang SW J Am Soc Nephrol 2003 14(8-3)

211-215

22 Sakaue T Kapral R Mikhailov A S Eur Phys J B 2010 75 381ndash387

23 Muddana H S Sengupta S Mallouk T E Sen A Butler P J J Am Chem Soc

2010 132 (7) 2110ndash2111

24 Baker CJ Deahl K Domek J Orlandi EW Arch Biochem Biophys 2000 382(2) 232-

237

25 V Vojinovic RH Carvalho F Lemos JMS Cabral LP Fonseca BS Ferreira

Biochemical Engineering Journal 2007 35 126-135

56

26

OV Lebedeva NN Ugarova Russian Chemical Bulletin 1996 45(1) 18-25

27 Nigel C Veitch Phytochemistry 2004 65(3) 249-259

28 Kenneth H Petersen Journal of Immunological Methods 2009 340(1) 86-89

29 httpwwwbrenda-enzymesorgphpresult_flatphp4ecno=11117

30 Goldenberg L M Wagner J Stumpe J Paulke B-R Goumlrnitz E Langmuir 2002 18

5627-5629

31 httpwwwgoldbiocompdf3761-Protocol201pdf

32 Av Rovisco Pais Lisboa Portugal T amp D Technology West Yorkshire UK Journal

of Molecular Catalysis B Enzymatic 2004 28(2-3) 129-135

33 httpwwwphysicsnyuedugrierlabmethodsnode11html

34 Yoon-Mee Lee1 O-Yul Kwon Yeo-Joon Yoon Keungarp Ryu Biotechnology Letters

2006 28 39ndash43

35 JC Pyun SD Kim JW Chung Analytical Biochemistry 2005 347(2) 227-233

36 George H Pimbley Jr Mathematical Biosciences 1974 20(1-2) 27-51

37 Andy Fenton Sarah E Perkins Parasitology 2010 137 1027-1038

38 Mead James G Brownell Robert L Mammal Species of the World A Taxonomic and

Geographic Reference (3rd ed) Johns Hopkins University Press 2005 2 2142

57

39

Krause KH Schweiz Med Wochenschr 2000 130(4) 97-100

40 Irina Kareva Faina Berezovskaya Carlos Castillo-Chavez Journal of Biological

Dynamics 2010 4(4)315 - 327

41 Delaittre G Reynhout I Cornelissen J Nolte R Chemistry ndash A European Journal

2009 15 12600ndash12603

42 C Buller DH Johnson RC Tschumper Investigative Ophthalmology amp Visual Science

1990 31 2156-2163

43 Robert M Mazo Brownian Motion Fluctuations Dynamics and Applications Oxford

Science Publications 2009 57

44 Vivek K Mutalik KV Venkatesh Theoretical Biology and Medical Modelling 2005 2

19

45 Satoshi Nakamura Kunimasa Koga Biochemical and Biophysical Research

Communications 1977 78(2) 806-810

46 Krishnananda Chattopadhyay Shyamalava Mazumdar Biochemistry 2000 39 (1)

263ndash270

47 Satoshi Nakamura Yasuyuki Ogura J Biochem 1968 63 (3) 308-316

48 van Dongen SF Nallani M Cornelissen JJ Nolte RJ van Hest JC Chemistry 2009

15(5) 1107-1114

49 Ronald Bentley Methods in Enzymology 1955 1 340-345

50 Vartiainen J Raumlttouml M Paulussen S Packaging Technology and Science 2005

18 243ndash251

58

51 GA Sherbeny AA Shindia YMMM Sheriff International Journal of Agriculture

and Biology 2005 7

52 D Rando G-W Kohring F Giffhorn Appl Microbiol Biotechnol 1997 48

53 Worthington enzyme manual Worthington Biochemical Corporation

httpworthington-biochemcomgopdefaulthtml

54 httpwwwbrenda-enzymesorgphpresult_flatphp4ecno=11117

55 httpwwwoxygenphwatercom

56 P Boon Chock Sue Goo Rhee and Earl R Stadtman Annu Rev Biochem 1980 49

813-841

57 Sybil Golden P A Wals Joseph Katz Analytical Biochemistry 1977 77(2) 436-445

58 R L Baehner S K Murrmann J Davis R B Johnston Jr J Clin Invest 1975 56(3)

571ndash576

59 I Bejarano M P Terroacuten S D Paredes C Barriga A B Rodriacuteguez J A Pariente

Molecular and Cellular Biochemistry 2007 296(1-2) 77-84

60 Michael C Moulton Laura K Braydich-Stolle Mallikarjuna N Nadagouda Samantha

Kunzelman Saber M Hussain Rajender S Varma Nanoscale 2010 2 763-770

61 Goya DV Matijevic E Colloids and Surfaces A 1999 146 139

62 Velikov KP Zegers GE van Blaanderen A Langmuir 2003 19 1384

63 Goldenberg L M Wagner J Stumpe J Paulke B-R Goumlrnitz E Langmuir 2002 18

5627-5629

59

64

Banerjee S Gopal J Muraleedharan P Tyagi A K Raj B Curr Sci 2006

901378ndash1383

65 Bogomolov V N Kudinov E K Firsov Y A Sov Phys Solid State 1968 35 555

66 Kudo A Miseki Y Chem Soc Rev 2009 38 253ndash278

67 Gaj T Meyer SC Ghosh I Protein expression and Purification 2007 56 54-61

2

Second part of this thesis consists of synthesis and motion of silver-titania nanomotors

which can move autonomously in hydrogen peroxide solution According to some recent

findings3 colloidal silver and hydrogen peroxide may prove to be an effective

combination for treatment of infections in human body Combination of silver particles

and hydrogen peroxide has been tested for disinfection of drinking water and may hold

promise as a secondary disinfecatant which can provide long lasting residual and biofilm

control that is required in water distribution systems4 Silver-titania nanomotors can open

interesting avenues in such applications

11 Motion at the nanoscale

Motion as we understand at macroscale is significantly different from motion at

micronnanoscale A definite discontinuity exists between the two This can be

emphasized by observing the biological systems surrounding us For example at the size

of a cell which is around 10 μm mixing of large molecules is instantaneous due to

diffusion and there is no need for additional convective mixing which is what we

generally think of when we picture macroscopic mixing For instance while convective

flow driven by the pumping of lungs or the use of gills is essential for transport of oxygen

inside an organism which is larger than 1mm in size such as a lizard or a man small

organisms receive nutrients and oxygen solely through diffusion through their tissues5

These differences in motion can be understood both physically and mathematically

Physically as the size of an object decreases the mass and weight decrease with the third

3

power of linear size A large body predominantly has gravitational pull of earth acting on

it due to its large mass and other forces like gravitational effect of surrounding objects are

relatively insignificant As the object gets smaller forces such as molecular attraction and

cohesion become more significant and the relative magnitude of the gravitational pull of

the earth on the object decreases due to reduction in mass5 Thus the forces that need to

be considered to cause motion at the macroscale are different than those at the

microscale

Mathematically the difference in motion at macro- and micro- scales can be explained by

means of a dimensionless number called Reynolds number Reynolds number is the ratio

of inertial forces of a system to its viscous forces As size decreases Reynolds number

decreases and the viscous forces become more dominant For a human swimming in

water Reynolds number is around 104 for goldfish it is around 10

2 whereas for an

organism of 1μm size it is around 10-4

to 10-5

[67]

What this means is that as we scale

down from the size of a goldfish to micron sized organisms the inertial forces become

insignificant and the familiar laws of macroscale motion lose their applicability Equation

11 gives the formula for calculating Reynolds number where ρ is density of the fluid

(kgmsup3) μ is the dynamic viscosity of the fluid (kg(mmiddots)) V is the mean fluid velocity

(ms) and D is diameter of particle (m)

Equation 1 1

To understand the implications of low-Reynolds-number systems we can consider the

motion of a macroscopic and an imaginary microscopic scallop Macroscopic scallop

4

moves by opening and closing of its shell Inertial thrust is created when a scallop closes

its shell and the scallop moves forward due to the clapping motion (Figure 1 1) This

however does not hold true at the nanoscale since the inertial forces are relatively

insignificant due to the large viscous forces If a microscopic scallop were to exhibit a

similar clapping motion it will just keep moving forward and backward in an oscillatory

kind of motion and will not experience a net motion This is an example of reciprocal

motion which can linearly move a body at macroscale however fails to cause motion at

the microscale

For motion at microscale nonreciprocal motion is necessary8 Nonreciprocal motion

occurs when the path chosen for going from Position A to Position B is different from the

path chosen to come back from Position B to Position A It can be better understood with

the help of Figure 1 1 which shows the change of shapes for a theoretical object going

from Position A to Position B and then returning to Position A by using a different set of

motions This kind of change can lead to motion at the nanomicron scale

5

Another factor which gains importance at nanoscale is the dominance and ubiquity of

Brownian motion13

The trajectory of a linearly propelled nanomotor would be

significantly affected by the continuous collision of randomly moving particles in the

environment These collisions deviate the nanomotor from its linear path and hence this

effect needs to be considered while projecting the path of a nanomotor

Motion at the nanoscale can be powered by different types of mechanisms such as

diffusiophoretic electrophoretic osmophoretic chemophoretic thermophoretic and

others Different types of mechanisms of motions may coexist or act individually to cause

Position A Position B

Figure 1 1 Reciprocal and Nonreciprocal motion8

Reciprocal motion

Nonreciprocal motion

6

motion Diffusiophoretic motion of particles is caused due to concentration gradients of

solutes in an electrolytenonelectrolyte solution9 Electrophoresis is the motion of a

particle relative to a fluid under the influence of an electric field10

This occurs as the

particle carries an electric surface charge which is acted upon by an electrostatic

coulombic force of the external electric field Osmophoresis is the motion of a

vesiclefluid particle encapsulated in a semi-permeable membrane towards lower

concentration gradient when it is placed in a solution containing solute concentration

gradient1112

Thus considering the numerous mechanisms of motion known to work at

the nanoscale several suitable options are available for the development of artificial

nanomotors

12 Existing nanomotors Mechanisms and uses

Several nanomotors have been developed in the past decade or so A general principle of

working of these nanomotors is that they function by catalytically decomposing the

dissolved fuel to generate asymmetry at their surface or at their interfacial region thereby

causing propulsion13

The self-electrophoretic bimetallic nanowires developed by

WPaxton and A Sen etal consisted of a 2 μm long rod with one tip made up of Au and

the other tip made up of Pt1 These motors were synthesized by electrodeposition in an

alumina template The rods moved autonomously in a solution of hydrogen peroxide

with platinum end forward Pt oxidizes H2O2 to produce H+ and O2 and Au utilizes H

+ to

reduce H2O2 to water This generates an electron flow through the rod and a proton flow

outside the rod resulting in an electric field around the rod in the solution This electric

7

field in solution acts on the unbalanced charges within the nanorodrsquos double layer This

moves the nanorod with respect to the surrounding fuel due to the ion flux that is

generated

Bubble propulsion has been predominantly used as mechanism of motion in the motors

developed by Feringa etal His group developed a micromotor by attaching catalase to a

microparticle and placing the particle in a solution containing hydrogen peroxide This

system allowed the microparticle to move by producing oxygen bubbles from the

decomposition of hydrogen peroxide14

A similar motor was later created by Feringarsquos

group which consisted of glucose oxidase and catalase immobilized on a carbon

nanotube in a substrate solution of hydrogen peroxide Glucose oxidase converted

glucose to H2O2 which was then utilized by catalase to produce water and oxygen

thereby propelling the carbon nanotube15

Another interesting example of a self-propelled motor is the polymerization driven motor

developed by Cameron et al16

It consists of polystyrene beads immobilized with ActA

protein which catalyzes actin polymerization leading to the formation of an actin

filament which resembles the flagella of bacteria This actin polymerization propels the

beads at the speed of 001-015 μms

A theoretical model has been proposed by Nafaji and Golestanian etal which consists

of three spheres linked by rigid rods in one dimension17

The length of the rods can

switch between two specific values Due to the nonreciprocal motion of the rod this

8

model can swim in low Reynolds number systems An actual model based on

nonreciprocal motion has been developed by Kim etal in which heart cells have been

linked to an asymmetric polydimethylsiloxane robot As the heart cells beat the

asymmetric structure of the device resembles a walking movement18

Most of the nanomicromotors mentioned above are either metallic or work in

environments not compatible with human body Enzymes being an integral part of the

body can solve this problem by operating in suitable substrates already present in the

body fluids The nonreciprocal conformation changes of the enzyme as well as

diffusiophoresis and electrophoresis can conceivably be used to generate motion in case

of enzyme driven nanomotors such motors will be discussed in detail in the following

chapter

9

Chapter 2

Immobilized enzymes on Janus particles as catalytic motors

21 Introduction

Enzymes are widely found in biological systems and act on a large variety of substrates

This can make enzymes an ideal choice for powering nanomotors which need to be

biocompatible Several fluids found in the body can act as substrates to the enzymes For

example dissolved glucose which is abundantly found in the body19

is a susbtrate for

enzyme glucose oxidase Urea which is found in the urinary tract20

is a substrate for

urease Hydrogen peroxide produced by phagocytic cells in the body21

is a substrate for

horseradish peroxidase Having motors driven by enzymes can open possibilities of

targeted drug delivery to these parts of the body containing such substrates

Enzymes have already been used to generate motion of carbon nanotubes by mechanism

of bubble propulsion by Feringa etal15

Two other possible mechanisms which may be

able to power motion of nanoparticles using enzymes are asymmetrical decomposition of

substrates into products leading to self-diffusiophoresis (Figure 2 1) and nonreciprocal

changes in enzyme conformation leading to propulsion (Figure 2 2) As seen in Figure 2

1 enzymes which are immobilized on a Janus particle can breakdown the available

substrate in the solution into products causing asymmetric distribution of solutes in the

surrounding fluid This can lead to movement of particle towards the region of lower

10

solute concentration by principle of self-diffusiophoresis Figure 2-2 shows the possible

nonreciprocal changes in the conformation of a theoretical protein which can lead to

motion of the protein22

These nonreciprocal changes in conformation are triggered by the

attachment of substrate molecule and release of product molecules from the enzymersquos

active site

Figure 2 1 Enzyme functionalized Janus particle moves upon catalyzing asymmetrical

decomposition of substrate into product

11

Figure 2 2 Schematic showing idealized nonreciprocal conformation changes in a

theoretical enzyme22

Enhanced diffusion of a single enzyme molecule by catalysis in a substrate solution has

been demonstrated23

The diffusion coefficient of a single urease molecule was measured

using fluorescence correlation spectroscopy and it was observed that the diffusion of the

urease molecule increases by 16-28 by varying urea concentrations from 0001 M to 1

M Addition of enzyme inhibitors to the system curbed the enhanced diffusion This

enhanced diffusion has been attributed to the force generated by self-electrophoresis by

the enzyme in substrate solution

12

Based on the above facts it might be possible to propel a nanoparticle by asymmetric

distribution of enzymes on its surface thereby leading to self-propulsion of the particles

in substrate solution (Figure 2 1) This propulsion may be due to self-

electrophoresisself-diffusiophoresis andor due to nonreciprocal changes in enzyme

conformations Here we aim to investigate a system consisting of polystyrene particles

half coated with enzyme introduced in a suitable substrate solution The enzyme can be

directly linked to the polystyrene particle or it can be linked through a long spacer

molecule (linker) to provide more degrees of freedom to the enzyme

Several factors need to be considered for choosing enzyme for powering motion at the

nanoscale Some of the important factors are the ratio of kcatKm which denotes the

catalytic efficiency of the enzyme possible substrates stability of enzymessubstrates

ease of immobilization relationship with other enzymes in cascade reactions and

commercial availability of the enzymes The enzyme being used in the present study is

horseradish peroxidase It acts on a molecule of hydrogen peroxide and reduces it while

simultaneously oxidizing another substrate molecule In the absence of alternate

oxidizable substrate HRP can oxidize water and oxygen back to hydrogen peroxide24

thus giving an equilibrium reaction

13

211 Horseradish peroxidase

Figure 2 3 Horseradish peroxidase

Horseradish peroxidase belongs to the group of peroxidases which are a group of heme-

containing oxidoreductases that act on peroxide as hydrogen donor25

It is a redox

enzyme which contains a protoheme a complex of trivalent iron with 1358-

tetramethyl-24-divinylporphin-67-dipropionic acid as a prosthetic group It is a 44 kDa

protein which includes 33900 Da polypeptide chain bound with eight oligosaccharide

chains The carbohydrate chains improve the stability of the enzyme by protecting it

against radicals formed in the course of the reaction it catalyzes26

Horseradish

peroxidase is found in horseradish and is used widely in bioanalytical applications such

as ELISA and Western blots It decomposes hydrogen peroxide into water and oxygen

while simultaneously oxidizing another substrate27

The oxidizable substrates can be

chromogenic substrates such as 22rsquo55rsquo-tetramethyl-benzidine (TMB) and 33-

diaminobenzidine (DMB) or chemoluminescent substrates like SuperSignal and ECL

luminal28

14

Figure 2 4 Reaction catalyzed by horseradish peroxidase

The functional parameters for horseradish peroxidase include Km of 015 mM and kcat of

790 s-1

at an optimum temperature of 20 ordmC and an optimum pH of 6429

Some of the

inhibitors of HRP are 110-phenanthroline aminotriazole and bromine

22 Experimental

221 Materials and methods

Uniform carboxyl modified polystyrene microspheres 05 microm in diameter were obtained

from Polysciences Inc The size of the purchased microspheres was verified by Field

Emission Scanning Electron Microscopy (FESEM) Peroxidase Type I from

Horseradish N-(3-dimethyl-aminopropyl)-Nrsquo-ethylcarbodiimide (EDC) and hydrogen

peroxide solution 50 wt in water were purchased from Sigma-Aldrich N-

hydroxysuccinimide (NHS) ge 97 was purchased from Fluka All chemicals were of

reagent grade and used without further purification Nanopure deionized water was used

for all experiments and preparation of solutions All measurements were made at room

15

temperature (25 oC) unless mentioned otherwise The UV-Visible spectroscopy

measurements were performed using Agilent 8453A diode-array UV-Visible

Spectrophotometer with ChemStation UV-Vis Software Diffusion coefficient

measurements were performed using Nanosight LM10

222 Synthesis of Janus particles

A monolayer of particles was prepared according to the procedure (Figure 2 5) by

Goldenberg L et al30

Glass slides were placed on the bottom of a petridish Water was

put into the petridish so that the entire slide was immersed in water A thin layer of

hexane was added on top of water in the petridish A suspension of microspheres in

ethanol (5 ww) was introduced to the water-hexane interface After the monolayer was

formed the glass slide was lifted up through the monolayer with tweezers The

microsphere-covered glass slides were left to dry overnight at room temperature

Following drying a thin film of silica was evaporated on the glass slides in Semicore

evaporator The spheres were partially coated with 30 nm of silica Particles were

removed from the glass slide by gentle sonication The polystyrene-silica (PS-SiO2)

Janus particles thus obtained were washed in deionized water several times before

further immobilization steps were performed FESEM was used to confirm synthesis of

Janus particles

16

Figure 2 5 Preparation of PS-SiO2 Janus particles by evaporation

223 Immobilization of enzymes using EDCNHS coupling method

1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide and N-hydroxysulfosuccinimide

(EDCNHS) coupling procedure was followed for immobilizing horseradish peroxidase

type I on 05 μm PS-SiO2 Janus particles (Figure 2 6) In this process EDC reacts with a

carboxyl groups on the Janus particles to form an amine-reactive O-acylisourea

intermediate If this intermediate does not encounter an amine it can hydrolyze and

regenerate the carboxyl group In the presence of NHS EDC can be used to convert

carboxyl groups to amine-reactive sulfo-NHS esters which are much more stable and do

not readily hydrolyze to regenerate carboxyl groups In the current experiment

immobilization was performed in the presence as well as absence of a long spacer

17

molecule (linker) between Janus particles and enzymes The chemical 6-aminohexanoic

acid was used as the linker (Figure 2 7) HRP immobilized PS-SiO2 Janus particles were

stored in deionized water until required for further experiments

Figure 2 6 EDCNHS coupling method for immobilization of enzyme on carboxylated

polystyrene particles

Figure 2 7 Immobilization of enzyme on Janus particles with and without 6-

aminohexanoic acid linker

18

224 Enzyme assay

Assay for activity of HRP was performed using a spectrophotometric method which

involved using 4-aminoantipyrene as hydrogen donor3132

The reaction rate was

determined by measuring increase in absorbance at 510 nm resulting from the

decomposition of hydrogen peroxide One unit represents the decomposition of 1 μM of

hydrogen peroxide per minute at 25 degC and pH 70 under the specified conditions

The spectrophotometer was set to the wavelength of 510 nm and temperature of 25 degC

Phenol4-aminoantipyrene solution (14 mL) and 00017 M H2O2 solution (15 mL) were

pipetted into each cuvette The cuvette was incubated for 3-4 mins at 25degC and blank was

established A 01 mL solution of diluted enzyme was added to the above mixture and

absorbance was recorded for 4-5 mins Standard graphs of absorbance versus time were

drawn for known concentration of the free enzyme and compared to that of the

immobilized enzyme on the Janus particles The estimate of the amount of enzyme

present on the Janus particles was deduced from the difference between the quantity of

the free enzyme previously added for immobilization and the amount that was measured

to be in the supernatant after some of the enzyme became attached to the Janus particles

The experiment was carried out in triplicate to assess reproducibility

19

The formula used to calculate the activity of enzyme is

where ∆Amin denotes the change in absorbance per minute

225 Diffusion coefficient measurements using Nanosight LM10

Diffusion coefficients for HRP immobilized particles were measured using Nanosight

LM 10 nanoparticle tracking instrument with Nanoparticle Tracking Analysis (NTA) 20

software in the presence of 003 H2O2 solution as the substrate and also in the presence

of only deionized water The videos were recorded for 30 seconds at the rate of 30 frames

per second Each experiment was carried out in triplicate to assess reproducibility

Particles

visualization

zone

Microscope with

LM 10 unit

LM 10 unit

Figure 2 8 Nanosight LM 10 instrumentation

20

Nanosight LM 10 (Figure 2 8) generates videos of population of moving nanoparticles in

a liquid when illuminated by laser light The particles which pass through the laser beam

path are seen by the instrument as small points of light undergoing Brownian motion

(Figure 2 9a)

The videos are further analyzed by NTA (Nanoparticle tracking analysis) software

(Figure 2 9b) which identifies and tracks each particle independently on a frame-by-

frame basis The software also calculates and subtracts any bulk flow in the system The

mean square displacement is calculated for each particle as long as it is visible If the

particle blinks that is if it disappears momentarily and then reappears on the screen the

software will consider the same particle as two separate ones All analysis data is

exported to the excel files in terms of diffusion coefficients of the tracked particles

B A

Figure 2 9 NTA video analysis (a) Particles as they are seen on the screen during

experimentation in Nanosight LM10 (b) NTA screen as the particles are being analyzed for

their diffusion coefficients

21

23 Results

231 Enzyme assay

The activity of free HRP enzyme reported by Sigma Aldrich is 150 unitsmg However

the activity of HRP upon analysis was found to be 52158 unitsmg which is much lower

than the reported value The activity of HRP was found to be further lowered to 2133

unitsmg when it was immobilized on the PS-SiO2 Janus particles in the presence of a

linker The following formula is used to calculate enzyme activity

where ΔAmin is the change in absorbance per minute

22

Figure 2 10 Graph for the assay of solution of free enzyme containing 05mgL of HRP

ΔAmin = 01716 Unitsmg of free enzyme = 52158

During EDCNHS coupling 04 mg enzyme was added to 1 ml of 025 vv suspension

of Janus particles Thus initial concentration of enzyme in the mixture was 04 mgml

Assuming retained activity of the enzyme in supernatant solution the amount of enzyme

in supernatant was calculated to be 03868 mgml from the assay Amount of enzyme

functionalized on the Janus particles was calculated as the difference between enzyme

previously added for immobilization (04 mgml) and the enzyme present in supernatant

(03868 mgml) and was found to be 00132 mgml Enzyme assay was performed on the

enzyme immobilized Janus particles to estimate the activity of immobilized enzyme

(Figure 2 11) and was found to be 2133 unitsmg

y = 0002x + 00516

0

01

02

03

04

05

06

07

08

0 50 100 150 200 250 300 350 400

Ab

sorb

an

ce

51

0 n

m

Time (sec)

Assay of free enzyme HRP

23

Figure 2 11 Graph for the assay of of immobilized enzyme on 05 μm Janus particles

(~025 vv) ΔAmin = 01853 Unitsmg of free enzyme = 2133

232 Diffusion coefficient measurements

No significant increase was observed in diffusion coefficient of HRP-immobilized Janus

particles in the presence of H2O2 as the substrate Similar results were obtained for

diffusion coefficient measurement with and without linker moieties bridging the enzymes

and particles(Table 2 1 Table 2 2) Thus the linker did not seem to assist in increasing

the particle diffusion by improving or retaining the activity of the enzyme

y = 00015x + 00953

0

01

02

03

04

05

06

07

0 50 100 150 200 250 300 350 400

Ab

sorb

an

ce

510 n

m

Time (sec)

Assay of HRP immobilized 05 μm Janus particles

24

Table 2 1 Diffusion coefficient measurements of HRP immobilized Janus particles

without linker

HRP immobilized

Janus particles in

003 H2O2

Control - Janus

particles in

003 H2O2

Control - HRP

immobilized Janus

particles in DI water

Diffusion coefficient

(10-10

cm2s)

1359416 1051777 1308675

Standard deviation 4534801 4122075 4918597

Table 2 2 Diffusion coefficient measurements of HRP immobilized Janus particles in

the presence of linker

HRP immobilized

Janus particles in

003 H2O2

Control - Janus

particles in

003 H2O2

Control - HRP

immobilized Janus

particles in DI water

Diffusion coefficient

(10-10

cm2s)

13838 1339829 1407891

Standard deviation 5644692 5649364 5797218

25

Figure 2 12 Diffusion coefficient measurements of PS-SiO2 Janus particles without and

with 6-aminohexanoic acid linker

24 Discussion

To compare the diffusion coefficients observed during the experiments theoretical

diffusion coefficient was calculated using following equation33

r

kTD altraslation

6 Equation 21

where r is the hydrodynamic radius of particle Dtranslational is translational diffusion

coefficient of the particle T is temperature of the system η is the viscosity of fluid

through which the particles are moving and k is the Boltzmann constant From FE-SEM

images the size of the particles used was determined to be 450 nm For 450 nm diameter

0

20

40

60

80

100

120

140

160

Without linker With linker

Dif

fusio

n c

oeff

icie

nt

(10

-10 c

m2s

)

HRP immobilized 05 μm Janus particles - Diffusion coefficient measurements

HRP immobilized Janus particles in 003 H2O2

Control - Janus particles in 003 H2O2

Control - HRP immobilized Janus particles in DI water

26

particle Dtranslational is equal to 109 μm2s which is comparable to the diffusion

coefficient measured for the enzyme immobilized particles

HRP was found to be active on the Janus particles however the activity of the enzyme

was reduced upon immobilization Lower enzyme activity can be due to the coupling

method used which in this case was EDCNHS covalent binding However EDCNHS

coupling method has been shown to significantly retain the enzyme activity when the

enzyme is immobilized on carboxylated multi-walled carbon nanotubes under appropriate

conditions34

Hence EDCNHS binding method by itself is not believed to be

responsible for reduction of enzyme activity upon immobilization

Direct binding of enzyme to the Janus particle may lead to reduction in the enzyme

activity because of space constraints for the enzyme and difficulty for the passage of

substrate to the active site of the enzyme due to stearic hindrance Hence a linker was

inserted between the enzyme and Janus particle during enzyme immobilization However

inserting a linker between the enzyme and the particle did not lead to enhanced diffusion

of Janus particles or increase in enzyme activity of immobilized enzyme

Lack of enhanced diffusion can be firstly attributed to the reduction in enzyme activity

upon immobilization Enzyme activity may be retained by modifying the conditions

during EDCNHS coupling or changing the binding method altogether The degree of

immobilization of HRP on carboxylated Janus particles can possibly be affected by

several factors such as temperature the concentration of the coupling reagent EDC

27

concentration HRP concentration in solution and incubation time34

Different

combinations of these factors can be tested to find optimum conditions for

immobilization of HRP Alternate binding method can involve thiolation of HRP by the

covalent conjugation of mercaptopropionic acid and then the thiolated HRP can then be

attached on the gold surface of a polystyrene-gold Janus particle35

Thus further

modifications can be made to the system in order to retain enzyme activity on the

immobilized particles and thereby power nanomotors using enzymes

28

Chapter 3

Predator-prey system of enzyme immobilized particles

31 Introduction

Predator-prey systems are widely found in nature and play a major role in biological

processes3637

Predator-prey systems include all the interactions in which one organism

pursues andor consumes another organism One familiar predator-prey system at the

macroscale is that of a tiger chasing and consuming a deer Another not so familiar one

includes Baleen whales which eat millions of microscopic planktons at once38

At the

microscale such kind of interactions can be observed in the case of phagocytes in the

body which are capable of engulfing foreign microorganisms and killing them with a

combination of microbicidal systems39

In the case of cancer the tumor is the prey and

the mature myeloid cells in the body are the predator However the tumor here has a

defense mechanism against the predator in which the tumor produces cytokines which

inhibits the maturation of the myeloid cells and thereby prevents predation40

One similarity that can be observed in all these systems is that the prey possesses some

attribute which is desired by the predator which makes the predator chase the prey A

similar relationship is seen in case of enzymes which are involved in cascade reactions41

In cascade reactions product synthesized by one enzyme is the reactant required by

another enzyme to carry out its own catalytic reaction As such there is a predator-prey

29

kind of dependence between the two enzymes which may cause one enzyme to chase the

other enzyme when placed in suitable substrate solutions However the mechanism

hypothesized for such a system is totally different than the mechanism involved in

biological systems In biological systems such as bacterial chemotaxis or phagocytes

chasing foreign particles in human body complex biochemical reactions are responsible

for the predator-prey behavior42

In the proposed enzyme cascade system there are no

complex biochemical reactions involved The principle hypothesized for predator-prey

behavior of enzyme cascade reactions can be explained as follows Motion of synthetic

motors at nanoscale involves two components translational diffusion and rotational

Brownian diffusion At very short time scales of less than 1Dr where Dr (s-1

) is

rotational Brownian diffusion coefficient the motion of particles is unidirectional For

longer time scales of greater than 1Dr the direction of motion is random giving

poweredactive diffusion43

If the chemical homogeneity is broken that is when there is

higher concentration of a substrate present in one direction of the enzyme the enzyme

might acquire a subtle bias which may lead to a spatial aggregation of the enzymes based

on the substrate concentration gradient The bias can be a result of the relationship

between the speed of enzyme diffusion and substrate concentration where higher

substrate concentration lead to higher enzyme diffusion As a result in an enzyme

cascade system one enzyme will migrate towards higher concentration of substrate

which is present near the other enzyme thus giving rise to a predator-prey behavior

Such enzyme cascades are widely found in signaling networks and metabolic pathways in

living cells and can be used to produce predator-prey like nanomotors systems44

The

30

enzymes being used in the present study are horseradish peroxidase and glucose oxidase

Both enzymes are extremely stable4546

Glucose is the substrate for glucose oxidase

which is widely available and highly stable47

Horseradish peroxidase acts on hydrogen

peroxide It is involved in a cascade reaction with glucose oxidase which produces

hydrogen peroxide by converting glucose into gluconic acid (Figure 3 1)48

Figure 3 1 Concept of particle aggregation due to cascade reactions of immobilized

enzymes

In this experiment glucose oxidase (GOx) and horseradish peroxidase (HRP) were

immobilized on polystyrene carboxylate beads of different sizes using EDCNHS

coupling method Glucose and 3355-Tetramethylbenzidine (TMB) were dissolved

together in water and this solution was used as the substrate solution All the components

were present in a single system in DI water Aggregation of particles and precipitation of

oxidized TMB from the solution was expected to be observed when all these components

were present in such a system

Properties of horseradish peroxidase have been discussed in the previous chapter (Section

211 Horseradish peroxidase)

GOxGOx HRPHRP

Glucose + O2Gluconic acid + H2O2

TMB TMB oxidizedBlue precipitate

H2O

Aggregation

31

311 Glucose oxidase

Figure 3 2 Glucose oxidase

Glucose oxidase (GOx) belongs to the group of oxidoreductases and is also called

glucose aerodehydrogenase49

It is obtained from fungi and has been identified as an

antimicrobial enzyme50

GOx binds to β-D-glucopyranose and catalyzes the oxidation

reaction to from gluconolactone while simultaneously reducing oxygen to hydrogen

peroxide51

GOx has high substrate affinity and specificity high stability and high

turnover number which makes it a suitable biocatalyst for industrial applications52

Figure 3 3 Redox reaction of glucose catalyzed by glucose oxidase53

32

GOx is a glycoprotein with molecular weight of 155 kDa and consists of two identical

polypeptide subunits linked together by a disulfide bond The optimum pH for GOx is

55 though it is stable in a broad pH range of 4 to 7 and can be stored for a long period of

time51

The functional parameters for glucose oxidase include Km of 151 mM and kcat of

337 s-1

at temperature of 20 ordmC and pH of 754

Some of the inhibitors of GOx are 2-

Deoxy-D-glucose adenine nucleotides and NaNO3

32 Experimental

321 Materials and methods

Uniform carboxyl modified polystyrene microspheres 05 microm in diameter were obtained

from Polysciences Inc The size of the purchased microspheres was verified by FESEM

Peroxidase Type I from horseradish glucose oxidase Type II-S from Aspergillus niger

N-(3-dimethyl-aminopropyl)-Nrsquo-ethylcarbodiimide (EDC) and Hydrogen peroxide

solution 50 wt in water were purchased from Sigma-Aldrich N-hydroxysuccinimide

(NHS) was purchased from Fluka D-Glucose (99) was purchased from VWR

International Secure-Seal imaging spacers (9mm diameter) for glass slides were obtained

from Sigma Aldrich All chemicals were of reagent grade and used without further

purification Nanopure deionized water was used for all experiments and preparation of

solutions All measurements were made at room temperature (25 oC) unless mentioned

otherwise The UV-Visible measurements were performed using Agilent 8453A diode-

array UV-Visible Spectrophotometer with ChemStation UV-Vis Software Optical

33

microscopy experiments were performed using Axiovert 200 MAT microscope by Carl

Zeiss Diffusion coefficient measurements were obtained using Nanosight LM10

322 Optical microscopy experiments

The enzyme substrate system was observed under optical microscope to check for

aggregation and precipitation due to cascade reactions Several such systems were

studied Each system in general consisted of GOx and HRP immobilized on different

polystyrene particles The substrate solutions consisted of 01 M glucose and different

concentrations of TMB solution The systems were studied in the presence of all the

constituents and in the absence of glucose GOxHRP and TMB Systems consisting of

different sized HRP immobilized particles in substrate solution of 003 hydrogen

peroxide and TMB were also studied Videos were recorded over a period of 24 hours to

observe changes in the system Each experiment was performed atleast thrice to assess

reproducibility of results

323 Diffusion coefficient measurements using Nanosight LM10

Diffusion coefficient of GOx and HRP immobilized particles were measured using

Nanosight LM10 nanoparticle tracking instrument with Nanoparticle Tracking Analysis

(NTA) 20 software Diffusion coefficients were measured for different systems namely

GOx particles in 01 M glucose and oxygen both GOx and HRP functionalized particles

in 01 M glucose and oxygen along with controls for the individual systems Videos were

34

recorded for 30 seconds at the rate of 30 frames per second Each experiment was carried

out in triplicate to assess reproducibility

33 Results

331 Optical microscopy

According to the enzyme cascade model precipitation of TMB was possible only upon

the interaction of GOx and HRP immobilized particles in presence of appropriate

substrate solution Hence in the first set of experiments precipitation of TMB was used

as an indication of the type of interaction between GOx and HRP immobilized particles

Fan-shaped structures were observed when both HRP and GOx immobilized particles

were present in a substrate solution consisting of glucose and TMB These structures

started forming as a single strand and then branched out and intertwined to form the fan-

shaped structures (Figure 3 4)

Table 3 1 Observations for formation of fan-shaped structures in enzyme cascade

system in presence of different components

GOx HRP Glucose TMB Observation

Yes Yes Yes Yes

Fan-shaped structures observed particles associated with

the fan-shaped structures

No Yes Yes Yes No fan-shaped structures

Yes No Yes Yes No fan-shaped structures

Yes Yes No Yes No fan-shaped structures

Yes Yes Yes No No fan-shaped structures

35

Fan-shaped structures can also be observed in the system consisting of only HRP

immobilized particles in substrate solution of hydrogen peroxide and TMB The size of

the fan-shaped structures depends on the size of the enzyme immobilized particles Free

enzymes show extremely tiny structures which are difficult to view under the optical

microscope

Figure 3 4 Formation of fan-shaped structures when both HRP and GOx

immobilized particles were present in a substrate solution consisting of glucose and

TMB A In the initial stages single strands form on the surface of liquid (5Ox) B

Single strands branch out and intertwine to form fan-shaped structures (50x) C Many

such structures form and settle down after 20 minutes (20x)

A

C

B

36

Experiments were performed in the absence of TMB that is the system consisted of GOx

and HRP immobilized particles in glucose substrate solution In the absence of TMB the

particles were expected to move closer to each other without any precipitation due to the

cascade reaction However there was no conclusive aggregation of particles observed in

the system

Free Enzyme 005 μm HRP - immobilized particle

10 μm HRP - immobilized particle 05 μm HRP - immobilized particle

Figure 3 5 Size dependence of fan-shaped structures on the size of HRP -

immobilized particles (20x)

37

Table 3 2 Observations for particle aggregation in enzyme cascade system in presence

and absence of TMB

Glucose GOx HRP TMB Observation

Present Present Present Present Aggregation and precipitate

Present Present Present Absent No conclusive aggregation

Present Absent Present Present No aggregation or ppt

Absent Present Present Present No aggregation or ppt

Present Present Absent Present No aggregation or ppt

332 Diffusion coefficient measurements

Diffusion coefficients were measured for GOx immobilized particles of 05 μm diameter

in substrate solution of 01 M glucose and oxygen Diffusion coefficients of particles

were also measured for system consisting of GOx and HRP functionalized particles in

glucose and oxygen Each experiment was done in triplicate to assess reproducibility

Nanosight visualization chamber being a closed chamber oxygen was purged into all the

solutions before starting the experiments to ensure sufficient supply of oxygen for

catalytic reaction of GOx

38

Table 3 3 Diffusion coefficients for 05 μm GOx immobilized particles in 01 M

glucose purged with O2

Mean

GOx

particles in

01 M

glucose

Control - GOx

particles in DI

water

Control - GOx

particles in 01 M

glucose without

oxygen purging

Control -

particles in 01

M Glucose

Diffusion

coefficient

(x10-10

cm2s)

13316 12888 13827 13702

Standard

deviation 6307 4926 4424 7468

Table 3 4 Diffusion coefficients for GOx and HRP immobilized particles in 01 M

glucose and purged with O2

Mean

GOx- HRP-

functionalized

particles in 01 M

glucose

Control ndash Gox-

HRP- functionalized

particles in DI water

Control - particles

in 01 M glucose

Diffusion coefficient

(x10-10

cm2s)

13005 14434 13702

Standard deviation 5263 6622 7468

No significant increase was observed in diffusion coefficient of enzyme immobilized

particles in any of the systems

39

34 Discussion

Formation of fan-shaped structures due to patterned deposition of oxidized TMB

indicates some kind of interaction between GOx and HRP immobilized particles Particle

diameter dependence of the size of structures can be explained with a simple model in

Figure 3 6 A larger particle will have a higher surface area as compared to a smaller

particle Hence more enzyme is immobilized on the larger particle As a result higher

quantity of TMB is oxidized and precipitated by the larger particle as compared to the

smaller particle This leads to thicker strands of fan-shaped structures eventually scaling

up the whole system to give bigger fan-shaped structures

The exact reason for the formation of such fan-shaped structures which are dependent on

particle size could not be determined It is hypothesized that hydrogen peroxide andor

Figure 3 6 Model for size dependence of fan-shaped structures on the particle

diameter of enzyme immobilized particles Larger the particle more surface area is

available for precipitation of TMB which eventually leads to bigger fan-shaped

structures

Larger particle Smaller particle

40

TMB gradient in the substrate solution might be responsible for such structures If there

is higher concentration of H2O2 on one side of the particle and assuming an even

concentration distribution of TMB there will be selective precipitation in the direction of

H2O2 When several particles come in the vicinity of higher concentration of H2O2 the

precipitation lines will converge to give fan-shaped structures (Figure 3 7)

Figure 3 7 Hypothesis for formation of fan-shaped structures

In the system consisting of GOx immobilized particles HRP immobilized particles

glucose and TMB it was observed that the fan-shaped structures formed at the liquid-air

interface and eventually settled down to the bottom of the glass slide This makes sense

as oxygen is necessary for the catalytic reaction of conversion of glucose to gluconic acid

and hydrogen peroxide which takes place on the surface of GOx immobilized particle As

a result H2O2 is primarily formed at the liquid-air interface and is then used by HRP for

precipitation of TMB

Following the observations that precipitation of TMB occurs at the surface GOx and

HRP immobilized particles are also expected to interact at the surface in the absence of

TMB Due the cascade reaction it was expected that HRP immobilized particles will be

41

attracted to GOx immobilized particles in appropriate substrate solution Experiments

were carried out to visualize inter-particle interactions at the liquid-air interface

However liquid-air interface not being a stable system to image the precise interaction

of GOx and HRP immobilized particles could not be determined Significant external

disturbances were involved at the interface To enable the cascade reaction in the bulk of

the liquid oxygen was purged into the reaction system before starting the experiment

However this did not lead to significant reaction in the bulk of the liquid and the reaction

prominently took place at the liquid-air interface where the oxygen concentration was

higher

In order to observe the interaction of GOx and HRP immobilized particles better imaging

techniques need to be used to observe diffusion of particles in absence of external

disturbances at the liquid-air interface Large quantity of oxygen needs to be purged into

the system and retained to make the reaction more pronounced in the bulk of the substrate

solution Higher concentration of oxygen in water may also be achieved by using oxygen

enriched water which is commercially available55

Alternatively a different enzyme cascade system may be used which does not require a

gaseous substrate56

For example glycogen synthase synthesizes glycogen from glucose-

1-phosphate and glycogen phosphorylase breaks down glycogen to glucose-1-

phosphate57

This system does not require a gaseous substrate and might be able to

overcome the deficiencies of GOx and HRP enzyme cascade system

42

Chapter 4

Silver-titania nanomotors

41 Introduction

Bimetallic nanowires made of platinum and gold have been developed by WPaxton

etal1 The rods move autonomously in a solution of hydrogen peroxide In the present

study silver-titania (Ag-TiO2) particles are being synthesized and investigated for motion

in presence of hydrogen peroxide and UV light With proper control of conditions and

UV light trigger it can be possible to control the speed and direction of motion of these

nanoparticles

Hydrogen peroxide is produced in the human body by phagocytes as an immune response

to foreign particles and pathogens5859

Also silver nanoparticles have been shown to be

biocompatible and capable of interacting with cells in human body without disrupting

cellular function60

Similarly it has been demonstrated that titania is compatible with

different cells DNA and proteins in human body and does not cause toxic effects Hence

silver-titania nanomotors which swim in hydrogen peroxide may open interesting

avenues in applications like targeted drug delivery in those parts of the body where

hydrogen peroxide is present

43

42 Experimental

421 Materials and methods

Silver nitrate (999995) was purchased from Alfa Aesar Gum arabic was purchased

from MP Biomedicals Hydrogen peroxide solution 50 wt in water and ascorbic acid

were purchased from Sigma-Aldrich Nanopure deionized water was used for all

experiments and preparation of all solutions Secure-Seal imaging spacers (9 mm

diameter) were obtained from Sigma Aldrich All experiments were carried out at room

temperature of 25ordmC unless otherwise mentioned Ag-TiO2 particles were synthesized by

evaporation of titania in Semicore electron beam evaporator Particles were characterized

using field-emission scanning electron microscopy Optical microscopy experiments

were performed using Axiovert 200 MAT microscope by Carl Zeiss Particles were

tracked using Physvis particle tracking software (Figure 4 1) in which the position

information of a particle on screen is stored on a frame by frame basis The data is stored

in a dat file as X and Y co-ordinates of particles tracked on the screen This information

can be converted to diffusion coefficients of the particles by further manipulation using

MathWorks-MATLABreg software

44

Figure 4 1 Physvis particle tracking software

422 Synthesis of silver colloidal particles

Silver particles were synthesized by a nucleationgrowth type reaction developed by

Goya and Matijevic61

and modified by Velikov62

Silver nitrate was used as precursor

metal salt for synthesis of silver particles Solution A was 75 ml of 50 mM AgNO3 and

035 by weight gum arabic Solution B was 75 mL of 100 mM 1-ascorbic acid and

035 by weight gum arabic Solution B was rapidly added to solution A in a 250 mL

reaction flask The reaction flask was covered and particles were stirred for 48 hrs After

48 hours particles were allowed to settle for 2-3 hours in refrigerator and supernatant

was separated from the particles Particles were then rinsed with DI water and centrifuged

thrice to obtain pure silver particles Zeta potential of these particles was reported to be

45

around -50 to -60 mV and the size was around 1μm The size of the particles can be

varied by varying the amounts of ascorbic acid and silver nitrate Using molar ratio of 31

of ascorbic acid silver nitrate can give particles of 200 nm diameter whereas ratio of

151 can give particles of upto 5 μm diameter

423 Preparation of silver-titania (Ag-TiO2) particles

A monolayer of silver particles was prepared according to the procedure by Goldenberg

et al63

Glass slides were placed on the bottom of a petridish Water was put into the

petridish so that the entire slide was immersed in water A thin layer of hexane was then

added on top of water in the petridish A suspension of silver particles in ethanol (5

ww) was slowly introduced to the water-hexane interface in the dish After the

monolayer was formed the glass slide was lifted up through the monolayer with tweezers

The silver particles-covered glass slides were left at room temperature to dry overnight

Following drying a thin film of titania was evaporated on the glass slides in Semicore

evaporator (Figure 2 7) The spheres were partially coated with a 10 nm film of titania

Ag-TiO2 particles were removed from the glass slide by gentle sonication FE-SEM was

used to characterize the particles (Figure 4 2) The thin titania layer was not easily

distinguishable due to roughness of the particles

46

Figure 4 2 FESEM image of Ag-TiO2 particles

424 Optical microscopy experiments

Imaging spacers were placed on a glass slide Suspension of Ag-TiO2 particles was

placed on the glass slide and the particles were allowed to settle Movement of particles

was observed in DI water and videos were recorded at 50x magnification of optical

microscope for 30 sec 05 H2O2 was added to the particle suspension on the slide

Videos were recorded at 50x ranging from 30 sec to 10 min to study the movement of

silver titania particles in hydrogen peroxide solution Particles in both DI water and

hydrogen peroxide were subjected to UV light and videos were recorded to study particle

dynamics in presence of UV radiation Particle tracking was performed using Physvis

particle tracking software Atleast 30 particles in every system were tracked for 10

seconds each Particle tracking data was obtained in terms of X and Y coordinates of

particles on screen This data was manipulated using MathWorks-Matlab software and

graphs of mean square displacement of particles versus time were plotted Diffusion

coefficients of particles were calculated from these graphs

47

43 Results and Discussion

Ag-TiO2 particles demonstrated autonomous motion in the presence of H2O2 No UV

light was required for this motion Motion of the particles was accelerated by addition of

fresh H2O2 to the system When subjected to UV radiation the particles displayed

momentary acceleration for 4-5 seconds

Figure 4 3 Mechanism of motion of Ag-TiO2 nanomotors

In the presence of H2O2 silver part of the Ag-TiO2 particle converts H2O2 to H2O and O2

This may lead to propulsion of the Ag-TiO2 particle due to bubble propulsion andor self-

diffusiophoresis In the presence of UV light additional reaction takes place on the

titania part of Ag-TiO2 particle which increases the velocity of the Ag-TiO2 particles

(Figure 4 4) This UV triggered photoactivity of titania is due to hole-electron separation

caused by photons having energy equal to or greater than the band gap of titania646566

Most of the holes-electrons recombine but a small fraction move to the surface and react

with the available redox species like water and oxygen to form protons (H+) superoxide

(O2-) and hydroxyl (OH) radicals

6566 This may lead to propulsion by diffusiophoresis

as well as electrophoresis

48

Figure 4 4 Silver-titania motors move faster when subjected to UV radiation

In addition to linear motion rotational motion is also observed in several particles and

particle aggregates Aggregation of particles can lead to a net circular propulsive force

which can rotate the aggregate as shown in Figure 4 5

Figure 4 5 Rotation due to particle aggregation of Ag-TiO2 particles

Particle tracking by Physvis software revealed a significant increase in diffusion

coefficient of Ag-TiO2 particles in hydrogen peroxide solution as compared to DI water

49

The Ag-TiO2 particles have a diffusion coefficient of 1491 μm2s in hydrogen peroxide

versus only 180 μm2s in DI water Thus the Ag-TiO2 particles move around more than

10 times their body length per second in hydrogen peroxide (Figure 4 6) When

subjected to UV light the particles experience a significant acceleration in hydrogen

peroxide which lasts only for a short duration of time (4-5 sec) after which the particles

return to their original velocity The diffusion coefficient of the particles during this UV

light trigger is found to be around 5093 μm2s UV light trigger does not have any visible

effect on the Ag-TiO2 particles in DI water

Table 4 1 Translational diffusion coefficients of Ag-TiO2 particles in hydrogen

peroxide and water

Mean 05 H2O2 solution DI water

Diffusion coefficient of Ag-TiO2 (μm2s)

without UV light

1491 180

Diffusion coefficient of Ag-TiO2 (μm2s)

in the presence of UV light

5093 113

50

Figure 4 6 Mean square displacement curves of Ag-TiO2 Janus particles in hydrogen

peroxide and DI water

When higher concentration (~30 vv) of Ag-TiO2 particles in hydrogen peroxide is

subjected to UV radiation the particles exhibit pattern formation (Figure 4 7)

Aggregation of Ag-TiO2 Janus particles is observed when UV light is turned on and the

particles disaggregated when UV light is switched off This can be due to predator-prey

kind of interaction between silver and titania half of the Janus particles in the presence of

UV light Oxygen and water that are produced at the silver end by decomposition of

hydrogen peroxide are used up by titania when subjected to UV radiation This can lead

to attraction between titania and silver parts of neighboring Janus particles causing

aggregation

y = 20373x

y = 59994x

y = 71848x

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10 12

Mean

sq

uare

dis

pla

cem

en

t (μ

m2s

)

Time (sec)

Mean square displacement vs Time for Ag-TiO2

particles

05 Hydrogen peroxide and UV05 Hydrogen peroxide

DI Water

51

A UV OFF B UV ON

C UV OFF

Figure 4 7 Higher concentrations of Ag-TiO2 particles showing aggregation when UV

is turned on

52

Chapter 5

Summary and Conclusion

In summary no significant enhancement in diffusion was observed in case of enzyme

immobilized Janus particles in substrate solution Presence of a linker between enzyme

and particle did not increase the diffusion coefficient of particles Alternative enzyme

immobilization methods need to be tested like thiolation of enzyme and subsequent

binding to gold surface of a Janus particle35

or expressing the enzyme with polyhistidine

or avidin tag67

This can result in higher enzyme activity upon immobilization and may

lead to enhanced diffusion of Janus particles

Predator-prey system of GOx and HRP immobilized particles showed some promising

results Aggregation of particles and formation of fan-shaped structures by TMB

oxidation were observed under optical microscope when GOx and HRP immobilized

particles glucose and TMB were present in the system The size of the fan-shaped

structures was dependent on the size of enzyme immobilized particles This can be

indicative of some kind of interaction between the GOx and HRP immobilized particles

No conclusive aggregation of GOx and HRP immobilized particles was observed in

absence of TMB Better imaging techniques are required to study the interaction of GOx

and HRP immobilized particles at the unstable liquid-air interface where majority of the

reaction takes place Other enzyme cascade systems not requiring gaseous reactants like

glycogen synthase and glycogen phosphorylase should be tested for similar activity

53

Ag-TiO2 particles were found to exhibit autonomous motion in the presence of hydrogen

peroxide solution The motion was momentarily accelerated upon exposure to UV

radiation The particles showed a diffusion coefficient of 1491 μm2s in hydrogen

peroxide versus only 180 μm2s in DI water Upon subjecting particles to UV radiation in

presence of H2O2 diffusion coefficient increased to 5093 μm2s for a period of 4-5

seconds In addition to linear motion rotating motion could also be observed in some

particle aggregates Further development of these aggregate structures can lead to

formation of rotors at nanoscale Higher concentration of silver-titania particles in

hydrogen peroxide displayed aggregation in presence of UV light and disaggregated

when UV light was switched off This may be attributed to the predator-prey kind of

interaction between silver and titania where oxygen and water that are produced by

silver by decomposition of hydrogen peroxide are used up by titania when subjected to

UV radiation This promises to be an interesting system and needs to be further

investigated to explore its properties

54

References

1 Paxton WF Sundararajan S Mallouk TE Sen A Angew Chem Int Ed Engl

2006 45 5420-5429

2 Golestanian R Liverpool TB Adjari A Phys Rev Lett 2005 94 220801

3 httpwwwsilvermedicineorgh2o2html

4 Rami Pedahzur Ovadia Lev Badri Fattal Hillel Shuval Water Science and Technology

1995 31(5-6) 123-129

5 W Went F American Scientist 1968 56

6 Adriano Cavalcanti Tad Hogg Bijan Shirinzadeh IEEE MHS 2006 International

Symposium on Micro-NanoMechatronics and Human Science 2006 226-232

7 EM Purcell American Journal of Physics 1977 45 3-11

8 Golestanian R Phys Rev Lett 2010 105 018103(1)-018103(4)

9 S S Dukhin Z R Ulberg G L Dvornichenko B V Deryagin Russian Chemical

Bulletin 1983 31(8) 1535-1544

10 Dukhin AS Goetz PJ Ultrasound for characterizing colloids Particle sizing zeta

potential rheology Elsevier 2002 59

11 HJKeh HJTu Trends in Colloid and Interface Science XIV Progress in Colloid and

Polymer Science 2000 115 174-180

12 D Zinemanas A Nir International Journal of Multiphase Flow 1995 21(5) 787-800

55

13 Stephen Ebbens Jonathan Howse Soft matter 2010 6 726-739

14 J Vicario R Eelkema W R Browne A Meetsma R M La Crois B L Feringa

Chem Commun 2005 31 3936

15 Davide Pantarotto Wesley R Browne Ben L Feringa Chem Commun 2008 1533 -

1535

16 Lisa A Cameron Matthew J Footer Alexander van Oudenaarden Julie A Theriot

PNAS 1999 96(9) 4908-4913

17 Ali Najafi Ramin Golestanian Phys Rev E 2004 69 062901

18 Jinseok Kim Jungyul Park Sungwook Yang Jeongeun Baek Byungkyu Kim Sang

Ho Lee Eui-Sung Yoon Kukjin Chun Sukho Park Lab Chip 2007 7 1504 - 1508

19 Kurt M Dubowski Clinical Chemistry 2008 5411 1919ndash1920

20 Spector DA Yang Q Wade JB Am J Physiol Renal Physiol 2007 292(1) 467-474

21 Rhee SG Chang TS Bae YS Lee SR Kang SW J Am Soc Nephrol 2003 14(8-3)

211-215

22 Sakaue T Kapral R Mikhailov A S Eur Phys J B 2010 75 381ndash387

23 Muddana H S Sengupta S Mallouk T E Sen A Butler P J J Am Chem Soc

2010 132 (7) 2110ndash2111

24 Baker CJ Deahl K Domek J Orlandi EW Arch Biochem Biophys 2000 382(2) 232-

237

25 V Vojinovic RH Carvalho F Lemos JMS Cabral LP Fonseca BS Ferreira

Biochemical Engineering Journal 2007 35 126-135

56

26

OV Lebedeva NN Ugarova Russian Chemical Bulletin 1996 45(1) 18-25

27 Nigel C Veitch Phytochemistry 2004 65(3) 249-259

28 Kenneth H Petersen Journal of Immunological Methods 2009 340(1) 86-89

29 httpwwwbrenda-enzymesorgphpresult_flatphp4ecno=11117

30 Goldenberg L M Wagner J Stumpe J Paulke B-R Goumlrnitz E Langmuir 2002 18

5627-5629

31 httpwwwgoldbiocompdf3761-Protocol201pdf

32 Av Rovisco Pais Lisboa Portugal T amp D Technology West Yorkshire UK Journal

of Molecular Catalysis B Enzymatic 2004 28(2-3) 129-135

33 httpwwwphysicsnyuedugrierlabmethodsnode11html

34 Yoon-Mee Lee1 O-Yul Kwon Yeo-Joon Yoon Keungarp Ryu Biotechnology Letters

2006 28 39ndash43

35 JC Pyun SD Kim JW Chung Analytical Biochemistry 2005 347(2) 227-233

36 George H Pimbley Jr Mathematical Biosciences 1974 20(1-2) 27-51

37 Andy Fenton Sarah E Perkins Parasitology 2010 137 1027-1038

38 Mead James G Brownell Robert L Mammal Species of the World A Taxonomic and

Geographic Reference (3rd ed) Johns Hopkins University Press 2005 2 2142

57

39

Krause KH Schweiz Med Wochenschr 2000 130(4) 97-100

40 Irina Kareva Faina Berezovskaya Carlos Castillo-Chavez Journal of Biological

Dynamics 2010 4(4)315 - 327

41 Delaittre G Reynhout I Cornelissen J Nolte R Chemistry ndash A European Journal

2009 15 12600ndash12603

42 C Buller DH Johnson RC Tschumper Investigative Ophthalmology amp Visual Science

1990 31 2156-2163

43 Robert M Mazo Brownian Motion Fluctuations Dynamics and Applications Oxford

Science Publications 2009 57

44 Vivek K Mutalik KV Venkatesh Theoretical Biology and Medical Modelling 2005 2

19

45 Satoshi Nakamura Kunimasa Koga Biochemical and Biophysical Research

Communications 1977 78(2) 806-810

46 Krishnananda Chattopadhyay Shyamalava Mazumdar Biochemistry 2000 39 (1)

263ndash270

47 Satoshi Nakamura Yasuyuki Ogura J Biochem 1968 63 (3) 308-316

48 van Dongen SF Nallani M Cornelissen JJ Nolte RJ van Hest JC Chemistry 2009

15(5) 1107-1114

49 Ronald Bentley Methods in Enzymology 1955 1 340-345

50 Vartiainen J Raumlttouml M Paulussen S Packaging Technology and Science 2005

18 243ndash251

58

51 GA Sherbeny AA Shindia YMMM Sheriff International Journal of Agriculture

and Biology 2005 7

52 D Rando G-W Kohring F Giffhorn Appl Microbiol Biotechnol 1997 48

53 Worthington enzyme manual Worthington Biochemical Corporation

httpworthington-biochemcomgopdefaulthtml

54 httpwwwbrenda-enzymesorgphpresult_flatphp4ecno=11117

55 httpwwwoxygenphwatercom

56 P Boon Chock Sue Goo Rhee and Earl R Stadtman Annu Rev Biochem 1980 49

813-841

57 Sybil Golden P A Wals Joseph Katz Analytical Biochemistry 1977 77(2) 436-445

58 R L Baehner S K Murrmann J Davis R B Johnston Jr J Clin Invest 1975 56(3)

571ndash576

59 I Bejarano M P Terroacuten S D Paredes C Barriga A B Rodriacuteguez J A Pariente

Molecular and Cellular Biochemistry 2007 296(1-2) 77-84

60 Michael C Moulton Laura K Braydich-Stolle Mallikarjuna N Nadagouda Samantha

Kunzelman Saber M Hussain Rajender S Varma Nanoscale 2010 2 763-770

61 Goya DV Matijevic E Colloids and Surfaces A 1999 146 139

62 Velikov KP Zegers GE van Blaanderen A Langmuir 2003 19 1384

63 Goldenberg L M Wagner J Stumpe J Paulke B-R Goumlrnitz E Langmuir 2002 18

5627-5629

59

64

Banerjee S Gopal J Muraleedharan P Tyagi A K Raj B Curr Sci 2006

901378ndash1383

65 Bogomolov V N Kudinov E K Firsov Y A Sov Phys Solid State 1968 35 555

66 Kudo A Miseki Y Chem Soc Rev 2009 38 253ndash278

67 Gaj T Meyer SC Ghosh I Protein expression and Purification 2007 56 54-61

3

power of linear size A large body predominantly has gravitational pull of earth acting on

it due to its large mass and other forces like gravitational effect of surrounding objects are

relatively insignificant As the object gets smaller forces such as molecular attraction and

cohesion become more significant and the relative magnitude of the gravitational pull of

the earth on the object decreases due to reduction in mass5 Thus the forces that need to

be considered to cause motion at the macroscale are different than those at the

microscale

Mathematically the difference in motion at macro- and micro- scales can be explained by

means of a dimensionless number called Reynolds number Reynolds number is the ratio

of inertial forces of a system to its viscous forces As size decreases Reynolds number

decreases and the viscous forces become more dominant For a human swimming in

water Reynolds number is around 104 for goldfish it is around 10

2 whereas for an

organism of 1μm size it is around 10-4

to 10-5

[67]

What this means is that as we scale

down from the size of a goldfish to micron sized organisms the inertial forces become

insignificant and the familiar laws of macroscale motion lose their applicability Equation

11 gives the formula for calculating Reynolds number where ρ is density of the fluid

(kgmsup3) μ is the dynamic viscosity of the fluid (kg(mmiddots)) V is the mean fluid velocity

(ms) and D is diameter of particle (m)

Equation 1 1

To understand the implications of low-Reynolds-number systems we can consider the

motion of a macroscopic and an imaginary microscopic scallop Macroscopic scallop

4

moves by opening and closing of its shell Inertial thrust is created when a scallop closes

its shell and the scallop moves forward due to the clapping motion (Figure 1 1) This

however does not hold true at the nanoscale since the inertial forces are relatively

insignificant due to the large viscous forces If a microscopic scallop were to exhibit a

similar clapping motion it will just keep moving forward and backward in an oscillatory

kind of motion and will not experience a net motion This is an example of reciprocal

motion which can linearly move a body at macroscale however fails to cause motion at

the microscale

For motion at microscale nonreciprocal motion is necessary8 Nonreciprocal motion

occurs when the path chosen for going from Position A to Position B is different from the

path chosen to come back from Position B to Position A It can be better understood with

the help of Figure 1 1 which shows the change of shapes for a theoretical object going

from Position A to Position B and then returning to Position A by using a different set of

motions This kind of change can lead to motion at the nanomicron scale

5

Another factor which gains importance at nanoscale is the dominance and ubiquity of

Brownian motion13

The trajectory of a linearly propelled nanomotor would be

significantly affected by the continuous collision of randomly moving particles in the

environment These collisions deviate the nanomotor from its linear path and hence this

effect needs to be considered while projecting the path of a nanomotor

Motion at the nanoscale can be powered by different types of mechanisms such as

diffusiophoretic electrophoretic osmophoretic chemophoretic thermophoretic and

others Different types of mechanisms of motions may coexist or act individually to cause

Position A Position B

Figure 1 1 Reciprocal and Nonreciprocal motion8

Reciprocal motion

Nonreciprocal motion

6

motion Diffusiophoretic motion of particles is caused due to concentration gradients of

solutes in an electrolytenonelectrolyte solution9 Electrophoresis is the motion of a

particle relative to a fluid under the influence of an electric field10

This occurs as the

particle carries an electric surface charge which is acted upon by an electrostatic

coulombic force of the external electric field Osmophoresis is the motion of a

vesiclefluid particle encapsulated in a semi-permeable membrane towards lower

concentration gradient when it is placed in a solution containing solute concentration

gradient1112

Thus considering the numerous mechanisms of motion known to work at

the nanoscale several suitable options are available for the development of artificial

nanomotors

12 Existing nanomotors Mechanisms and uses

Several nanomotors have been developed in the past decade or so A general principle of

working of these nanomotors is that they function by catalytically decomposing the

dissolved fuel to generate asymmetry at their surface or at their interfacial region thereby

causing propulsion13

The self-electrophoretic bimetallic nanowires developed by

WPaxton and A Sen etal consisted of a 2 μm long rod with one tip made up of Au and

the other tip made up of Pt1 These motors were synthesized by electrodeposition in an

alumina template The rods moved autonomously in a solution of hydrogen peroxide

with platinum end forward Pt oxidizes H2O2 to produce H+ and O2 and Au utilizes H

+ to

reduce H2O2 to water This generates an electron flow through the rod and a proton flow

outside the rod resulting in an electric field around the rod in the solution This electric

7

field in solution acts on the unbalanced charges within the nanorodrsquos double layer This

moves the nanorod with respect to the surrounding fuel due to the ion flux that is

generated

Bubble propulsion has been predominantly used as mechanism of motion in the motors

developed by Feringa etal His group developed a micromotor by attaching catalase to a

microparticle and placing the particle in a solution containing hydrogen peroxide This

system allowed the microparticle to move by producing oxygen bubbles from the

decomposition of hydrogen peroxide14

A similar motor was later created by Feringarsquos

group which consisted of glucose oxidase and catalase immobilized on a carbon

nanotube in a substrate solution of hydrogen peroxide Glucose oxidase converted

glucose to H2O2 which was then utilized by catalase to produce water and oxygen

thereby propelling the carbon nanotube15

Another interesting example of a self-propelled motor is the polymerization driven motor

developed by Cameron et al16

It consists of polystyrene beads immobilized with ActA

protein which catalyzes actin polymerization leading to the formation of an actin

filament which resembles the flagella of bacteria This actin polymerization propels the

beads at the speed of 001-015 μms

A theoretical model has been proposed by Nafaji and Golestanian etal which consists

of three spheres linked by rigid rods in one dimension17

The length of the rods can

switch between two specific values Due to the nonreciprocal motion of the rod this

8

model can swim in low Reynolds number systems An actual model based on

nonreciprocal motion has been developed by Kim etal in which heart cells have been

linked to an asymmetric polydimethylsiloxane robot As the heart cells beat the

asymmetric structure of the device resembles a walking movement18

Most of the nanomicromotors mentioned above are either metallic or work in

environments not compatible with human body Enzymes being an integral part of the

body can solve this problem by operating in suitable substrates already present in the

body fluids The nonreciprocal conformation changes of the enzyme as well as

diffusiophoresis and electrophoresis can conceivably be used to generate motion in case

of enzyme driven nanomotors such motors will be discussed in detail in the following

chapter

9

Chapter 2

Immobilized enzymes on Janus particles as catalytic motors

21 Introduction

Enzymes are widely found in biological systems and act on a large variety of substrates

This can make enzymes an ideal choice for powering nanomotors which need to be

biocompatible Several fluids found in the body can act as substrates to the enzymes For

example dissolved glucose which is abundantly found in the body19

is a susbtrate for

enzyme glucose oxidase Urea which is found in the urinary tract20

is a substrate for

urease Hydrogen peroxide produced by phagocytic cells in the body21

is a substrate for

horseradish peroxidase Having motors driven by enzymes can open possibilities of

targeted drug delivery to these parts of the body containing such substrates

Enzymes have already been used to generate motion of carbon nanotubes by mechanism

of bubble propulsion by Feringa etal15

Two other possible mechanisms which may be

able to power motion of nanoparticles using enzymes are asymmetrical decomposition of

substrates into products leading to self-diffusiophoresis (Figure 2 1) and nonreciprocal

changes in enzyme conformation leading to propulsion (Figure 2 2) As seen in Figure 2

1 enzymes which are immobilized on a Janus particle can breakdown the available

substrate in the solution into products causing asymmetric distribution of solutes in the

surrounding fluid This can lead to movement of particle towards the region of lower

10

solute concentration by principle of self-diffusiophoresis Figure 2-2 shows the possible

nonreciprocal changes in the conformation of a theoretical protein which can lead to

motion of the protein22

These nonreciprocal changes in conformation are triggered by the

attachment of substrate molecule and release of product molecules from the enzymersquos

active site

Figure 2 1 Enzyme functionalized Janus particle moves upon catalyzing asymmetrical

decomposition of substrate into product

11

Figure 2 2 Schematic showing idealized nonreciprocal conformation changes in a

theoretical enzyme22

Enhanced diffusion of a single enzyme molecule by catalysis in a substrate solution has

been demonstrated23

The diffusion coefficient of a single urease molecule was measured

using fluorescence correlation spectroscopy and it was observed that the diffusion of the

urease molecule increases by 16-28 by varying urea concentrations from 0001 M to 1

M Addition of enzyme inhibitors to the system curbed the enhanced diffusion This

enhanced diffusion has been attributed to the force generated by self-electrophoresis by

the enzyme in substrate solution

12

Based on the above facts it might be possible to propel a nanoparticle by asymmetric

distribution of enzymes on its surface thereby leading to self-propulsion of the particles

in substrate solution (Figure 2 1) This propulsion may be due to self-

electrophoresisself-diffusiophoresis andor due to nonreciprocal changes in enzyme

conformations Here we aim to investigate a system consisting of polystyrene particles

half coated with enzyme introduced in a suitable substrate solution The enzyme can be

directly linked to the polystyrene particle or it can be linked through a long spacer

molecule (linker) to provide more degrees of freedom to the enzyme

Several factors need to be considered for choosing enzyme for powering motion at the

nanoscale Some of the important factors are the ratio of kcatKm which denotes the

catalytic efficiency of the enzyme possible substrates stability of enzymessubstrates

ease of immobilization relationship with other enzymes in cascade reactions and

commercial availability of the enzymes The enzyme being used in the present study is

horseradish peroxidase It acts on a molecule of hydrogen peroxide and reduces it while

simultaneously oxidizing another substrate molecule In the absence of alternate

oxidizable substrate HRP can oxidize water and oxygen back to hydrogen peroxide24

thus giving an equilibrium reaction

13

211 Horseradish peroxidase

Figure 2 3 Horseradish peroxidase

Horseradish peroxidase belongs to the group of peroxidases which are a group of heme-

containing oxidoreductases that act on peroxide as hydrogen donor25

It is a redox

enzyme which contains a protoheme a complex of trivalent iron with 1358-

tetramethyl-24-divinylporphin-67-dipropionic acid as a prosthetic group It is a 44 kDa

protein which includes 33900 Da polypeptide chain bound with eight oligosaccharide

chains The carbohydrate chains improve the stability of the enzyme by protecting it

against radicals formed in the course of the reaction it catalyzes26

Horseradish

peroxidase is found in horseradish and is used widely in bioanalytical applications such

as ELISA and Western blots It decomposes hydrogen peroxide into water and oxygen

while simultaneously oxidizing another substrate27

The oxidizable substrates can be

chromogenic substrates such as 22rsquo55rsquo-tetramethyl-benzidine (TMB) and 33-

diaminobenzidine (DMB) or chemoluminescent substrates like SuperSignal and ECL

luminal28

14

Figure 2 4 Reaction catalyzed by horseradish peroxidase

The functional parameters for horseradish peroxidase include Km of 015 mM and kcat of

790 s-1

at an optimum temperature of 20 ordmC and an optimum pH of 6429

Some of the

inhibitors of HRP are 110-phenanthroline aminotriazole and bromine

22 Experimental

221 Materials and methods

Uniform carboxyl modified polystyrene microspheres 05 microm in diameter were obtained

from Polysciences Inc The size of the purchased microspheres was verified by Field

Emission Scanning Electron Microscopy (FESEM) Peroxidase Type I from

Horseradish N-(3-dimethyl-aminopropyl)-Nrsquo-ethylcarbodiimide (EDC) and hydrogen

peroxide solution 50 wt in water were purchased from Sigma-Aldrich N-

hydroxysuccinimide (NHS) ge 97 was purchased from Fluka All chemicals were of

reagent grade and used without further purification Nanopure deionized water was used

for all experiments and preparation of solutions All measurements were made at room

15

temperature (25 oC) unless mentioned otherwise The UV-Visible spectroscopy

measurements were performed using Agilent 8453A diode-array UV-Visible

Spectrophotometer with ChemStation UV-Vis Software Diffusion coefficient

measurements were performed using Nanosight LM10

222 Synthesis of Janus particles

A monolayer of particles was prepared according to the procedure (Figure 2 5) by

Goldenberg L et al30

Glass slides were placed on the bottom of a petridish Water was

put into the petridish so that the entire slide was immersed in water A thin layer of

hexane was added on top of water in the petridish A suspension of microspheres in

ethanol (5 ww) was introduced to the water-hexane interface After the monolayer was

formed the glass slide was lifted up through the monolayer with tweezers The

microsphere-covered glass slides were left to dry overnight at room temperature

Following drying a thin film of silica was evaporated on the glass slides in Semicore

evaporator The spheres were partially coated with 30 nm of silica Particles were

removed from the glass slide by gentle sonication The polystyrene-silica (PS-SiO2)

Janus particles thus obtained were washed in deionized water several times before

further immobilization steps were performed FESEM was used to confirm synthesis of

Janus particles

16

Figure 2 5 Preparation of PS-SiO2 Janus particles by evaporation

223 Immobilization of enzymes using EDCNHS coupling method

1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide and N-hydroxysulfosuccinimide

(EDCNHS) coupling procedure was followed for immobilizing horseradish peroxidase

type I on 05 μm PS-SiO2 Janus particles (Figure 2 6) In this process EDC reacts with a

carboxyl groups on the Janus particles to form an amine-reactive O-acylisourea

intermediate If this intermediate does not encounter an amine it can hydrolyze and

regenerate the carboxyl group In the presence of NHS EDC can be used to convert

carboxyl groups to amine-reactive sulfo-NHS esters which are much more stable and do

not readily hydrolyze to regenerate carboxyl groups In the current experiment

immobilization was performed in the presence as well as absence of a long spacer

17

molecule (linker) between Janus particles and enzymes The chemical 6-aminohexanoic

acid was used as the linker (Figure 2 7) HRP immobilized PS-SiO2 Janus particles were

stored in deionized water until required for further experiments

Figure 2 6 EDCNHS coupling method for immobilization of enzyme on carboxylated

polystyrene particles

Figure 2 7 Immobilization of enzyme on Janus particles with and without 6-

aminohexanoic acid linker

18

224 Enzyme assay

Assay for activity of HRP was performed using a spectrophotometric method which

involved using 4-aminoantipyrene as hydrogen donor3132

The reaction rate was

determined by measuring increase in absorbance at 510 nm resulting from the

decomposition of hydrogen peroxide One unit represents the decomposition of 1 μM of

hydrogen peroxide per minute at 25 degC and pH 70 under the specified conditions

The spectrophotometer was set to the wavelength of 510 nm and temperature of 25 degC

Phenol4-aminoantipyrene solution (14 mL) and 00017 M H2O2 solution (15 mL) were

pipetted into each cuvette The cuvette was incubated for 3-4 mins at 25degC and blank was

established A 01 mL solution of diluted enzyme was added to the above mixture and

absorbance was recorded for 4-5 mins Standard graphs of absorbance versus time were

drawn for known concentration of the free enzyme and compared to that of the

immobilized enzyme on the Janus particles The estimate of the amount of enzyme

present on the Janus particles was deduced from the difference between the quantity of

the free enzyme previously added for immobilization and the amount that was measured

to be in the supernatant after some of the enzyme became attached to the Janus particles

The experiment was carried out in triplicate to assess reproducibility

19

The formula used to calculate the activity of enzyme is

where ∆Amin denotes the change in absorbance per minute

225 Diffusion coefficient measurements using Nanosight LM10

Diffusion coefficients for HRP immobilized particles were measured using Nanosight

LM 10 nanoparticle tracking instrument with Nanoparticle Tracking Analysis (NTA) 20

software in the presence of 003 H2O2 solution as the substrate and also in the presence

of only deionized water The videos were recorded for 30 seconds at the rate of 30 frames

per second Each experiment was carried out in triplicate to assess reproducibility

Particles

visualization

zone

Microscope with

LM 10 unit

LM 10 unit

Figure 2 8 Nanosight LM 10 instrumentation

20

Nanosight LM 10 (Figure 2 8) generates videos of population of moving nanoparticles in

a liquid when illuminated by laser light The particles which pass through the laser beam

path are seen by the instrument as small points of light undergoing Brownian motion

(Figure 2 9a)

The videos are further analyzed by NTA (Nanoparticle tracking analysis) software

(Figure 2 9b) which identifies and tracks each particle independently on a frame-by-

frame basis The software also calculates and subtracts any bulk flow in the system The

mean square displacement is calculated for each particle as long as it is visible If the

particle blinks that is if it disappears momentarily and then reappears on the screen the

software will consider the same particle as two separate ones All analysis data is

exported to the excel files in terms of diffusion coefficients of the tracked particles

B A

Figure 2 9 NTA video analysis (a) Particles as they are seen on the screen during

experimentation in Nanosight LM10 (b) NTA screen as the particles are being analyzed for

their diffusion coefficients

21

23 Results

231 Enzyme assay

The activity of free HRP enzyme reported by Sigma Aldrich is 150 unitsmg However

the activity of HRP upon analysis was found to be 52158 unitsmg which is much lower

than the reported value The activity of HRP was found to be further lowered to 2133

unitsmg when it was immobilized on the PS-SiO2 Janus particles in the presence of a

linker The following formula is used to calculate enzyme activity

where ΔAmin is the change in absorbance per minute

22

Figure 2 10 Graph for the assay of solution of free enzyme containing 05mgL of HRP

ΔAmin = 01716 Unitsmg of free enzyme = 52158

During EDCNHS coupling 04 mg enzyme was added to 1 ml of 025 vv suspension

of Janus particles Thus initial concentration of enzyme in the mixture was 04 mgml

Assuming retained activity of the enzyme in supernatant solution the amount of enzyme

in supernatant was calculated to be 03868 mgml from the assay Amount of enzyme

functionalized on the Janus particles was calculated as the difference between enzyme

previously added for immobilization (04 mgml) and the enzyme present in supernatant

(03868 mgml) and was found to be 00132 mgml Enzyme assay was performed on the

enzyme immobilized Janus particles to estimate the activity of immobilized enzyme

(Figure 2 11) and was found to be 2133 unitsmg

y = 0002x + 00516

0

01

02

03

04

05

06

07

08

0 50 100 150 200 250 300 350 400

Ab

sorb

an

ce

51

0 n

m

Time (sec)

Assay of free enzyme HRP

23

Figure 2 11 Graph for the assay of of immobilized enzyme on 05 μm Janus particles

(~025 vv) ΔAmin = 01853 Unitsmg of free enzyme = 2133

232 Diffusion coefficient measurements

No significant increase was observed in diffusion coefficient of HRP-immobilized Janus

particles in the presence of H2O2 as the substrate Similar results were obtained for

diffusion coefficient measurement with and without linker moieties bridging the enzymes

and particles(Table 2 1 Table 2 2) Thus the linker did not seem to assist in increasing

the particle diffusion by improving or retaining the activity of the enzyme

y = 00015x + 00953

0

01

02

03

04

05

06

07

0 50 100 150 200 250 300 350 400

Ab

sorb

an

ce

510 n

m

Time (sec)

Assay of HRP immobilized 05 μm Janus particles

24

Table 2 1 Diffusion coefficient measurements of HRP immobilized Janus particles

without linker

HRP immobilized

Janus particles in

003 H2O2

Control - Janus

particles in

003 H2O2

Control - HRP

immobilized Janus

particles in DI water

Diffusion coefficient

(10-10

cm2s)

1359416 1051777 1308675

Standard deviation 4534801 4122075 4918597

Table 2 2 Diffusion coefficient measurements of HRP immobilized Janus particles in

the presence of linker

HRP immobilized

Janus particles in

003 H2O2

Control - Janus

particles in

003 H2O2

Control - HRP

immobilized Janus

particles in DI water

Diffusion coefficient

(10-10

cm2s)

13838 1339829 1407891

Standard deviation 5644692 5649364 5797218

25

Figure 2 12 Diffusion coefficient measurements of PS-SiO2 Janus particles without and

with 6-aminohexanoic acid linker

24 Discussion

To compare the diffusion coefficients observed during the experiments theoretical

diffusion coefficient was calculated using following equation33

r

kTD altraslation

6 Equation 21

where r is the hydrodynamic radius of particle Dtranslational is translational diffusion

coefficient of the particle T is temperature of the system η is the viscosity of fluid

through which the particles are moving and k is the Boltzmann constant From FE-SEM

images the size of the particles used was determined to be 450 nm For 450 nm diameter

0

20

40

60

80

100

120

140

160

Without linker With linker

Dif

fusio

n c

oeff

icie

nt

(10

-10 c

m2s

)

HRP immobilized 05 μm Janus particles - Diffusion coefficient measurements

HRP immobilized Janus particles in 003 H2O2

Control - Janus particles in 003 H2O2

Control - HRP immobilized Janus particles in DI water

26

particle Dtranslational is equal to 109 μm2s which is comparable to the diffusion

coefficient measured for the enzyme immobilized particles

HRP was found to be active on the Janus particles however the activity of the enzyme

was reduced upon immobilization Lower enzyme activity can be due to the coupling

method used which in this case was EDCNHS covalent binding However EDCNHS

coupling method has been shown to significantly retain the enzyme activity when the

enzyme is immobilized on carboxylated multi-walled carbon nanotubes under appropriate

conditions34

Hence EDCNHS binding method by itself is not believed to be

responsible for reduction of enzyme activity upon immobilization

Direct binding of enzyme to the Janus particle may lead to reduction in the enzyme

activity because of space constraints for the enzyme and difficulty for the passage of

substrate to the active site of the enzyme due to stearic hindrance Hence a linker was

inserted between the enzyme and Janus particle during enzyme immobilization However

inserting a linker between the enzyme and the particle did not lead to enhanced diffusion

of Janus particles or increase in enzyme activity of immobilized enzyme

Lack of enhanced diffusion can be firstly attributed to the reduction in enzyme activity

upon immobilization Enzyme activity may be retained by modifying the conditions

during EDCNHS coupling or changing the binding method altogether The degree of

immobilization of HRP on carboxylated Janus particles can possibly be affected by

several factors such as temperature the concentration of the coupling reagent EDC

27

concentration HRP concentration in solution and incubation time34

Different

combinations of these factors can be tested to find optimum conditions for

immobilization of HRP Alternate binding method can involve thiolation of HRP by the

covalent conjugation of mercaptopropionic acid and then the thiolated HRP can then be

attached on the gold surface of a polystyrene-gold Janus particle35

Thus further

modifications can be made to the system in order to retain enzyme activity on the

immobilized particles and thereby power nanomotors using enzymes

28

Chapter 3

Predator-prey system of enzyme immobilized particles

31 Introduction

Predator-prey systems are widely found in nature and play a major role in biological

processes3637

Predator-prey systems include all the interactions in which one organism

pursues andor consumes another organism One familiar predator-prey system at the

macroscale is that of a tiger chasing and consuming a deer Another not so familiar one

includes Baleen whales which eat millions of microscopic planktons at once38

At the

microscale such kind of interactions can be observed in the case of phagocytes in the

body which are capable of engulfing foreign microorganisms and killing them with a

combination of microbicidal systems39

In the case of cancer the tumor is the prey and

the mature myeloid cells in the body are the predator However the tumor here has a

defense mechanism against the predator in which the tumor produces cytokines which

inhibits the maturation of the myeloid cells and thereby prevents predation40

One similarity that can be observed in all these systems is that the prey possesses some

attribute which is desired by the predator which makes the predator chase the prey A

similar relationship is seen in case of enzymes which are involved in cascade reactions41

In cascade reactions product synthesized by one enzyme is the reactant required by

another enzyme to carry out its own catalytic reaction As such there is a predator-prey

29

kind of dependence between the two enzymes which may cause one enzyme to chase the

other enzyme when placed in suitable substrate solutions However the mechanism

hypothesized for such a system is totally different than the mechanism involved in

biological systems In biological systems such as bacterial chemotaxis or phagocytes

chasing foreign particles in human body complex biochemical reactions are responsible

for the predator-prey behavior42

In the proposed enzyme cascade system there are no

complex biochemical reactions involved The principle hypothesized for predator-prey

behavior of enzyme cascade reactions can be explained as follows Motion of synthetic

motors at nanoscale involves two components translational diffusion and rotational

Brownian diffusion At very short time scales of less than 1Dr where Dr (s-1

) is

rotational Brownian diffusion coefficient the motion of particles is unidirectional For

longer time scales of greater than 1Dr the direction of motion is random giving

poweredactive diffusion43

If the chemical homogeneity is broken that is when there is

higher concentration of a substrate present in one direction of the enzyme the enzyme

might acquire a subtle bias which may lead to a spatial aggregation of the enzymes based

on the substrate concentration gradient The bias can be a result of the relationship

between the speed of enzyme diffusion and substrate concentration where higher

substrate concentration lead to higher enzyme diffusion As a result in an enzyme

cascade system one enzyme will migrate towards higher concentration of substrate

which is present near the other enzyme thus giving rise to a predator-prey behavior

Such enzyme cascades are widely found in signaling networks and metabolic pathways in

living cells and can be used to produce predator-prey like nanomotors systems44

The

30

enzymes being used in the present study are horseradish peroxidase and glucose oxidase

Both enzymes are extremely stable4546

Glucose is the substrate for glucose oxidase

which is widely available and highly stable47

Horseradish peroxidase acts on hydrogen

peroxide It is involved in a cascade reaction with glucose oxidase which produces

hydrogen peroxide by converting glucose into gluconic acid (Figure 3 1)48

Figure 3 1 Concept of particle aggregation due to cascade reactions of immobilized

enzymes

In this experiment glucose oxidase (GOx) and horseradish peroxidase (HRP) were

immobilized on polystyrene carboxylate beads of different sizes using EDCNHS

coupling method Glucose and 3355-Tetramethylbenzidine (TMB) were dissolved

together in water and this solution was used as the substrate solution All the components

were present in a single system in DI water Aggregation of particles and precipitation of

oxidized TMB from the solution was expected to be observed when all these components

were present in such a system

Properties of horseradish peroxidase have been discussed in the previous chapter (Section

211 Horseradish peroxidase)

GOxGOx HRPHRP

Glucose + O2Gluconic acid + H2O2

TMB TMB oxidizedBlue precipitate

H2O

Aggregation

31

311 Glucose oxidase

Figure 3 2 Glucose oxidase

Glucose oxidase (GOx) belongs to the group of oxidoreductases and is also called

glucose aerodehydrogenase49

It is obtained from fungi and has been identified as an

antimicrobial enzyme50

GOx binds to β-D-glucopyranose and catalyzes the oxidation

reaction to from gluconolactone while simultaneously reducing oxygen to hydrogen

peroxide51

GOx has high substrate affinity and specificity high stability and high

turnover number which makes it a suitable biocatalyst for industrial applications52

Figure 3 3 Redox reaction of glucose catalyzed by glucose oxidase53

32

GOx is a glycoprotein with molecular weight of 155 kDa and consists of two identical

polypeptide subunits linked together by a disulfide bond The optimum pH for GOx is

55 though it is stable in a broad pH range of 4 to 7 and can be stored for a long period of

time51

The functional parameters for glucose oxidase include Km of 151 mM and kcat of

337 s-1

at temperature of 20 ordmC and pH of 754

Some of the inhibitors of GOx are 2-

Deoxy-D-glucose adenine nucleotides and NaNO3

32 Experimental

321 Materials and methods

Uniform carboxyl modified polystyrene microspheres 05 microm in diameter were obtained

from Polysciences Inc The size of the purchased microspheres was verified by FESEM

Peroxidase Type I from horseradish glucose oxidase Type II-S from Aspergillus niger

N-(3-dimethyl-aminopropyl)-Nrsquo-ethylcarbodiimide (EDC) and Hydrogen peroxide

solution 50 wt in water were purchased from Sigma-Aldrich N-hydroxysuccinimide

(NHS) was purchased from Fluka D-Glucose (99) was purchased from VWR

International Secure-Seal imaging spacers (9mm diameter) for glass slides were obtained

from Sigma Aldrich All chemicals were of reagent grade and used without further

purification Nanopure deionized water was used for all experiments and preparation of

solutions All measurements were made at room temperature (25 oC) unless mentioned

otherwise The UV-Visible measurements were performed using Agilent 8453A diode-

array UV-Visible Spectrophotometer with ChemStation UV-Vis Software Optical

33

microscopy experiments were performed using Axiovert 200 MAT microscope by Carl

Zeiss Diffusion coefficient measurements were obtained using Nanosight LM10

322 Optical microscopy experiments

The enzyme substrate system was observed under optical microscope to check for

aggregation and precipitation due to cascade reactions Several such systems were

studied Each system in general consisted of GOx and HRP immobilized on different

polystyrene particles The substrate solutions consisted of 01 M glucose and different

concentrations of TMB solution The systems were studied in the presence of all the

constituents and in the absence of glucose GOxHRP and TMB Systems consisting of

different sized HRP immobilized particles in substrate solution of 003 hydrogen

peroxide and TMB were also studied Videos were recorded over a period of 24 hours to

observe changes in the system Each experiment was performed atleast thrice to assess

reproducibility of results

323 Diffusion coefficient measurements using Nanosight LM10

Diffusion coefficient of GOx and HRP immobilized particles were measured using

Nanosight LM10 nanoparticle tracking instrument with Nanoparticle Tracking Analysis

(NTA) 20 software Diffusion coefficients were measured for different systems namely

GOx particles in 01 M glucose and oxygen both GOx and HRP functionalized particles

in 01 M glucose and oxygen along with controls for the individual systems Videos were

34

recorded for 30 seconds at the rate of 30 frames per second Each experiment was carried

out in triplicate to assess reproducibility

33 Results

331 Optical microscopy

According to the enzyme cascade model precipitation of TMB was possible only upon

the interaction of GOx and HRP immobilized particles in presence of appropriate

substrate solution Hence in the first set of experiments precipitation of TMB was used

as an indication of the type of interaction between GOx and HRP immobilized particles

Fan-shaped structures were observed when both HRP and GOx immobilized particles

were present in a substrate solution consisting of glucose and TMB These structures

started forming as a single strand and then branched out and intertwined to form the fan-

shaped structures (Figure 3 4)

Table 3 1 Observations for formation of fan-shaped structures in enzyme cascade

system in presence of different components

GOx HRP Glucose TMB Observation

Yes Yes Yes Yes

Fan-shaped structures observed particles associated with

the fan-shaped structures

No Yes Yes Yes No fan-shaped structures

Yes No Yes Yes No fan-shaped structures

Yes Yes No Yes No fan-shaped structures

Yes Yes Yes No No fan-shaped structures

35

Fan-shaped structures can also be observed in the system consisting of only HRP

immobilized particles in substrate solution of hydrogen peroxide and TMB The size of

the fan-shaped structures depends on the size of the enzyme immobilized particles Free

enzymes show extremely tiny structures which are difficult to view under the optical

microscope

Figure 3 4 Formation of fan-shaped structures when both HRP and GOx

immobilized particles were present in a substrate solution consisting of glucose and

TMB A In the initial stages single strands form on the surface of liquid (5Ox) B

Single strands branch out and intertwine to form fan-shaped structures (50x) C Many

such structures form and settle down after 20 minutes (20x)

A

C

B

36

Experiments were performed in the absence of TMB that is the system consisted of GOx

and HRP immobilized particles in glucose substrate solution In the absence of TMB the

particles were expected to move closer to each other without any precipitation due to the

cascade reaction However there was no conclusive aggregation of particles observed in

the system

Free Enzyme 005 μm HRP - immobilized particle

10 μm HRP - immobilized particle 05 μm HRP - immobilized particle

Figure 3 5 Size dependence of fan-shaped structures on the size of HRP -

immobilized particles (20x)

37

Table 3 2 Observations for particle aggregation in enzyme cascade system in presence

and absence of TMB

Glucose GOx HRP TMB Observation

Present Present Present Present Aggregation and precipitate

Present Present Present Absent No conclusive aggregation

Present Absent Present Present No aggregation or ppt

Absent Present Present Present No aggregation or ppt

Present Present Absent Present No aggregation or ppt

332 Diffusion coefficient measurements

Diffusion coefficients were measured for GOx immobilized particles of 05 μm diameter

in substrate solution of 01 M glucose and oxygen Diffusion coefficients of particles

were also measured for system consisting of GOx and HRP functionalized particles in

glucose and oxygen Each experiment was done in triplicate to assess reproducibility

Nanosight visualization chamber being a closed chamber oxygen was purged into all the

solutions before starting the experiments to ensure sufficient supply of oxygen for

catalytic reaction of GOx

38

Table 3 3 Diffusion coefficients for 05 μm GOx immobilized particles in 01 M

glucose purged with O2

Mean

GOx

particles in

01 M

glucose

Control - GOx

particles in DI

water

Control - GOx

particles in 01 M

glucose without

oxygen purging

Control -

particles in 01

M Glucose

Diffusion

coefficient

(x10-10

cm2s)

13316 12888 13827 13702

Standard

deviation 6307 4926 4424 7468

Table 3 4 Diffusion coefficients for GOx and HRP immobilized particles in 01 M

glucose and purged with O2

Mean

GOx- HRP-

functionalized

particles in 01 M

glucose

Control ndash Gox-

HRP- functionalized

particles in DI water

Control - particles

in 01 M glucose

Diffusion coefficient

(x10-10

cm2s)

13005 14434 13702

Standard deviation 5263 6622 7468

No significant increase was observed in diffusion coefficient of enzyme immobilized

particles in any of the systems

39

34 Discussion

Formation of fan-shaped structures due to patterned deposition of oxidized TMB

indicates some kind of interaction between GOx and HRP immobilized particles Particle

diameter dependence of the size of structures can be explained with a simple model in

Figure 3 6 A larger particle will have a higher surface area as compared to a smaller

particle Hence more enzyme is immobilized on the larger particle As a result higher

quantity of TMB is oxidized and precipitated by the larger particle as compared to the

smaller particle This leads to thicker strands of fan-shaped structures eventually scaling

up the whole system to give bigger fan-shaped structures

The exact reason for the formation of such fan-shaped structures which are dependent on

particle size could not be determined It is hypothesized that hydrogen peroxide andor

Figure 3 6 Model for size dependence of fan-shaped structures on the particle

diameter of enzyme immobilized particles Larger the particle more surface area is

available for precipitation of TMB which eventually leads to bigger fan-shaped

structures

Larger particle Smaller particle

40

TMB gradient in the substrate solution might be responsible for such structures If there

is higher concentration of H2O2 on one side of the particle and assuming an even

concentration distribution of TMB there will be selective precipitation in the direction of

H2O2 When several particles come in the vicinity of higher concentration of H2O2 the

precipitation lines will converge to give fan-shaped structures (Figure 3 7)

Figure 3 7 Hypothesis for formation of fan-shaped structures

In the system consisting of GOx immobilized particles HRP immobilized particles

glucose and TMB it was observed that the fan-shaped structures formed at the liquid-air

interface and eventually settled down to the bottom of the glass slide This makes sense

as oxygen is necessary for the catalytic reaction of conversion of glucose to gluconic acid

and hydrogen peroxide which takes place on the surface of GOx immobilized particle As

a result H2O2 is primarily formed at the liquid-air interface and is then used by HRP for

precipitation of TMB

Following the observations that precipitation of TMB occurs at the surface GOx and

HRP immobilized particles are also expected to interact at the surface in the absence of

TMB Due the cascade reaction it was expected that HRP immobilized particles will be

41

attracted to GOx immobilized particles in appropriate substrate solution Experiments

were carried out to visualize inter-particle interactions at the liquid-air interface

However liquid-air interface not being a stable system to image the precise interaction

of GOx and HRP immobilized particles could not be determined Significant external

disturbances were involved at the interface To enable the cascade reaction in the bulk of

the liquid oxygen was purged into the reaction system before starting the experiment

However this did not lead to significant reaction in the bulk of the liquid and the reaction

prominently took place at the liquid-air interface where the oxygen concentration was

higher

In order to observe the interaction of GOx and HRP immobilized particles better imaging

techniques need to be used to observe diffusion of particles in absence of external

disturbances at the liquid-air interface Large quantity of oxygen needs to be purged into

the system and retained to make the reaction more pronounced in the bulk of the substrate

solution Higher concentration of oxygen in water may also be achieved by using oxygen

enriched water which is commercially available55

Alternatively a different enzyme cascade system may be used which does not require a

gaseous substrate56

For example glycogen synthase synthesizes glycogen from glucose-

1-phosphate and glycogen phosphorylase breaks down glycogen to glucose-1-

phosphate57

This system does not require a gaseous substrate and might be able to

overcome the deficiencies of GOx and HRP enzyme cascade system

42

Chapter 4

Silver-titania nanomotors

41 Introduction

Bimetallic nanowires made of platinum and gold have been developed by WPaxton

etal1 The rods move autonomously in a solution of hydrogen peroxide In the present

study silver-titania (Ag-TiO2) particles are being synthesized and investigated for motion

in presence of hydrogen peroxide and UV light With proper control of conditions and

UV light trigger it can be possible to control the speed and direction of motion of these

nanoparticles

Hydrogen peroxide is produced in the human body by phagocytes as an immune response

to foreign particles and pathogens5859

Also silver nanoparticles have been shown to be

biocompatible and capable of interacting with cells in human body without disrupting

cellular function60

Similarly it has been demonstrated that titania is compatible with

different cells DNA and proteins in human body and does not cause toxic effects Hence

silver-titania nanomotors which swim in hydrogen peroxide may open interesting

avenues in applications like targeted drug delivery in those parts of the body where

hydrogen peroxide is present

43

42 Experimental

421 Materials and methods

Silver nitrate (999995) was purchased from Alfa Aesar Gum arabic was purchased

from MP Biomedicals Hydrogen peroxide solution 50 wt in water and ascorbic acid

were purchased from Sigma-Aldrich Nanopure deionized water was used for all

experiments and preparation of all solutions Secure-Seal imaging spacers (9 mm

diameter) were obtained from Sigma Aldrich All experiments were carried out at room

temperature of 25ordmC unless otherwise mentioned Ag-TiO2 particles were synthesized by

evaporation of titania in Semicore electron beam evaporator Particles were characterized

using field-emission scanning electron microscopy Optical microscopy experiments

were performed using Axiovert 200 MAT microscope by Carl Zeiss Particles were

tracked using Physvis particle tracking software (Figure 4 1) in which the position

information of a particle on screen is stored on a frame by frame basis The data is stored

in a dat file as X and Y co-ordinates of particles tracked on the screen This information

can be converted to diffusion coefficients of the particles by further manipulation using

MathWorks-MATLABreg software

44

Figure 4 1 Physvis particle tracking software

422 Synthesis of silver colloidal particles

Silver particles were synthesized by a nucleationgrowth type reaction developed by

Goya and Matijevic61

and modified by Velikov62

Silver nitrate was used as precursor

metal salt for synthesis of silver particles Solution A was 75 ml of 50 mM AgNO3 and

035 by weight gum arabic Solution B was 75 mL of 100 mM 1-ascorbic acid and

035 by weight gum arabic Solution B was rapidly added to solution A in a 250 mL

reaction flask The reaction flask was covered and particles were stirred for 48 hrs After

48 hours particles were allowed to settle for 2-3 hours in refrigerator and supernatant

was separated from the particles Particles were then rinsed with DI water and centrifuged

thrice to obtain pure silver particles Zeta potential of these particles was reported to be

45

around -50 to -60 mV and the size was around 1μm The size of the particles can be

varied by varying the amounts of ascorbic acid and silver nitrate Using molar ratio of 31

of ascorbic acid silver nitrate can give particles of 200 nm diameter whereas ratio of

151 can give particles of upto 5 μm diameter

423 Preparation of silver-titania (Ag-TiO2) particles

A monolayer of silver particles was prepared according to the procedure by Goldenberg

et al63

Glass slides were placed on the bottom of a petridish Water was put into the

petridish so that the entire slide was immersed in water A thin layer of hexane was then

added on top of water in the petridish A suspension of silver particles in ethanol (5

ww) was slowly introduced to the water-hexane interface in the dish After the

monolayer was formed the glass slide was lifted up through the monolayer with tweezers

The silver particles-covered glass slides were left at room temperature to dry overnight

Following drying a thin film of titania was evaporated on the glass slides in Semicore

evaporator (Figure 2 7) The spheres were partially coated with a 10 nm film of titania

Ag-TiO2 particles were removed from the glass slide by gentle sonication FE-SEM was

used to characterize the particles (Figure 4 2) The thin titania layer was not easily

distinguishable due to roughness of the particles

46

Figure 4 2 FESEM image of Ag-TiO2 particles

424 Optical microscopy experiments

Imaging spacers were placed on a glass slide Suspension of Ag-TiO2 particles was

placed on the glass slide and the particles were allowed to settle Movement of particles

was observed in DI water and videos were recorded at 50x magnification of optical

microscope for 30 sec 05 H2O2 was added to the particle suspension on the slide

Videos were recorded at 50x ranging from 30 sec to 10 min to study the movement of

silver titania particles in hydrogen peroxide solution Particles in both DI water and

hydrogen peroxide were subjected to UV light and videos were recorded to study particle

dynamics in presence of UV radiation Particle tracking was performed using Physvis

particle tracking software Atleast 30 particles in every system were tracked for 10

seconds each Particle tracking data was obtained in terms of X and Y coordinates of

particles on screen This data was manipulated using MathWorks-Matlab software and

graphs of mean square displacement of particles versus time were plotted Diffusion

coefficients of particles were calculated from these graphs

47

43 Results and Discussion

Ag-TiO2 particles demonstrated autonomous motion in the presence of H2O2 No UV

light was required for this motion Motion of the particles was accelerated by addition of

fresh H2O2 to the system When subjected to UV radiation the particles displayed

momentary acceleration for 4-5 seconds

Figure 4 3 Mechanism of motion of Ag-TiO2 nanomotors

In the presence of H2O2 silver part of the Ag-TiO2 particle converts H2O2 to H2O and O2

This may lead to propulsion of the Ag-TiO2 particle due to bubble propulsion andor self-

diffusiophoresis In the presence of UV light additional reaction takes place on the

titania part of Ag-TiO2 particle which increases the velocity of the Ag-TiO2 particles

(Figure 4 4) This UV triggered photoactivity of titania is due to hole-electron separation

caused by photons having energy equal to or greater than the band gap of titania646566

Most of the holes-electrons recombine but a small fraction move to the surface and react

with the available redox species like water and oxygen to form protons (H+) superoxide

(O2-) and hydroxyl (OH) radicals

6566 This may lead to propulsion by diffusiophoresis

as well as electrophoresis

48

Figure 4 4 Silver-titania motors move faster when subjected to UV radiation

In addition to linear motion rotational motion is also observed in several particles and

particle aggregates Aggregation of particles can lead to a net circular propulsive force

which can rotate the aggregate as shown in Figure 4 5

Figure 4 5 Rotation due to particle aggregation of Ag-TiO2 particles

Particle tracking by Physvis software revealed a significant increase in diffusion

coefficient of Ag-TiO2 particles in hydrogen peroxide solution as compared to DI water

49

The Ag-TiO2 particles have a diffusion coefficient of 1491 μm2s in hydrogen peroxide

versus only 180 μm2s in DI water Thus the Ag-TiO2 particles move around more than

10 times their body length per second in hydrogen peroxide (Figure 4 6) When

subjected to UV light the particles experience a significant acceleration in hydrogen

peroxide which lasts only for a short duration of time (4-5 sec) after which the particles

return to their original velocity The diffusion coefficient of the particles during this UV

light trigger is found to be around 5093 μm2s UV light trigger does not have any visible

effect on the Ag-TiO2 particles in DI water

Table 4 1 Translational diffusion coefficients of Ag-TiO2 particles in hydrogen

peroxide and water

Mean 05 H2O2 solution DI water

Diffusion coefficient of Ag-TiO2 (μm2s)

without UV light

1491 180

Diffusion coefficient of Ag-TiO2 (μm2s)

in the presence of UV light

5093 113

50

Figure 4 6 Mean square displacement curves of Ag-TiO2 Janus particles in hydrogen

peroxide and DI water

When higher concentration (~30 vv) of Ag-TiO2 particles in hydrogen peroxide is

subjected to UV radiation the particles exhibit pattern formation (Figure 4 7)

Aggregation of Ag-TiO2 Janus particles is observed when UV light is turned on and the

particles disaggregated when UV light is switched off This can be due to predator-prey

kind of interaction between silver and titania half of the Janus particles in the presence of

UV light Oxygen and water that are produced at the silver end by decomposition of

hydrogen peroxide are used up by titania when subjected to UV radiation This can lead

to attraction between titania and silver parts of neighboring Janus particles causing

aggregation

y = 20373x

y = 59994x

y = 71848x

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10 12

Mean

sq

uare

dis

pla

cem

en

t (μ

m2s

)

Time (sec)

Mean square displacement vs Time for Ag-TiO2

particles

05 Hydrogen peroxide and UV05 Hydrogen peroxide

DI Water

51

A UV OFF B UV ON

C UV OFF

Figure 4 7 Higher concentrations of Ag-TiO2 particles showing aggregation when UV

is turned on

52

Chapter 5

Summary and Conclusion

In summary no significant enhancement in diffusion was observed in case of enzyme

immobilized Janus particles in substrate solution Presence of a linker between enzyme

and particle did not increase the diffusion coefficient of particles Alternative enzyme

immobilization methods need to be tested like thiolation of enzyme and subsequent

binding to gold surface of a Janus particle35

or expressing the enzyme with polyhistidine

or avidin tag67

This can result in higher enzyme activity upon immobilization and may

lead to enhanced diffusion of Janus particles

Predator-prey system of GOx and HRP immobilized particles showed some promising

results Aggregation of particles and formation of fan-shaped structures by TMB

oxidation were observed under optical microscope when GOx and HRP immobilized

particles glucose and TMB were present in the system The size of the fan-shaped

structures was dependent on the size of enzyme immobilized particles This can be

indicative of some kind of interaction between the GOx and HRP immobilized particles

No conclusive aggregation of GOx and HRP immobilized particles was observed in

absence of TMB Better imaging techniques are required to study the interaction of GOx

and HRP immobilized particles at the unstable liquid-air interface where majority of the

reaction takes place Other enzyme cascade systems not requiring gaseous reactants like

glycogen synthase and glycogen phosphorylase should be tested for similar activity

53

Ag-TiO2 particles were found to exhibit autonomous motion in the presence of hydrogen

peroxide solution The motion was momentarily accelerated upon exposure to UV

radiation The particles showed a diffusion coefficient of 1491 μm2s in hydrogen

peroxide versus only 180 μm2s in DI water Upon subjecting particles to UV radiation in

presence of H2O2 diffusion coefficient increased to 5093 μm2s for a period of 4-5

seconds In addition to linear motion rotating motion could also be observed in some

particle aggregates Further development of these aggregate structures can lead to

formation of rotors at nanoscale Higher concentration of silver-titania particles in

hydrogen peroxide displayed aggregation in presence of UV light and disaggregated

when UV light was switched off This may be attributed to the predator-prey kind of

interaction between silver and titania where oxygen and water that are produced by

silver by decomposition of hydrogen peroxide are used up by titania when subjected to

UV radiation This promises to be an interesting system and needs to be further

investigated to explore its properties

54

References

1 Paxton WF Sundararajan S Mallouk TE Sen A Angew Chem Int Ed Engl

2006 45 5420-5429

2 Golestanian R Liverpool TB Adjari A Phys Rev Lett 2005 94 220801

3 httpwwwsilvermedicineorgh2o2html

4 Rami Pedahzur Ovadia Lev Badri Fattal Hillel Shuval Water Science and Technology

1995 31(5-6) 123-129

5 W Went F American Scientist 1968 56

6 Adriano Cavalcanti Tad Hogg Bijan Shirinzadeh IEEE MHS 2006 International

Symposium on Micro-NanoMechatronics and Human Science 2006 226-232

7 EM Purcell American Journal of Physics 1977 45 3-11

8 Golestanian R Phys Rev Lett 2010 105 018103(1)-018103(4)

9 S S Dukhin Z R Ulberg G L Dvornichenko B V Deryagin Russian Chemical

Bulletin 1983 31(8) 1535-1544

10 Dukhin AS Goetz PJ Ultrasound for characterizing colloids Particle sizing zeta

potential rheology Elsevier 2002 59

11 HJKeh HJTu Trends in Colloid and Interface Science XIV Progress in Colloid and

Polymer Science 2000 115 174-180

12 D Zinemanas A Nir International Journal of Multiphase Flow 1995 21(5) 787-800

55

13 Stephen Ebbens Jonathan Howse Soft matter 2010 6 726-739

14 J Vicario R Eelkema W R Browne A Meetsma R M La Crois B L Feringa

Chem Commun 2005 31 3936

15 Davide Pantarotto Wesley R Browne Ben L Feringa Chem Commun 2008 1533 -

1535

16 Lisa A Cameron Matthew J Footer Alexander van Oudenaarden Julie A Theriot

PNAS 1999 96(9) 4908-4913

17 Ali Najafi Ramin Golestanian Phys Rev E 2004 69 062901

18 Jinseok Kim Jungyul Park Sungwook Yang Jeongeun Baek Byungkyu Kim Sang

Ho Lee Eui-Sung Yoon Kukjin Chun Sukho Park Lab Chip 2007 7 1504 - 1508

19 Kurt M Dubowski Clinical Chemistry 2008 5411 1919ndash1920

20 Spector DA Yang Q Wade JB Am J Physiol Renal Physiol 2007 292(1) 467-474

21 Rhee SG Chang TS Bae YS Lee SR Kang SW J Am Soc Nephrol 2003 14(8-3)

211-215

22 Sakaue T Kapral R Mikhailov A S Eur Phys J B 2010 75 381ndash387

23 Muddana H S Sengupta S Mallouk T E Sen A Butler P J J Am Chem Soc

2010 132 (7) 2110ndash2111

24 Baker CJ Deahl K Domek J Orlandi EW Arch Biochem Biophys 2000 382(2) 232-

237

25 V Vojinovic RH Carvalho F Lemos JMS Cabral LP Fonseca BS Ferreira

Biochemical Engineering Journal 2007 35 126-135

56

26

OV Lebedeva NN Ugarova Russian Chemical Bulletin 1996 45(1) 18-25

27 Nigel C Veitch Phytochemistry 2004 65(3) 249-259

28 Kenneth H Petersen Journal of Immunological Methods 2009 340(1) 86-89

29 httpwwwbrenda-enzymesorgphpresult_flatphp4ecno=11117

30 Goldenberg L M Wagner J Stumpe J Paulke B-R Goumlrnitz E Langmuir 2002 18

5627-5629

31 httpwwwgoldbiocompdf3761-Protocol201pdf

32 Av Rovisco Pais Lisboa Portugal T amp D Technology West Yorkshire UK Journal

of Molecular Catalysis B Enzymatic 2004 28(2-3) 129-135

33 httpwwwphysicsnyuedugrierlabmethodsnode11html

34 Yoon-Mee Lee1 O-Yul Kwon Yeo-Joon Yoon Keungarp Ryu Biotechnology Letters

2006 28 39ndash43

35 JC Pyun SD Kim JW Chung Analytical Biochemistry 2005 347(2) 227-233

36 George H Pimbley Jr Mathematical Biosciences 1974 20(1-2) 27-51

37 Andy Fenton Sarah E Perkins Parasitology 2010 137 1027-1038

38 Mead James G Brownell Robert L Mammal Species of the World A Taxonomic and

Geographic Reference (3rd ed) Johns Hopkins University Press 2005 2 2142

57

39

Krause KH Schweiz Med Wochenschr 2000 130(4) 97-100

40 Irina Kareva Faina Berezovskaya Carlos Castillo-Chavez Journal of Biological

Dynamics 2010 4(4)315 - 327

41 Delaittre G Reynhout I Cornelissen J Nolte R Chemistry ndash A European Journal

2009 15 12600ndash12603

42 C Buller DH Johnson RC Tschumper Investigative Ophthalmology amp Visual Science

1990 31 2156-2163

43 Robert M Mazo Brownian Motion Fluctuations Dynamics and Applications Oxford

Science Publications 2009 57

44 Vivek K Mutalik KV Venkatesh Theoretical Biology and Medical Modelling 2005 2

19

45 Satoshi Nakamura Kunimasa Koga Biochemical and Biophysical Research

Communications 1977 78(2) 806-810

46 Krishnananda Chattopadhyay Shyamalava Mazumdar Biochemistry 2000 39 (1)

263ndash270

47 Satoshi Nakamura Yasuyuki Ogura J Biochem 1968 63 (3) 308-316

48 van Dongen SF Nallani M Cornelissen JJ Nolte RJ van Hest JC Chemistry 2009

15(5) 1107-1114

49 Ronald Bentley Methods in Enzymology 1955 1 340-345

50 Vartiainen J Raumlttouml M Paulussen S Packaging Technology and Science 2005

18 243ndash251

58

51 GA Sherbeny AA Shindia YMMM Sheriff International Journal of Agriculture

and Biology 2005 7

52 D Rando G-W Kohring F Giffhorn Appl Microbiol Biotechnol 1997 48

53 Worthington enzyme manual Worthington Biochemical Corporation

httpworthington-biochemcomgopdefaulthtml

54 httpwwwbrenda-enzymesorgphpresult_flatphp4ecno=11117

55 httpwwwoxygenphwatercom

56 P Boon Chock Sue Goo Rhee and Earl R Stadtman Annu Rev Biochem 1980 49

813-841

57 Sybil Golden P A Wals Joseph Katz Analytical Biochemistry 1977 77(2) 436-445

58 R L Baehner S K Murrmann J Davis R B Johnston Jr J Clin Invest 1975 56(3)

571ndash576

59 I Bejarano M P Terroacuten S D Paredes C Barriga A B Rodriacuteguez J A Pariente

Molecular and Cellular Biochemistry 2007 296(1-2) 77-84

60 Michael C Moulton Laura K Braydich-Stolle Mallikarjuna N Nadagouda Samantha

Kunzelman Saber M Hussain Rajender S Varma Nanoscale 2010 2 763-770

61 Goya DV Matijevic E Colloids and Surfaces A 1999 146 139

62 Velikov KP Zegers GE van Blaanderen A Langmuir 2003 19 1384

63 Goldenberg L M Wagner J Stumpe J Paulke B-R Goumlrnitz E Langmuir 2002 18

5627-5629

59

64

Banerjee S Gopal J Muraleedharan P Tyagi A K Raj B Curr Sci 2006

901378ndash1383

65 Bogomolov V N Kudinov E K Firsov Y A Sov Phys Solid State 1968 35 555

66 Kudo A Miseki Y Chem Soc Rev 2009 38 253ndash278

67 Gaj T Meyer SC Ghosh I Protein expression and Purification 2007 56 54-61

4

moves by opening and closing of its shell Inertial thrust is created when a scallop closes

its shell and the scallop moves forward due to the clapping motion (Figure 1 1) This

however does not hold true at the nanoscale since the inertial forces are relatively

insignificant due to the large viscous forces If a microscopic scallop were to exhibit a

similar clapping motion it will just keep moving forward and backward in an oscillatory

kind of motion and will not experience a net motion This is an example of reciprocal

motion which can linearly move a body at macroscale however fails to cause motion at

the microscale

For motion at microscale nonreciprocal motion is necessary8 Nonreciprocal motion

occurs when the path chosen for going from Position A to Position B is different from the

path chosen to come back from Position B to Position A It can be better understood with

the help of Figure 1 1 which shows the change of shapes for a theoretical object going

from Position A to Position B and then returning to Position A by using a different set of

motions This kind of change can lead to motion at the nanomicron scale

5

Another factor which gains importance at nanoscale is the dominance and ubiquity of

Brownian motion13

The trajectory of a linearly propelled nanomotor would be

significantly affected by the continuous collision of randomly moving particles in the

environment These collisions deviate the nanomotor from its linear path and hence this

effect needs to be considered while projecting the path of a nanomotor

Motion at the nanoscale can be powered by different types of mechanisms such as

diffusiophoretic electrophoretic osmophoretic chemophoretic thermophoretic and

others Different types of mechanisms of motions may coexist or act individually to cause

Position A Position B

Figure 1 1 Reciprocal and Nonreciprocal motion8

Reciprocal motion

Nonreciprocal motion

6

motion Diffusiophoretic motion of particles is caused due to concentration gradients of

solutes in an electrolytenonelectrolyte solution9 Electrophoresis is the motion of a

particle relative to a fluid under the influence of an electric field10

This occurs as the

particle carries an electric surface charge which is acted upon by an electrostatic

coulombic force of the external electric field Osmophoresis is the motion of a

vesiclefluid particle encapsulated in a semi-permeable membrane towards lower

concentration gradient when it is placed in a solution containing solute concentration

gradient1112

Thus considering the numerous mechanisms of motion known to work at

the nanoscale several suitable options are available for the development of artificial

nanomotors

12 Existing nanomotors Mechanisms and uses

Several nanomotors have been developed in the past decade or so A general principle of

working of these nanomotors is that they function by catalytically decomposing the

dissolved fuel to generate asymmetry at their surface or at their interfacial region thereby

causing propulsion13

The self-electrophoretic bimetallic nanowires developed by

WPaxton and A Sen etal consisted of a 2 μm long rod with one tip made up of Au and

the other tip made up of Pt1 These motors were synthesized by electrodeposition in an

alumina template The rods moved autonomously in a solution of hydrogen peroxide

with platinum end forward Pt oxidizes H2O2 to produce H+ and O2 and Au utilizes H

+ to

reduce H2O2 to water This generates an electron flow through the rod and a proton flow

outside the rod resulting in an electric field around the rod in the solution This electric

7

field in solution acts on the unbalanced charges within the nanorodrsquos double layer This

moves the nanorod with respect to the surrounding fuel due to the ion flux that is

generated

Bubble propulsion has been predominantly used as mechanism of motion in the motors

developed by Feringa etal His group developed a micromotor by attaching catalase to a

microparticle and placing the particle in a solution containing hydrogen peroxide This

system allowed the microparticle to move by producing oxygen bubbles from the

decomposition of hydrogen peroxide14

A similar motor was later created by Feringarsquos

group which consisted of glucose oxidase and catalase immobilized on a carbon

nanotube in a substrate solution of hydrogen peroxide Glucose oxidase converted

glucose to H2O2 which was then utilized by catalase to produce water and oxygen

thereby propelling the carbon nanotube15

Another interesting example of a self-propelled motor is the polymerization driven motor

developed by Cameron et al16

It consists of polystyrene beads immobilized with ActA

protein which catalyzes actin polymerization leading to the formation of an actin

filament which resembles the flagella of bacteria This actin polymerization propels the

beads at the speed of 001-015 μms

A theoretical model has been proposed by Nafaji and Golestanian etal which consists

of three spheres linked by rigid rods in one dimension17

The length of the rods can

switch between two specific values Due to the nonreciprocal motion of the rod this

8

model can swim in low Reynolds number systems An actual model based on

nonreciprocal motion has been developed by Kim etal in which heart cells have been

linked to an asymmetric polydimethylsiloxane robot As the heart cells beat the

asymmetric structure of the device resembles a walking movement18

Most of the nanomicromotors mentioned above are either metallic or work in

environments not compatible with human body Enzymes being an integral part of the

body can solve this problem by operating in suitable substrates already present in the

body fluids The nonreciprocal conformation changes of the enzyme as well as

diffusiophoresis and electrophoresis can conceivably be used to generate motion in case

of enzyme driven nanomotors such motors will be discussed in detail in the following

chapter

9

Chapter 2

Immobilized enzymes on Janus particles as catalytic motors

21 Introduction

Enzymes are widely found in biological systems and act on a large variety of substrates

This can make enzymes an ideal choice for powering nanomotors which need to be

biocompatible Several fluids found in the body can act as substrates to the enzymes For

example dissolved glucose which is abundantly found in the body19

is a susbtrate for

enzyme glucose oxidase Urea which is found in the urinary tract20

is a substrate for

urease Hydrogen peroxide produced by phagocytic cells in the body21

is a substrate for

horseradish peroxidase Having motors driven by enzymes can open possibilities of

targeted drug delivery to these parts of the body containing such substrates

Enzymes have already been used to generate motion of carbon nanotubes by mechanism

of bubble propulsion by Feringa etal15

Two other possible mechanisms which may be

able to power motion of nanoparticles using enzymes are asymmetrical decomposition of

substrates into products leading to self-diffusiophoresis (Figure 2 1) and nonreciprocal

changes in enzyme conformation leading to propulsion (Figure 2 2) As seen in Figure 2

1 enzymes which are immobilized on a Janus particle can breakdown the available

substrate in the solution into products causing asymmetric distribution of solutes in the

surrounding fluid This can lead to movement of particle towards the region of lower

10

solute concentration by principle of self-diffusiophoresis Figure 2-2 shows the possible

nonreciprocal changes in the conformation of a theoretical protein which can lead to

motion of the protein22

These nonreciprocal changes in conformation are triggered by the

attachment of substrate molecule and release of product molecules from the enzymersquos

active site

Figure 2 1 Enzyme functionalized Janus particle moves upon catalyzing asymmetrical

decomposition of substrate into product

11

Figure 2 2 Schematic showing idealized nonreciprocal conformation changes in a

theoretical enzyme22

Enhanced diffusion of a single enzyme molecule by catalysis in a substrate solution has

been demonstrated23

The diffusion coefficient of a single urease molecule was measured

using fluorescence correlation spectroscopy and it was observed that the diffusion of the

urease molecule increases by 16-28 by varying urea concentrations from 0001 M to 1

M Addition of enzyme inhibitors to the system curbed the enhanced diffusion This

enhanced diffusion has been attributed to the force generated by self-electrophoresis by

the enzyme in substrate solution

12

Based on the above facts it might be possible to propel a nanoparticle by asymmetric

distribution of enzymes on its surface thereby leading to self-propulsion of the particles

in substrate solution (Figure 2 1) This propulsion may be due to self-

electrophoresisself-diffusiophoresis andor due to nonreciprocal changes in enzyme

conformations Here we aim to investigate a system consisting of polystyrene particles

half coated with enzyme introduced in a suitable substrate solution The enzyme can be

directly linked to the polystyrene particle or it can be linked through a long spacer

molecule (linker) to provide more degrees of freedom to the enzyme

Several factors need to be considered for choosing enzyme for powering motion at the

nanoscale Some of the important factors are the ratio of kcatKm which denotes the

catalytic efficiency of the enzyme possible substrates stability of enzymessubstrates

ease of immobilization relationship with other enzymes in cascade reactions and

commercial availability of the enzymes The enzyme being used in the present study is

horseradish peroxidase It acts on a molecule of hydrogen peroxide and reduces it while

simultaneously oxidizing another substrate molecule In the absence of alternate

oxidizable substrate HRP can oxidize water and oxygen back to hydrogen peroxide24

thus giving an equilibrium reaction

13

211 Horseradish peroxidase

Figure 2 3 Horseradish peroxidase

Horseradish peroxidase belongs to the group of peroxidases which are a group of heme-

containing oxidoreductases that act on peroxide as hydrogen donor25

It is a redox

enzyme which contains a protoheme a complex of trivalent iron with 1358-

tetramethyl-24-divinylporphin-67-dipropionic acid as a prosthetic group It is a 44 kDa

protein which includes 33900 Da polypeptide chain bound with eight oligosaccharide

chains The carbohydrate chains improve the stability of the enzyme by protecting it

against radicals formed in the course of the reaction it catalyzes26

Horseradish

peroxidase is found in horseradish and is used widely in bioanalytical applications such

as ELISA and Western blots It decomposes hydrogen peroxide into water and oxygen

while simultaneously oxidizing another substrate27

The oxidizable substrates can be

chromogenic substrates such as 22rsquo55rsquo-tetramethyl-benzidine (TMB) and 33-

diaminobenzidine (DMB) or chemoluminescent substrates like SuperSignal and ECL

luminal28

14

Figure 2 4 Reaction catalyzed by horseradish peroxidase

The functional parameters for horseradish peroxidase include Km of 015 mM and kcat of

790 s-1

at an optimum temperature of 20 ordmC and an optimum pH of 6429

Some of the

inhibitors of HRP are 110-phenanthroline aminotriazole and bromine

22 Experimental

221 Materials and methods

Uniform carboxyl modified polystyrene microspheres 05 microm in diameter were obtained

from Polysciences Inc The size of the purchased microspheres was verified by Field

Emission Scanning Electron Microscopy (FESEM) Peroxidase Type I from

Horseradish N-(3-dimethyl-aminopropyl)-Nrsquo-ethylcarbodiimide (EDC) and hydrogen

peroxide solution 50 wt in water were purchased from Sigma-Aldrich N-

hydroxysuccinimide (NHS) ge 97 was purchased from Fluka All chemicals were of

reagent grade and used without further purification Nanopure deionized water was used

for all experiments and preparation of solutions All measurements were made at room

15

temperature (25 oC) unless mentioned otherwise The UV-Visible spectroscopy

measurements were performed using Agilent 8453A diode-array UV-Visible

Spectrophotometer with ChemStation UV-Vis Software Diffusion coefficient

measurements were performed using Nanosight LM10

222 Synthesis of Janus particles

A monolayer of particles was prepared according to the procedure (Figure 2 5) by

Goldenberg L et al30

Glass slides were placed on the bottom of a petridish Water was

put into the petridish so that the entire slide was immersed in water A thin layer of

hexane was added on top of water in the petridish A suspension of microspheres in

ethanol (5 ww) was introduced to the water-hexane interface After the monolayer was

formed the glass slide was lifted up through the monolayer with tweezers The

microsphere-covered glass slides were left to dry overnight at room temperature

Following drying a thin film of silica was evaporated on the glass slides in Semicore

evaporator The spheres were partially coated with 30 nm of silica Particles were

removed from the glass slide by gentle sonication The polystyrene-silica (PS-SiO2)

Janus particles thus obtained were washed in deionized water several times before

further immobilization steps were performed FESEM was used to confirm synthesis of

Janus particles

16

Figure 2 5 Preparation of PS-SiO2 Janus particles by evaporation

223 Immobilization of enzymes using EDCNHS coupling method

1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide and N-hydroxysulfosuccinimide

(EDCNHS) coupling procedure was followed for immobilizing horseradish peroxidase

type I on 05 μm PS-SiO2 Janus particles (Figure 2 6) In this process EDC reacts with a

carboxyl groups on the Janus particles to form an amine-reactive O-acylisourea

intermediate If this intermediate does not encounter an amine it can hydrolyze and

regenerate the carboxyl group In the presence of NHS EDC can be used to convert

carboxyl groups to amine-reactive sulfo-NHS esters which are much more stable and do

not readily hydrolyze to regenerate carboxyl groups In the current experiment

immobilization was performed in the presence as well as absence of a long spacer

17

molecule (linker) between Janus particles and enzymes The chemical 6-aminohexanoic

acid was used as the linker (Figure 2 7) HRP immobilized PS-SiO2 Janus particles were

stored in deionized water until required for further experiments

Figure 2 6 EDCNHS coupling method for immobilization of enzyme on carboxylated

polystyrene particles

Figure 2 7 Immobilization of enzyme on Janus particles with and without 6-

aminohexanoic acid linker

18

224 Enzyme assay

Assay for activity of HRP was performed using a spectrophotometric method which

involved using 4-aminoantipyrene as hydrogen donor3132

The reaction rate was

determined by measuring increase in absorbance at 510 nm resulting from the

decomposition of hydrogen peroxide One unit represents the decomposition of 1 μM of

hydrogen peroxide per minute at 25 degC and pH 70 under the specified conditions

The spectrophotometer was set to the wavelength of 510 nm and temperature of 25 degC

Phenol4-aminoantipyrene solution (14 mL) and 00017 M H2O2 solution (15 mL) were

pipetted into each cuvette The cuvette was incubated for 3-4 mins at 25degC and blank was

established A 01 mL solution of diluted enzyme was added to the above mixture and

absorbance was recorded for 4-5 mins Standard graphs of absorbance versus time were

drawn for known concentration of the free enzyme and compared to that of the

immobilized enzyme on the Janus particles The estimate of the amount of enzyme

present on the Janus particles was deduced from the difference between the quantity of

the free enzyme previously added for immobilization and the amount that was measured

to be in the supernatant after some of the enzyme became attached to the Janus particles

The experiment was carried out in triplicate to assess reproducibility

19

The formula used to calculate the activity of enzyme is

where ∆Amin denotes the change in absorbance per minute

225 Diffusion coefficient measurements using Nanosight LM10

Diffusion coefficients for HRP immobilized particles were measured using Nanosight

LM 10 nanoparticle tracking instrument with Nanoparticle Tracking Analysis (NTA) 20

software in the presence of 003 H2O2 solution as the substrate and also in the presence

of only deionized water The videos were recorded for 30 seconds at the rate of 30 frames

per second Each experiment was carried out in triplicate to assess reproducibility

Particles

visualization

zone

Microscope with

LM 10 unit

LM 10 unit

Figure 2 8 Nanosight LM 10 instrumentation

20

Nanosight LM 10 (Figure 2 8) generates videos of population of moving nanoparticles in

a liquid when illuminated by laser light The particles which pass through the laser beam

path are seen by the instrument as small points of light undergoing Brownian motion

(Figure 2 9a)

The videos are further analyzed by NTA (Nanoparticle tracking analysis) software

(Figure 2 9b) which identifies and tracks each particle independently on a frame-by-

frame basis The software also calculates and subtracts any bulk flow in the system The

mean square displacement is calculated for each particle as long as it is visible If the

particle blinks that is if it disappears momentarily and then reappears on the screen the

software will consider the same particle as two separate ones All analysis data is

exported to the excel files in terms of diffusion coefficients of the tracked particles

B A

Figure 2 9 NTA video analysis (a) Particles as they are seen on the screen during

experimentation in Nanosight LM10 (b) NTA screen as the particles are being analyzed for

their diffusion coefficients

21

23 Results

231 Enzyme assay

The activity of free HRP enzyme reported by Sigma Aldrich is 150 unitsmg However

the activity of HRP upon analysis was found to be 52158 unitsmg which is much lower

than the reported value The activity of HRP was found to be further lowered to 2133

unitsmg when it was immobilized on the PS-SiO2 Janus particles in the presence of a

linker The following formula is used to calculate enzyme activity

where ΔAmin is the change in absorbance per minute

22

Figure 2 10 Graph for the assay of solution of free enzyme containing 05mgL of HRP

ΔAmin = 01716 Unitsmg of free enzyme = 52158

During EDCNHS coupling 04 mg enzyme was added to 1 ml of 025 vv suspension

of Janus particles Thus initial concentration of enzyme in the mixture was 04 mgml

Assuming retained activity of the enzyme in supernatant solution the amount of enzyme

in supernatant was calculated to be 03868 mgml from the assay Amount of enzyme

functionalized on the Janus particles was calculated as the difference between enzyme

previously added for immobilization (04 mgml) and the enzyme present in supernatant

(03868 mgml) and was found to be 00132 mgml Enzyme assay was performed on the

enzyme immobilized Janus particles to estimate the activity of immobilized enzyme

(Figure 2 11) and was found to be 2133 unitsmg

y = 0002x + 00516

0

01

02

03

04

05

06

07

08

0 50 100 150 200 250 300 350 400

Ab

sorb

an

ce

51

0 n

m

Time (sec)

Assay of free enzyme HRP

23

Figure 2 11 Graph for the assay of of immobilized enzyme on 05 μm Janus particles

(~025 vv) ΔAmin = 01853 Unitsmg of free enzyme = 2133

232 Diffusion coefficient measurements

No significant increase was observed in diffusion coefficient of HRP-immobilized Janus

particles in the presence of H2O2 as the substrate Similar results were obtained for

diffusion coefficient measurement with and without linker moieties bridging the enzymes

and particles(Table 2 1 Table 2 2) Thus the linker did not seem to assist in increasing

the particle diffusion by improving or retaining the activity of the enzyme

y = 00015x + 00953

0

01

02

03

04

05

06

07

0 50 100 150 200 250 300 350 400

Ab

sorb

an

ce

510 n

m

Time (sec)

Assay of HRP immobilized 05 μm Janus particles

24

Table 2 1 Diffusion coefficient measurements of HRP immobilized Janus particles

without linker

HRP immobilized

Janus particles in

003 H2O2

Control - Janus

particles in

003 H2O2

Control - HRP

immobilized Janus

particles in DI water

Diffusion coefficient

(10-10

cm2s)

1359416 1051777 1308675

Standard deviation 4534801 4122075 4918597

Table 2 2 Diffusion coefficient measurements of HRP immobilized Janus particles in

the presence of linker

HRP immobilized

Janus particles in

003 H2O2

Control - Janus

particles in

003 H2O2

Control - HRP

immobilized Janus

particles in DI water

Diffusion coefficient

(10-10

cm2s)

13838 1339829 1407891

Standard deviation 5644692 5649364 5797218

25

Figure 2 12 Diffusion coefficient measurements of PS-SiO2 Janus particles without and

with 6-aminohexanoic acid linker

24 Discussion

To compare the diffusion coefficients observed during the experiments theoretical

diffusion coefficient was calculated using following equation33

r

kTD altraslation

6 Equation 21

where r is the hydrodynamic radius of particle Dtranslational is translational diffusion

coefficient of the particle T is temperature of the system η is the viscosity of fluid

through which the particles are moving and k is the Boltzmann constant From FE-SEM

images the size of the particles used was determined to be 450 nm For 450 nm diameter

0

20

40

60

80

100

120

140

160

Without linker With linker

Dif

fusio

n c

oeff

icie

nt

(10

-10 c

m2s

)

HRP immobilized 05 μm Janus particles - Diffusion coefficient measurements

HRP immobilized Janus particles in 003 H2O2

Control - Janus particles in 003 H2O2

Control - HRP immobilized Janus particles in DI water

26

particle Dtranslational is equal to 109 μm2s which is comparable to the diffusion

coefficient measured for the enzyme immobilized particles

HRP was found to be active on the Janus particles however the activity of the enzyme

was reduced upon immobilization Lower enzyme activity can be due to the coupling

method used which in this case was EDCNHS covalent binding However EDCNHS

coupling method has been shown to significantly retain the enzyme activity when the

enzyme is immobilized on carboxylated multi-walled carbon nanotubes under appropriate

conditions34

Hence EDCNHS binding method by itself is not believed to be

responsible for reduction of enzyme activity upon immobilization

Direct binding of enzyme to the Janus particle may lead to reduction in the enzyme

activity because of space constraints for the enzyme and difficulty for the passage of

substrate to the active site of the enzyme due to stearic hindrance Hence a linker was

inserted between the enzyme and Janus particle during enzyme immobilization However

inserting a linker between the enzyme and the particle did not lead to enhanced diffusion

of Janus particles or increase in enzyme activity of immobilized enzyme

Lack of enhanced diffusion can be firstly attributed to the reduction in enzyme activity

upon immobilization Enzyme activity may be retained by modifying the conditions

during EDCNHS coupling or changing the binding method altogether The degree of

immobilization of HRP on carboxylated Janus particles can possibly be affected by

several factors such as temperature the concentration of the coupling reagent EDC

27

concentration HRP concentration in solution and incubation time34

Different

combinations of these factors can be tested to find optimum conditions for

immobilization of HRP Alternate binding method can involve thiolation of HRP by the

covalent conjugation of mercaptopropionic acid and then the thiolated HRP can then be

attached on the gold surface of a polystyrene-gold Janus particle35

Thus further

modifications can be made to the system in order to retain enzyme activity on the

immobilized particles and thereby power nanomotors using enzymes

28

Chapter 3

Predator-prey system of enzyme immobilized particles

31 Introduction

Predator-prey systems are widely found in nature and play a major role in biological

processes3637

Predator-prey systems include all the interactions in which one organism

pursues andor consumes another organism One familiar predator-prey system at the

macroscale is that of a tiger chasing and consuming a deer Another not so familiar one

includes Baleen whales which eat millions of microscopic planktons at once38

At the

microscale such kind of interactions can be observed in the case of phagocytes in the

body which are capable of engulfing foreign microorganisms and killing them with a

combination of microbicidal systems39

In the case of cancer the tumor is the prey and

the mature myeloid cells in the body are the predator However the tumor here has a

defense mechanism against the predator in which the tumor produces cytokines which

inhibits the maturation of the myeloid cells and thereby prevents predation40

One similarity that can be observed in all these systems is that the prey possesses some

attribute which is desired by the predator which makes the predator chase the prey A

similar relationship is seen in case of enzymes which are involved in cascade reactions41

In cascade reactions product synthesized by one enzyme is the reactant required by

another enzyme to carry out its own catalytic reaction As such there is a predator-prey

29

kind of dependence between the two enzymes which may cause one enzyme to chase the

other enzyme when placed in suitable substrate solutions However the mechanism

hypothesized for such a system is totally different than the mechanism involved in

biological systems In biological systems such as bacterial chemotaxis or phagocytes

chasing foreign particles in human body complex biochemical reactions are responsible

for the predator-prey behavior42

In the proposed enzyme cascade system there are no

complex biochemical reactions involved The principle hypothesized for predator-prey

behavior of enzyme cascade reactions can be explained as follows Motion of synthetic

motors at nanoscale involves two components translational diffusion and rotational

Brownian diffusion At very short time scales of less than 1Dr where Dr (s-1

) is

rotational Brownian diffusion coefficient the motion of particles is unidirectional For

longer time scales of greater than 1Dr the direction of motion is random giving

poweredactive diffusion43

If the chemical homogeneity is broken that is when there is

higher concentration of a substrate present in one direction of the enzyme the enzyme

might acquire a subtle bias which may lead to a spatial aggregation of the enzymes based

on the substrate concentration gradient The bias can be a result of the relationship

between the speed of enzyme diffusion and substrate concentration where higher

substrate concentration lead to higher enzyme diffusion As a result in an enzyme

cascade system one enzyme will migrate towards higher concentration of substrate

which is present near the other enzyme thus giving rise to a predator-prey behavior

Such enzyme cascades are widely found in signaling networks and metabolic pathways in

living cells and can be used to produce predator-prey like nanomotors systems44

The

30

enzymes being used in the present study are horseradish peroxidase and glucose oxidase

Both enzymes are extremely stable4546

Glucose is the substrate for glucose oxidase

which is widely available and highly stable47

Horseradish peroxidase acts on hydrogen

peroxide It is involved in a cascade reaction with glucose oxidase which produces

hydrogen peroxide by converting glucose into gluconic acid (Figure 3 1)48

Figure 3 1 Concept of particle aggregation due to cascade reactions of immobilized

enzymes

In this experiment glucose oxidase (GOx) and horseradish peroxidase (HRP) were

immobilized on polystyrene carboxylate beads of different sizes using EDCNHS

coupling method Glucose and 3355-Tetramethylbenzidine (TMB) were dissolved

together in water and this solution was used as the substrate solution All the components

were present in a single system in DI water Aggregation of particles and precipitation of

oxidized TMB from the solution was expected to be observed when all these components

were present in such a system

Properties of horseradish peroxidase have been discussed in the previous chapter (Section

211 Horseradish peroxidase)

GOxGOx HRPHRP

Glucose + O2Gluconic acid + H2O2

TMB TMB oxidizedBlue precipitate

H2O

Aggregation

31

311 Glucose oxidase

Figure 3 2 Glucose oxidase

Glucose oxidase (GOx) belongs to the group of oxidoreductases and is also called

glucose aerodehydrogenase49

It is obtained from fungi and has been identified as an

antimicrobial enzyme50

GOx binds to β-D-glucopyranose and catalyzes the oxidation

reaction to from gluconolactone while simultaneously reducing oxygen to hydrogen

peroxide51

GOx has high substrate affinity and specificity high stability and high

turnover number which makes it a suitable biocatalyst for industrial applications52

Figure 3 3 Redox reaction of glucose catalyzed by glucose oxidase53

32

GOx is a glycoprotein with molecular weight of 155 kDa and consists of two identical

polypeptide subunits linked together by a disulfide bond The optimum pH for GOx is

55 though it is stable in a broad pH range of 4 to 7 and can be stored for a long period of

time51

The functional parameters for glucose oxidase include Km of 151 mM and kcat of

337 s-1

at temperature of 20 ordmC and pH of 754

Some of the inhibitors of GOx are 2-

Deoxy-D-glucose adenine nucleotides and NaNO3

32 Experimental

321 Materials and methods

Uniform carboxyl modified polystyrene microspheres 05 microm in diameter were obtained

from Polysciences Inc The size of the purchased microspheres was verified by FESEM

Peroxidase Type I from horseradish glucose oxidase Type II-S from Aspergillus niger

N-(3-dimethyl-aminopropyl)-Nrsquo-ethylcarbodiimide (EDC) and Hydrogen peroxide

solution 50 wt in water were purchased from Sigma-Aldrich N-hydroxysuccinimide

(NHS) was purchased from Fluka D-Glucose (99) was purchased from VWR

International Secure-Seal imaging spacers (9mm diameter) for glass slides were obtained

from Sigma Aldrich All chemicals were of reagent grade and used without further

purification Nanopure deionized water was used for all experiments and preparation of

solutions All measurements were made at room temperature (25 oC) unless mentioned

otherwise The UV-Visible measurements were performed using Agilent 8453A diode-

array UV-Visible Spectrophotometer with ChemStation UV-Vis Software Optical

33

microscopy experiments were performed using Axiovert 200 MAT microscope by Carl

Zeiss Diffusion coefficient measurements were obtained using Nanosight LM10

322 Optical microscopy experiments

The enzyme substrate system was observed under optical microscope to check for

aggregation and precipitation due to cascade reactions Several such systems were

studied Each system in general consisted of GOx and HRP immobilized on different

polystyrene particles The substrate solutions consisted of 01 M glucose and different

concentrations of TMB solution The systems were studied in the presence of all the

constituents and in the absence of glucose GOxHRP and TMB Systems consisting of

different sized HRP immobilized particles in substrate solution of 003 hydrogen

peroxide and TMB were also studied Videos were recorded over a period of 24 hours to

observe changes in the system Each experiment was performed atleast thrice to assess

reproducibility of results

323 Diffusion coefficient measurements using Nanosight LM10

Diffusion coefficient of GOx and HRP immobilized particles were measured using

Nanosight LM10 nanoparticle tracking instrument with Nanoparticle Tracking Analysis

(NTA) 20 software Diffusion coefficients were measured for different systems namely

GOx particles in 01 M glucose and oxygen both GOx and HRP functionalized particles

in 01 M glucose and oxygen along with controls for the individual systems Videos were

34

recorded for 30 seconds at the rate of 30 frames per second Each experiment was carried

out in triplicate to assess reproducibility

33 Results

331 Optical microscopy

According to the enzyme cascade model precipitation of TMB was possible only upon

the interaction of GOx and HRP immobilized particles in presence of appropriate

substrate solution Hence in the first set of experiments precipitation of TMB was used

as an indication of the type of interaction between GOx and HRP immobilized particles

Fan-shaped structures were observed when both HRP and GOx immobilized particles

were present in a substrate solution consisting of glucose and TMB These structures

started forming as a single strand and then branched out and intertwined to form the fan-

shaped structures (Figure 3 4)

Table 3 1 Observations for formation of fan-shaped structures in enzyme cascade

system in presence of different components

GOx HRP Glucose TMB Observation

Yes Yes Yes Yes

Fan-shaped structures observed particles associated with

the fan-shaped structures

No Yes Yes Yes No fan-shaped structures

Yes No Yes Yes No fan-shaped structures

Yes Yes No Yes No fan-shaped structures

Yes Yes Yes No No fan-shaped structures

35

Fan-shaped structures can also be observed in the system consisting of only HRP

immobilized particles in substrate solution of hydrogen peroxide and TMB The size of

the fan-shaped structures depends on the size of the enzyme immobilized particles Free

enzymes show extremely tiny structures which are difficult to view under the optical

microscope

Figure 3 4 Formation of fan-shaped structures when both HRP and GOx

immobilized particles were present in a substrate solution consisting of glucose and

TMB A In the initial stages single strands form on the surface of liquid (5Ox) B

Single strands branch out and intertwine to form fan-shaped structures (50x) C Many

such structures form and settle down after 20 minutes (20x)

A

C

B

36

Experiments were performed in the absence of TMB that is the system consisted of GOx

and HRP immobilized particles in glucose substrate solution In the absence of TMB the

particles were expected to move closer to each other without any precipitation due to the

cascade reaction However there was no conclusive aggregation of particles observed in

the system

Free Enzyme 005 μm HRP - immobilized particle

10 μm HRP - immobilized particle 05 μm HRP - immobilized particle

Figure 3 5 Size dependence of fan-shaped structures on the size of HRP -

immobilized particles (20x)

37

Table 3 2 Observations for particle aggregation in enzyme cascade system in presence

and absence of TMB

Glucose GOx HRP TMB Observation

Present Present Present Present Aggregation and precipitate

Present Present Present Absent No conclusive aggregation

Present Absent Present Present No aggregation or ppt

Absent Present Present Present No aggregation or ppt

Present Present Absent Present No aggregation or ppt

332 Diffusion coefficient measurements

Diffusion coefficients were measured for GOx immobilized particles of 05 μm diameter

in substrate solution of 01 M glucose and oxygen Diffusion coefficients of particles

were also measured for system consisting of GOx and HRP functionalized particles in

glucose and oxygen Each experiment was done in triplicate to assess reproducibility

Nanosight visualization chamber being a closed chamber oxygen was purged into all the

solutions before starting the experiments to ensure sufficient supply of oxygen for

catalytic reaction of GOx

38

Table 3 3 Diffusion coefficients for 05 μm GOx immobilized particles in 01 M

glucose purged with O2

Mean

GOx

particles in

01 M

glucose

Control - GOx

particles in DI

water

Control - GOx

particles in 01 M

glucose without

oxygen purging

Control -

particles in 01

M Glucose

Diffusion

coefficient

(x10-10

cm2s)

13316 12888 13827 13702

Standard

deviation 6307 4926 4424 7468

Table 3 4 Diffusion coefficients for GOx and HRP immobilized particles in 01 M

glucose and purged with O2

Mean

GOx- HRP-

functionalized

particles in 01 M

glucose

Control ndash Gox-

HRP- functionalized

particles in DI water

Control - particles

in 01 M glucose

Diffusion coefficient

(x10-10

cm2s)

13005 14434 13702

Standard deviation 5263 6622 7468

No significant increase was observed in diffusion coefficient of enzyme immobilized

particles in any of the systems

39

34 Discussion

Formation of fan-shaped structures due to patterned deposition of oxidized TMB

indicates some kind of interaction between GOx and HRP immobilized particles Particle

diameter dependence of the size of structures can be explained with a simple model in

Figure 3 6 A larger particle will have a higher surface area as compared to a smaller

particle Hence more enzyme is immobilized on the larger particle As a result higher

quantity of TMB is oxidized and precipitated by the larger particle as compared to the

smaller particle This leads to thicker strands of fan-shaped structures eventually scaling

up the whole system to give bigger fan-shaped structures

The exact reason for the formation of such fan-shaped structures which are dependent on

particle size could not be determined It is hypothesized that hydrogen peroxide andor

Figure 3 6 Model for size dependence of fan-shaped structures on the particle

diameter of enzyme immobilized particles Larger the particle more surface area is

available for precipitation of TMB which eventually leads to bigger fan-shaped

structures

Larger particle Smaller particle

40

TMB gradient in the substrate solution might be responsible for such structures If there

is higher concentration of H2O2 on one side of the particle and assuming an even

concentration distribution of TMB there will be selective precipitation in the direction of

H2O2 When several particles come in the vicinity of higher concentration of H2O2 the

precipitation lines will converge to give fan-shaped structures (Figure 3 7)

Figure 3 7 Hypothesis for formation of fan-shaped structures

In the system consisting of GOx immobilized particles HRP immobilized particles

glucose and TMB it was observed that the fan-shaped structures formed at the liquid-air

interface and eventually settled down to the bottom of the glass slide This makes sense

as oxygen is necessary for the catalytic reaction of conversion of glucose to gluconic acid

and hydrogen peroxide which takes place on the surface of GOx immobilized particle As

a result H2O2 is primarily formed at the liquid-air interface and is then used by HRP for

precipitation of TMB

Following the observations that precipitation of TMB occurs at the surface GOx and

HRP immobilized particles are also expected to interact at the surface in the absence of

TMB Due the cascade reaction it was expected that HRP immobilized particles will be

41

attracted to GOx immobilized particles in appropriate substrate solution Experiments

were carried out to visualize inter-particle interactions at the liquid-air interface

However liquid-air interface not being a stable system to image the precise interaction

of GOx and HRP immobilized particles could not be determined Significant external

disturbances were involved at the interface To enable the cascade reaction in the bulk of

the liquid oxygen was purged into the reaction system before starting the experiment

However this did not lead to significant reaction in the bulk of the liquid and the reaction

prominently took place at the liquid-air interface where the oxygen concentration was

higher

In order to observe the interaction of GOx and HRP immobilized particles better imaging

techniques need to be used to observe diffusion of particles in absence of external

disturbances at the liquid-air interface Large quantity of oxygen needs to be purged into

the system and retained to make the reaction more pronounced in the bulk of the substrate

solution Higher concentration of oxygen in water may also be achieved by using oxygen

enriched water which is commercially available55

Alternatively a different enzyme cascade system may be used which does not require a

gaseous substrate56

For example glycogen synthase synthesizes glycogen from glucose-

1-phosphate and glycogen phosphorylase breaks down glycogen to glucose-1-

phosphate57

This system does not require a gaseous substrate and might be able to

overcome the deficiencies of GOx and HRP enzyme cascade system

42

Chapter 4

Silver-titania nanomotors

41 Introduction

Bimetallic nanowires made of platinum and gold have been developed by WPaxton

etal1 The rods move autonomously in a solution of hydrogen peroxide In the present

study silver-titania (Ag-TiO2) particles are being synthesized and investigated for motion

in presence of hydrogen peroxide and UV light With proper control of conditions and

UV light trigger it can be possible to control the speed and direction of motion of these

nanoparticles

Hydrogen peroxide is produced in the human body by phagocytes as an immune response

to foreign particles and pathogens5859

Also silver nanoparticles have been shown to be

biocompatible and capable of interacting with cells in human body without disrupting

cellular function60

Similarly it has been demonstrated that titania is compatible with

different cells DNA and proteins in human body and does not cause toxic effects Hence

silver-titania nanomotors which swim in hydrogen peroxide may open interesting

avenues in applications like targeted drug delivery in those parts of the body where

hydrogen peroxide is present

43

42 Experimental

421 Materials and methods

Silver nitrate (999995) was purchased from Alfa Aesar Gum arabic was purchased

from MP Biomedicals Hydrogen peroxide solution 50 wt in water and ascorbic acid

were purchased from Sigma-Aldrich Nanopure deionized water was used for all

experiments and preparation of all solutions Secure-Seal imaging spacers (9 mm

diameter) were obtained from Sigma Aldrich All experiments were carried out at room

temperature of 25ordmC unless otherwise mentioned Ag-TiO2 particles were synthesized by

evaporation of titania in Semicore electron beam evaporator Particles were characterized

using field-emission scanning electron microscopy Optical microscopy experiments

were performed using Axiovert 200 MAT microscope by Carl Zeiss Particles were

tracked using Physvis particle tracking software (Figure 4 1) in which the position

information of a particle on screen is stored on a frame by frame basis The data is stored

in a dat file as X and Y co-ordinates of particles tracked on the screen This information

can be converted to diffusion coefficients of the particles by further manipulation using

MathWorks-MATLABreg software

44

Figure 4 1 Physvis particle tracking software

422 Synthesis of silver colloidal particles

Silver particles were synthesized by a nucleationgrowth type reaction developed by

Goya and Matijevic61

and modified by Velikov62

Silver nitrate was used as precursor

metal salt for synthesis of silver particles Solution A was 75 ml of 50 mM AgNO3 and

035 by weight gum arabic Solution B was 75 mL of 100 mM 1-ascorbic acid and

035 by weight gum arabic Solution B was rapidly added to solution A in a 250 mL

reaction flask The reaction flask was covered and particles were stirred for 48 hrs After

48 hours particles were allowed to settle for 2-3 hours in refrigerator and supernatant

was separated from the particles Particles were then rinsed with DI water and centrifuged

thrice to obtain pure silver particles Zeta potential of these particles was reported to be

45

around -50 to -60 mV and the size was around 1μm The size of the particles can be

varied by varying the amounts of ascorbic acid and silver nitrate Using molar ratio of 31

of ascorbic acid silver nitrate can give particles of 200 nm diameter whereas ratio of

151 can give particles of upto 5 μm diameter

423 Preparation of silver-titania (Ag-TiO2) particles

A monolayer of silver particles was prepared according to the procedure by Goldenberg

et al63

Glass slides were placed on the bottom of a petridish Water was put into the

petridish so that the entire slide was immersed in water A thin layer of hexane was then

added on top of water in the petridish A suspension of silver particles in ethanol (5

ww) was slowly introduced to the water-hexane interface in the dish After the

monolayer was formed the glass slide was lifted up through the monolayer with tweezers

The silver particles-covered glass slides were left at room temperature to dry overnight

Following drying a thin film of titania was evaporated on the glass slides in Semicore

evaporator (Figure 2 7) The spheres were partially coated with a 10 nm film of titania

Ag-TiO2 particles were removed from the glass slide by gentle sonication FE-SEM was

used to characterize the particles (Figure 4 2) The thin titania layer was not easily

distinguishable due to roughness of the particles

46

Figure 4 2 FESEM image of Ag-TiO2 particles

424 Optical microscopy experiments

Imaging spacers were placed on a glass slide Suspension of Ag-TiO2 particles was

placed on the glass slide and the particles were allowed to settle Movement of particles

was observed in DI water and videos were recorded at 50x magnification of optical

microscope for 30 sec 05 H2O2 was added to the particle suspension on the slide

Videos were recorded at 50x ranging from 30 sec to 10 min to study the movement of

silver titania particles in hydrogen peroxide solution Particles in both DI water and

hydrogen peroxide were subjected to UV light and videos were recorded to study particle

dynamics in presence of UV radiation Particle tracking was performed using Physvis

particle tracking software Atleast 30 particles in every system were tracked for 10

seconds each Particle tracking data was obtained in terms of X and Y coordinates of

particles on screen This data was manipulated using MathWorks-Matlab software and

graphs of mean square displacement of particles versus time were plotted Diffusion

coefficients of particles were calculated from these graphs

47

43 Results and Discussion

Ag-TiO2 particles demonstrated autonomous motion in the presence of H2O2 No UV

light was required for this motion Motion of the particles was accelerated by addition of

fresh H2O2 to the system When subjected to UV radiation the particles displayed

momentary acceleration for 4-5 seconds

Figure 4 3 Mechanism of motion of Ag-TiO2 nanomotors

In the presence of H2O2 silver part of the Ag-TiO2 particle converts H2O2 to H2O and O2

This may lead to propulsion of the Ag-TiO2 particle due to bubble propulsion andor self-

diffusiophoresis In the presence of UV light additional reaction takes place on the

titania part of Ag-TiO2 particle which increases the velocity of the Ag-TiO2 particles

(Figure 4 4) This UV triggered photoactivity of titania is due to hole-electron separation

caused by photons having energy equal to or greater than the band gap of titania646566

Most of the holes-electrons recombine but a small fraction move to the surface and react

with the available redox species like water and oxygen to form protons (H+) superoxide

(O2-) and hydroxyl (OH) radicals

6566 This may lead to propulsion by diffusiophoresis

as well as electrophoresis

48

Figure 4 4 Silver-titania motors move faster when subjected to UV radiation

In addition to linear motion rotational motion is also observed in several particles and

particle aggregates Aggregation of particles can lead to a net circular propulsive force

which can rotate the aggregate as shown in Figure 4 5

Figure 4 5 Rotation due to particle aggregation of Ag-TiO2 particles

Particle tracking by Physvis software revealed a significant increase in diffusion

coefficient of Ag-TiO2 particles in hydrogen peroxide solution as compared to DI water

49

The Ag-TiO2 particles have a diffusion coefficient of 1491 μm2s in hydrogen peroxide

versus only 180 μm2s in DI water Thus the Ag-TiO2 particles move around more than

10 times their body length per second in hydrogen peroxide (Figure 4 6) When

subjected to UV light the particles experience a significant acceleration in hydrogen

peroxide which lasts only for a short duration of time (4-5 sec) after which the particles

return to their original velocity The diffusion coefficient of the particles during this UV

light trigger is found to be around 5093 μm2s UV light trigger does not have any visible

effect on the Ag-TiO2 particles in DI water

Table 4 1 Translational diffusion coefficients of Ag-TiO2 particles in hydrogen

peroxide and water

Mean 05 H2O2 solution DI water

Diffusion coefficient of Ag-TiO2 (μm2s)

without UV light

1491 180

Diffusion coefficient of Ag-TiO2 (μm2s)

in the presence of UV light

5093 113

50

Figure 4 6 Mean square displacement curves of Ag-TiO2 Janus particles in hydrogen

peroxide and DI water

When higher concentration (~30 vv) of Ag-TiO2 particles in hydrogen peroxide is

subjected to UV radiation the particles exhibit pattern formation (Figure 4 7)

Aggregation of Ag-TiO2 Janus particles is observed when UV light is turned on and the

particles disaggregated when UV light is switched off This can be due to predator-prey

kind of interaction between silver and titania half of the Janus particles in the presence of

UV light Oxygen and water that are produced at the silver end by decomposition of

hydrogen peroxide are used up by titania when subjected to UV radiation This can lead

to attraction between titania and silver parts of neighboring Janus particles causing

aggregation

y = 20373x

y = 59994x

y = 71848x

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10 12

Mean

sq

uare

dis

pla

cem

en

t (μ

m2s

)

Time (sec)

Mean square displacement vs Time for Ag-TiO2

particles

05 Hydrogen peroxide and UV05 Hydrogen peroxide

DI Water

51

A UV OFF B UV ON

C UV OFF

Figure 4 7 Higher concentrations of Ag-TiO2 particles showing aggregation when UV

is turned on

52

Chapter 5

Summary and Conclusion

In summary no significant enhancement in diffusion was observed in case of enzyme

immobilized Janus particles in substrate solution Presence of a linker between enzyme

and particle did not increase the diffusion coefficient of particles Alternative enzyme

immobilization methods need to be tested like thiolation of enzyme and subsequent

binding to gold surface of a Janus particle35

or expressing the enzyme with polyhistidine

or avidin tag67

This can result in higher enzyme activity upon immobilization and may

lead to enhanced diffusion of Janus particles

Predator-prey system of GOx and HRP immobilized particles showed some promising

results Aggregation of particles and formation of fan-shaped structures by TMB

oxidation were observed under optical microscope when GOx and HRP immobilized

particles glucose and TMB were present in the system The size of the fan-shaped

structures was dependent on the size of enzyme immobilized particles This can be

indicative of some kind of interaction between the GOx and HRP immobilized particles

No conclusive aggregation of GOx and HRP immobilized particles was observed in

absence of TMB Better imaging techniques are required to study the interaction of GOx

and HRP immobilized particles at the unstable liquid-air interface where majority of the

reaction takes place Other enzyme cascade systems not requiring gaseous reactants like

glycogen synthase and glycogen phosphorylase should be tested for similar activity

53

Ag-TiO2 particles were found to exhibit autonomous motion in the presence of hydrogen

peroxide solution The motion was momentarily accelerated upon exposure to UV

radiation The particles showed a diffusion coefficient of 1491 μm2s in hydrogen

peroxide versus only 180 μm2s in DI water Upon subjecting particles to UV radiation in

presence of H2O2 diffusion coefficient increased to 5093 μm2s for a period of 4-5

seconds In addition to linear motion rotating motion could also be observed in some

particle aggregates Further development of these aggregate structures can lead to

formation of rotors at nanoscale Higher concentration of silver-titania particles in

hydrogen peroxide displayed aggregation in presence of UV light and disaggregated

when UV light was switched off This may be attributed to the predator-prey kind of

interaction between silver and titania where oxygen and water that are produced by

silver by decomposition of hydrogen peroxide are used up by titania when subjected to

UV radiation This promises to be an interesting system and needs to be further

investigated to explore its properties

54

References

1 Paxton WF Sundararajan S Mallouk TE Sen A Angew Chem Int Ed Engl

2006 45 5420-5429

2 Golestanian R Liverpool TB Adjari A Phys Rev Lett 2005 94 220801

3 httpwwwsilvermedicineorgh2o2html

4 Rami Pedahzur Ovadia Lev Badri Fattal Hillel Shuval Water Science and Technology

1995 31(5-6) 123-129

5 W Went F American Scientist 1968 56

6 Adriano Cavalcanti Tad Hogg Bijan Shirinzadeh IEEE MHS 2006 International

Symposium on Micro-NanoMechatronics and Human Science 2006 226-232

7 EM Purcell American Journal of Physics 1977 45 3-11

8 Golestanian R Phys Rev Lett 2010 105 018103(1)-018103(4)

9 S S Dukhin Z R Ulberg G L Dvornichenko B V Deryagin Russian Chemical

Bulletin 1983 31(8) 1535-1544

10 Dukhin AS Goetz PJ Ultrasound for characterizing colloids Particle sizing zeta

potential rheology Elsevier 2002 59

11 HJKeh HJTu Trends in Colloid and Interface Science XIV Progress in Colloid and

Polymer Science 2000 115 174-180

12 D Zinemanas A Nir International Journal of Multiphase Flow 1995 21(5) 787-800

55

13 Stephen Ebbens Jonathan Howse Soft matter 2010 6 726-739

14 J Vicario R Eelkema W R Browne A Meetsma R M La Crois B L Feringa

Chem Commun 2005 31 3936

15 Davide Pantarotto Wesley R Browne Ben L Feringa Chem Commun 2008 1533 -

1535

16 Lisa A Cameron Matthew J Footer Alexander van Oudenaarden Julie A Theriot

PNAS 1999 96(9) 4908-4913

17 Ali Najafi Ramin Golestanian Phys Rev E 2004 69 062901

18 Jinseok Kim Jungyul Park Sungwook Yang Jeongeun Baek Byungkyu Kim Sang

Ho Lee Eui-Sung Yoon Kukjin Chun Sukho Park Lab Chip 2007 7 1504 - 1508

19 Kurt M Dubowski Clinical Chemistry 2008 5411 1919ndash1920

20 Spector DA Yang Q Wade JB Am J Physiol Renal Physiol 2007 292(1) 467-474

21 Rhee SG Chang TS Bae YS Lee SR Kang SW J Am Soc Nephrol 2003 14(8-3)

211-215

22 Sakaue T Kapral R Mikhailov A S Eur Phys J B 2010 75 381ndash387

23 Muddana H S Sengupta S Mallouk T E Sen A Butler P J J Am Chem Soc

2010 132 (7) 2110ndash2111

24 Baker CJ Deahl K Domek J Orlandi EW Arch Biochem Biophys 2000 382(2) 232-

237

25 V Vojinovic RH Carvalho F Lemos JMS Cabral LP Fonseca BS Ferreira

Biochemical Engineering Journal 2007 35 126-135

56

26

OV Lebedeva NN Ugarova Russian Chemical Bulletin 1996 45(1) 18-25

27 Nigel C Veitch Phytochemistry 2004 65(3) 249-259

28 Kenneth H Petersen Journal of Immunological Methods 2009 340(1) 86-89

29 httpwwwbrenda-enzymesorgphpresult_flatphp4ecno=11117

30 Goldenberg L M Wagner J Stumpe J Paulke B-R Goumlrnitz E Langmuir 2002 18

5627-5629

31 httpwwwgoldbiocompdf3761-Protocol201pdf

32 Av Rovisco Pais Lisboa Portugal T amp D Technology West Yorkshire UK Journal

of Molecular Catalysis B Enzymatic 2004 28(2-3) 129-135

33 httpwwwphysicsnyuedugrierlabmethodsnode11html

34 Yoon-Mee Lee1 O-Yul Kwon Yeo-Joon Yoon Keungarp Ryu Biotechnology Letters

2006 28 39ndash43

35 JC Pyun SD Kim JW Chung Analytical Biochemistry 2005 347(2) 227-233

36 George H Pimbley Jr Mathematical Biosciences 1974 20(1-2) 27-51

37 Andy Fenton Sarah E Perkins Parasitology 2010 137 1027-1038

38 Mead James G Brownell Robert L Mammal Species of the World A Taxonomic and

Geographic Reference (3rd ed) Johns Hopkins University Press 2005 2 2142

57

39

Krause KH Schweiz Med Wochenschr 2000 130(4) 97-100

40 Irina Kareva Faina Berezovskaya Carlos Castillo-Chavez Journal of Biological

Dynamics 2010 4(4)315 - 327

41 Delaittre G Reynhout I Cornelissen J Nolte R Chemistry ndash A European Journal

2009 15 12600ndash12603

42 C Buller DH Johnson RC Tschumper Investigative Ophthalmology amp Visual Science

1990 31 2156-2163

43 Robert M Mazo Brownian Motion Fluctuations Dynamics and Applications Oxford

Science Publications 2009 57

44 Vivek K Mutalik KV Venkatesh Theoretical Biology and Medical Modelling 2005 2

19

45 Satoshi Nakamura Kunimasa Koga Biochemical and Biophysical Research

Communications 1977 78(2) 806-810

46 Krishnananda Chattopadhyay Shyamalava Mazumdar Biochemistry 2000 39 (1)

263ndash270

47 Satoshi Nakamura Yasuyuki Ogura J Biochem 1968 63 (3) 308-316

48 van Dongen SF Nallani M Cornelissen JJ Nolte RJ van Hest JC Chemistry 2009

15(5) 1107-1114

49 Ronald Bentley Methods in Enzymology 1955 1 340-345

50 Vartiainen J Raumlttouml M Paulussen S Packaging Technology and Science 2005

18 243ndash251

58

51 GA Sherbeny AA Shindia YMMM Sheriff International Journal of Agriculture

and Biology 2005 7

52 D Rando G-W Kohring F Giffhorn Appl Microbiol Biotechnol 1997 48

53 Worthington enzyme manual Worthington Biochemical Corporation

httpworthington-biochemcomgopdefaulthtml

54 httpwwwbrenda-enzymesorgphpresult_flatphp4ecno=11117

55 httpwwwoxygenphwatercom

56 P Boon Chock Sue Goo Rhee and Earl R Stadtman Annu Rev Biochem 1980 49

813-841

57 Sybil Golden P A Wals Joseph Katz Analytical Biochemistry 1977 77(2) 436-445

58 R L Baehner S K Murrmann J Davis R B Johnston Jr J Clin Invest 1975 56(3)

571ndash576

59 I Bejarano M P Terroacuten S D Paredes C Barriga A B Rodriacuteguez J A Pariente

Molecular and Cellular Biochemistry 2007 296(1-2) 77-84

60 Michael C Moulton Laura K Braydich-Stolle Mallikarjuna N Nadagouda Samantha

Kunzelman Saber M Hussain Rajender S Varma Nanoscale 2010 2 763-770

61 Goya DV Matijevic E Colloids and Surfaces A 1999 146 139

62 Velikov KP Zegers GE van Blaanderen A Langmuir 2003 19 1384

63 Goldenberg L M Wagner J Stumpe J Paulke B-R Goumlrnitz E Langmuir 2002 18

5627-5629

59

64

Banerjee S Gopal J Muraleedharan P Tyagi A K Raj B Curr Sci 2006

901378ndash1383

65 Bogomolov V N Kudinov E K Firsov Y A Sov Phys Solid State 1968 35 555

66 Kudo A Miseki Y Chem Soc Rev 2009 38 253ndash278

67 Gaj T Meyer SC Ghosh I Protein expression and Purification 2007 56 54-61

5

Another factor which gains importance at nanoscale is the dominance and ubiquity of

Brownian motion13

The trajectory of a linearly propelled nanomotor would be

significantly affected by the continuous collision of randomly moving particles in the

environment These collisions deviate the nanomotor from its linear path and hence this

effect needs to be considered while projecting the path of a nanomotor

Motion at the nanoscale can be powered by different types of mechanisms such as

diffusiophoretic electrophoretic osmophoretic chemophoretic thermophoretic and

others Different types of mechanisms of motions may coexist or act individually to cause

Position A Position B

Figure 1 1 Reciprocal and Nonreciprocal motion8

Reciprocal motion

Nonreciprocal motion

6

motion Diffusiophoretic motion of particles is caused due to concentration gradients of

solutes in an electrolytenonelectrolyte solution9 Electrophoresis is the motion of a

particle relative to a fluid under the influence of an electric field10

This occurs as the

particle carries an electric surface charge which is acted upon by an electrostatic

coulombic force of the external electric field Osmophoresis is the motion of a

vesiclefluid particle encapsulated in a semi-permeable membrane towards lower

concentration gradient when it is placed in a solution containing solute concentration

gradient1112

Thus considering the numerous mechanisms of motion known to work at

the nanoscale several suitable options are available for the development of artificial

nanomotors

12 Existing nanomotors Mechanisms and uses

Several nanomotors have been developed in the past decade or so A general principle of

working of these nanomotors is that they function by catalytically decomposing the

dissolved fuel to generate asymmetry at their surface or at their interfacial region thereby

causing propulsion13

The self-electrophoretic bimetallic nanowires developed by

WPaxton and A Sen etal consisted of a 2 μm long rod with one tip made up of Au and

the other tip made up of Pt1 These motors were synthesized by electrodeposition in an

alumina template The rods moved autonomously in a solution of hydrogen peroxide

with platinum end forward Pt oxidizes H2O2 to produce H+ and O2 and Au utilizes H

+ to

reduce H2O2 to water This generates an electron flow through the rod and a proton flow

outside the rod resulting in an electric field around the rod in the solution This electric

7

field in solution acts on the unbalanced charges within the nanorodrsquos double layer This

moves the nanorod with respect to the surrounding fuel due to the ion flux that is

generated

Bubble propulsion has been predominantly used as mechanism of motion in the motors

developed by Feringa etal His group developed a micromotor by attaching catalase to a

microparticle and placing the particle in a solution containing hydrogen peroxide This

system allowed the microparticle to move by producing oxygen bubbles from the

decomposition of hydrogen peroxide14

A similar motor was later created by Feringarsquos

group which consisted of glucose oxidase and catalase immobilized on a carbon

nanotube in a substrate solution of hydrogen peroxide Glucose oxidase converted

glucose to H2O2 which was then utilized by catalase to produce water and oxygen

thereby propelling the carbon nanotube15

Another interesting example of a self-propelled motor is the polymerization driven motor

developed by Cameron et al16

It consists of polystyrene beads immobilized with ActA

protein which catalyzes actin polymerization leading to the formation of an actin

filament which resembles the flagella of bacteria This actin polymerization propels the

beads at the speed of 001-015 μms

A theoretical model has been proposed by Nafaji and Golestanian etal which consists

of three spheres linked by rigid rods in one dimension17

The length of the rods can

switch between two specific values Due to the nonreciprocal motion of the rod this

8

model can swim in low Reynolds number systems An actual model based on

nonreciprocal motion has been developed by Kim etal in which heart cells have been

linked to an asymmetric polydimethylsiloxane robot As the heart cells beat the

asymmetric structure of the device resembles a walking movement18

Most of the nanomicromotors mentioned above are either metallic or work in

environments not compatible with human body Enzymes being an integral part of the

body can solve this problem by operating in suitable substrates already present in the

body fluids The nonreciprocal conformation changes of the enzyme as well as

diffusiophoresis and electrophoresis can conceivably be used to generate motion in case

of enzyme driven nanomotors such motors will be discussed in detail in the following

chapter

9

Chapter 2

Immobilized enzymes on Janus particles as catalytic motors

21 Introduction

Enzymes are widely found in biological systems and act on a large variety of substrates

This can make enzymes an ideal choice for powering nanomotors which need to be

biocompatible Several fluids found in the body can act as substrates to the enzymes For

example dissolved glucose which is abundantly found in the body19

is a susbtrate for

enzyme glucose oxidase Urea which is found in the urinary tract20

is a substrate for

urease Hydrogen peroxide produced by phagocytic cells in the body21

is a substrate for

horseradish peroxidase Having motors driven by enzymes can open possibilities of

targeted drug delivery to these parts of the body containing such substrates

Enzymes have already been used to generate motion of carbon nanotubes by mechanism

of bubble propulsion by Feringa etal15

Two other possible mechanisms which may be

able to power motion of nanoparticles using enzymes are asymmetrical decomposition of

substrates into products leading to self-diffusiophoresis (Figure 2 1) and nonreciprocal

changes in enzyme conformation leading to propulsion (Figure 2 2) As seen in Figure 2

1 enzymes which are immobilized on a Janus particle can breakdown the available

substrate in the solution into products causing asymmetric distribution of solutes in the

surrounding fluid This can lead to movement of particle towards the region of lower

10

solute concentration by principle of self-diffusiophoresis Figure 2-2 shows the possible

nonreciprocal changes in the conformation of a theoretical protein which can lead to

motion of the protein22

These nonreciprocal changes in conformation are triggered by the

attachment of substrate molecule and release of product molecules from the enzymersquos

active site

Figure 2 1 Enzyme functionalized Janus particle moves upon catalyzing asymmetrical

decomposition of substrate into product

11

Figure 2 2 Schematic showing idealized nonreciprocal conformation changes in a

theoretical enzyme22

Enhanced diffusion of a single enzyme molecule by catalysis in a substrate solution has

been demonstrated23

The diffusion coefficient of a single urease molecule was measured

using fluorescence correlation spectroscopy and it was observed that the diffusion of the

urease molecule increases by 16-28 by varying urea concentrations from 0001 M to 1

M Addition of enzyme inhibitors to the system curbed the enhanced diffusion This

enhanced diffusion has been attributed to the force generated by self-electrophoresis by

the enzyme in substrate solution

12

Based on the above facts it might be possible to propel a nanoparticle by asymmetric

distribution of enzymes on its surface thereby leading to self-propulsion of the particles

in substrate solution (Figure 2 1) This propulsion may be due to self-

electrophoresisself-diffusiophoresis andor due to nonreciprocal changes in enzyme

conformations Here we aim to investigate a system consisting of polystyrene particles

half coated with enzyme introduced in a suitable substrate solution The enzyme can be

directly linked to the polystyrene particle or it can be linked through a long spacer

molecule (linker) to provide more degrees of freedom to the enzyme

Several factors need to be considered for choosing enzyme for powering motion at the

nanoscale Some of the important factors are the ratio of kcatKm which denotes the

catalytic efficiency of the enzyme possible substrates stability of enzymessubstrates

ease of immobilization relationship with other enzymes in cascade reactions and

commercial availability of the enzymes The enzyme being used in the present study is

horseradish peroxidase It acts on a molecule of hydrogen peroxide and reduces it while

simultaneously oxidizing another substrate molecule In the absence of alternate

oxidizable substrate HRP can oxidize water and oxygen back to hydrogen peroxide24

thus giving an equilibrium reaction

13

211 Horseradish peroxidase

Figure 2 3 Horseradish peroxidase

Horseradish peroxidase belongs to the group of peroxidases which are a group of heme-

containing oxidoreductases that act on peroxide as hydrogen donor25

It is a redox

enzyme which contains a protoheme a complex of trivalent iron with 1358-

tetramethyl-24-divinylporphin-67-dipropionic acid as a prosthetic group It is a 44 kDa

protein which includes 33900 Da polypeptide chain bound with eight oligosaccharide

chains The carbohydrate chains improve the stability of the enzyme by protecting it

against radicals formed in the course of the reaction it catalyzes26

Horseradish

peroxidase is found in horseradish and is used widely in bioanalytical applications such

as ELISA and Western blots It decomposes hydrogen peroxide into water and oxygen

while simultaneously oxidizing another substrate27

The oxidizable substrates can be

chromogenic substrates such as 22rsquo55rsquo-tetramethyl-benzidine (TMB) and 33-

diaminobenzidine (DMB) or chemoluminescent substrates like SuperSignal and ECL

luminal28

14

Figure 2 4 Reaction catalyzed by horseradish peroxidase

The functional parameters for horseradish peroxidase include Km of 015 mM and kcat of

790 s-1

at an optimum temperature of 20 ordmC and an optimum pH of 6429

Some of the

inhibitors of HRP are 110-phenanthroline aminotriazole and bromine

22 Experimental

221 Materials and methods

Uniform carboxyl modified polystyrene microspheres 05 microm in diameter were obtained

from Polysciences Inc The size of the purchased microspheres was verified by Field

Emission Scanning Electron Microscopy (FESEM) Peroxidase Type I from

Horseradish N-(3-dimethyl-aminopropyl)-Nrsquo-ethylcarbodiimide (EDC) and hydrogen

peroxide solution 50 wt in water were purchased from Sigma-Aldrich N-

hydroxysuccinimide (NHS) ge 97 was purchased from Fluka All chemicals were of

reagent grade and used without further purification Nanopure deionized water was used

for all experiments and preparation of solutions All measurements were made at room

15

temperature (25 oC) unless mentioned otherwise The UV-Visible spectroscopy

measurements were performed using Agilent 8453A diode-array UV-Visible

Spectrophotometer with ChemStation UV-Vis Software Diffusion coefficient

measurements were performed using Nanosight LM10

222 Synthesis of Janus particles

A monolayer of particles was prepared according to the procedure (Figure 2 5) by

Goldenberg L et al30

Glass slides were placed on the bottom of a petridish Water was

put into the petridish so that the entire slide was immersed in water A thin layer of

hexane was added on top of water in the petridish A suspension of microspheres in

ethanol (5 ww) was introduced to the water-hexane interface After the monolayer was

formed the glass slide was lifted up through the monolayer with tweezers The

microsphere-covered glass slides were left to dry overnight at room temperature

Following drying a thin film of silica was evaporated on the glass slides in Semicore

evaporator The spheres were partially coated with 30 nm of silica Particles were

removed from the glass slide by gentle sonication The polystyrene-silica (PS-SiO2)

Janus particles thus obtained were washed in deionized water several times before

further immobilization steps were performed FESEM was used to confirm synthesis of

Janus particles

16

Figure 2 5 Preparation of PS-SiO2 Janus particles by evaporation

223 Immobilization of enzymes using EDCNHS coupling method

1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide and N-hydroxysulfosuccinimide

(EDCNHS) coupling procedure was followed for immobilizing horseradish peroxidase

type I on 05 μm PS-SiO2 Janus particles (Figure 2 6) In this process EDC reacts with a

carboxyl groups on the Janus particles to form an amine-reactive O-acylisourea

intermediate If this intermediate does not encounter an amine it can hydrolyze and

regenerate the carboxyl group In the presence of NHS EDC can be used to convert

carboxyl groups to amine-reactive sulfo-NHS esters which are much more stable and do

not readily hydrolyze to regenerate carboxyl groups In the current experiment

immobilization was performed in the presence as well as absence of a long spacer

17

molecule (linker) between Janus particles and enzymes The chemical 6-aminohexanoic

acid was used as the linker (Figure 2 7) HRP immobilized PS-SiO2 Janus particles were

stored in deionized water until required for further experiments

Figure 2 6 EDCNHS coupling method for immobilization of enzyme on carboxylated

polystyrene particles

Figure 2 7 Immobilization of enzyme on Janus particles with and without 6-

aminohexanoic acid linker

18

224 Enzyme assay

Assay for activity of HRP was performed using a spectrophotometric method which

involved using 4-aminoantipyrene as hydrogen donor3132

The reaction rate was

determined by measuring increase in absorbance at 510 nm resulting from the

decomposition of hydrogen peroxide One unit represents the decomposition of 1 μM of

hydrogen peroxide per minute at 25 degC and pH 70 under the specified conditions

The spectrophotometer was set to the wavelength of 510 nm and temperature of 25 degC

Phenol4-aminoantipyrene solution (14 mL) and 00017 M H2O2 solution (15 mL) were

pipetted into each cuvette The cuvette was incubated for 3-4 mins at 25degC and blank was

established A 01 mL solution of diluted enzyme was added to the above mixture and

absorbance was recorded for 4-5 mins Standard graphs of absorbance versus time were

drawn for known concentration of the free enzyme and compared to that of the

immobilized enzyme on the Janus particles The estimate of the amount of enzyme

present on the Janus particles was deduced from the difference between the quantity of

the free enzyme previously added for immobilization and the amount that was measured

to be in the supernatant after some of the enzyme became attached to the Janus particles

The experiment was carried out in triplicate to assess reproducibility

19

The formula used to calculate the activity of enzyme is

where ∆Amin denotes the change in absorbance per minute

225 Diffusion coefficient measurements using Nanosight LM10

Diffusion coefficients for HRP immobilized particles were measured using Nanosight

LM 10 nanoparticle tracking instrument with Nanoparticle Tracking Analysis (NTA) 20

software in the presence of 003 H2O2 solution as the substrate and also in the presence

of only deionized water The videos were recorded for 30 seconds at the rate of 30 frames

per second Each experiment was carried out in triplicate to assess reproducibility

Particles

visualization

zone

Microscope with

LM 10 unit

LM 10 unit

Figure 2 8 Nanosight LM 10 instrumentation

20

Nanosight LM 10 (Figure 2 8) generates videos of population of moving nanoparticles in

a liquid when illuminated by laser light The particles which pass through the laser beam

path are seen by the instrument as small points of light undergoing Brownian motion

(Figure 2 9a)

The videos are further analyzed by NTA (Nanoparticle tracking analysis) software

(Figure 2 9b) which identifies and tracks each particle independently on a frame-by-

frame basis The software also calculates and subtracts any bulk flow in the system The

mean square displacement is calculated for each particle as long as it is visible If the

particle blinks that is if it disappears momentarily and then reappears on the screen the

software will consider the same particle as two separate ones All analysis data is

exported to the excel files in terms of diffusion coefficients of the tracked particles

B A

Figure 2 9 NTA video analysis (a) Particles as they are seen on the screen during

experimentation in Nanosight LM10 (b) NTA screen as the particles are being analyzed for

their diffusion coefficients

21

23 Results

231 Enzyme assay

The activity of free HRP enzyme reported by Sigma Aldrich is 150 unitsmg However

the activity of HRP upon analysis was found to be 52158 unitsmg which is much lower

than the reported value The activity of HRP was found to be further lowered to 2133

unitsmg when it was immobilized on the PS-SiO2 Janus particles in the presence of a

linker The following formula is used to calculate enzyme activity

where ΔAmin is the change in absorbance per minute

22

Figure 2 10 Graph for the assay of solution of free enzyme containing 05mgL of HRP

ΔAmin = 01716 Unitsmg of free enzyme = 52158

During EDCNHS coupling 04 mg enzyme was added to 1 ml of 025 vv suspension

of Janus particles Thus initial concentration of enzyme in the mixture was 04 mgml

Assuming retained activity of the enzyme in supernatant solution the amount of enzyme

in supernatant was calculated to be 03868 mgml from the assay Amount of enzyme

functionalized on the Janus particles was calculated as the difference between enzyme

previously added for immobilization (04 mgml) and the enzyme present in supernatant

(03868 mgml) and was found to be 00132 mgml Enzyme assay was performed on the

enzyme immobilized Janus particles to estimate the activity of immobilized enzyme

(Figure 2 11) and was found to be 2133 unitsmg

y = 0002x + 00516

0

01

02

03

04

05

06

07

08

0 50 100 150 200 250 300 350 400

Ab

sorb

an

ce

51

0 n

m

Time (sec)

Assay of free enzyme HRP

23

Figure 2 11 Graph for the assay of of immobilized enzyme on 05 μm Janus particles

(~025 vv) ΔAmin = 01853 Unitsmg of free enzyme = 2133

232 Diffusion coefficient measurements

No significant increase was observed in diffusion coefficient of HRP-immobilized Janus

particles in the presence of H2O2 as the substrate Similar results were obtained for

diffusion coefficient measurement with and without linker moieties bridging the enzymes

and particles(Table 2 1 Table 2 2) Thus the linker did not seem to assist in increasing

the particle diffusion by improving or retaining the activity of the enzyme

y = 00015x + 00953

0

01

02

03

04

05

06

07

0 50 100 150 200 250 300 350 400

Ab

sorb

an

ce

510 n

m

Time (sec)

Assay of HRP immobilized 05 μm Janus particles

24

Table 2 1 Diffusion coefficient measurements of HRP immobilized Janus particles

without linker

HRP immobilized

Janus particles in

003 H2O2

Control - Janus

particles in

003 H2O2

Control - HRP

immobilized Janus

particles in DI water

Diffusion coefficient

(10-10

cm2s)

1359416 1051777 1308675

Standard deviation 4534801 4122075 4918597

Table 2 2 Diffusion coefficient measurements of HRP immobilized Janus particles in

the presence of linker

HRP immobilized

Janus particles in

003 H2O2

Control - Janus

particles in

003 H2O2

Control - HRP

immobilized Janus

particles in DI water

Diffusion coefficient

(10-10

cm2s)

13838 1339829 1407891

Standard deviation 5644692 5649364 5797218

25

Figure 2 12 Diffusion coefficient measurements of PS-SiO2 Janus particles without and

with 6-aminohexanoic acid linker

24 Discussion

To compare the diffusion coefficients observed during the experiments theoretical

diffusion coefficient was calculated using following equation33

r

kTD altraslation

6 Equation 21

where r is the hydrodynamic radius of particle Dtranslational is translational diffusion

coefficient of the particle T is temperature of the system η is the viscosity of fluid

through which the particles are moving and k is the Boltzmann constant From FE-SEM

images the size of the particles used was determined to be 450 nm For 450 nm diameter

0

20

40

60

80

100

120

140

160

Without linker With linker

Dif

fusio

n c

oeff

icie

nt

(10

-10 c

m2s

)

HRP immobilized 05 μm Janus particles - Diffusion coefficient measurements

HRP immobilized Janus particles in 003 H2O2

Control - Janus particles in 003 H2O2

Control - HRP immobilized Janus particles in DI water

26

particle Dtranslational is equal to 109 μm2s which is comparable to the diffusion

coefficient measured for the enzyme immobilized particles

HRP was found to be active on the Janus particles however the activity of the enzyme

was reduced upon immobilization Lower enzyme activity can be due to the coupling

method used which in this case was EDCNHS covalent binding However EDCNHS

coupling method has been shown to significantly retain the enzyme activity when the

enzyme is immobilized on carboxylated multi-walled carbon nanotubes under appropriate

conditions34

Hence EDCNHS binding method by itself is not believed to be

responsible for reduction of enzyme activity upon immobilization

Direct binding of enzyme to the Janus particle may lead to reduction in the enzyme

activity because of space constraints for the enzyme and difficulty for the passage of

substrate to the active site of the enzyme due to stearic hindrance Hence a linker was

inserted between the enzyme and Janus particle during enzyme immobilization However

inserting a linker between the enzyme and the particle did not lead to enhanced diffusion

of Janus particles or increase in enzyme activity of immobilized enzyme

Lack of enhanced diffusion can be firstly attributed to the reduction in enzyme activity

upon immobilization Enzyme activity may be retained by modifying the conditions

during EDCNHS coupling or changing the binding method altogether The degree of

immobilization of HRP on carboxylated Janus particles can possibly be affected by

several factors such as temperature the concentration of the coupling reagent EDC

27

concentration HRP concentration in solution and incubation time34

Different

combinations of these factors can be tested to find optimum conditions for

immobilization of HRP Alternate binding method can involve thiolation of HRP by the

covalent conjugation of mercaptopropionic acid and then the thiolated HRP can then be

attached on the gold surface of a polystyrene-gold Janus particle35

Thus further

modifications can be made to the system in order to retain enzyme activity on the

immobilized particles and thereby power nanomotors using enzymes

28

Chapter 3

Predator-prey system of enzyme immobilized particles

31 Introduction

Predator-prey systems are widely found in nature and play a major role in biological

processes3637

Predator-prey systems include all the interactions in which one organism

pursues andor consumes another organism One familiar predator-prey system at the

macroscale is that of a tiger chasing and consuming a deer Another not so familiar one

includes Baleen whales which eat millions of microscopic planktons at once38

At the

microscale such kind of interactions can be observed in the case of phagocytes in the

body which are capable of engulfing foreign microorganisms and killing them with a

combination of microbicidal systems39

In the case of cancer the tumor is the prey and

the mature myeloid cells in the body are the predator However the tumor here has a

defense mechanism against the predator in which the tumor produces cytokines which

inhibits the maturation of the myeloid cells and thereby prevents predation40

One similarity that can be observed in all these systems is that the prey possesses some

attribute which is desired by the predator which makes the predator chase the prey A

similar relationship is seen in case of enzymes which are involved in cascade reactions41

In cascade reactions product synthesized by one enzyme is the reactant required by

another enzyme to carry out its own catalytic reaction As such there is a predator-prey

29

kind of dependence between the two enzymes which may cause one enzyme to chase the

other enzyme when placed in suitable substrate solutions However the mechanism

hypothesized for such a system is totally different than the mechanism involved in

biological systems In biological systems such as bacterial chemotaxis or phagocytes

chasing foreign particles in human body complex biochemical reactions are responsible

for the predator-prey behavior42

In the proposed enzyme cascade system there are no

complex biochemical reactions involved The principle hypothesized for predator-prey

behavior of enzyme cascade reactions can be explained as follows Motion of synthetic

motors at nanoscale involves two components translational diffusion and rotational

Brownian diffusion At very short time scales of less than 1Dr where Dr (s-1

) is

rotational Brownian diffusion coefficient the motion of particles is unidirectional For

longer time scales of greater than 1Dr the direction of motion is random giving

poweredactive diffusion43

If the chemical homogeneity is broken that is when there is

higher concentration of a substrate present in one direction of the enzyme the enzyme

might acquire a subtle bias which may lead to a spatial aggregation of the enzymes based

on the substrate concentration gradient The bias can be a result of the relationship

between the speed of enzyme diffusion and substrate concentration where higher

substrate concentration lead to higher enzyme diffusion As a result in an enzyme

cascade system one enzyme will migrate towards higher concentration of substrate

which is present near the other enzyme thus giving rise to a predator-prey behavior

Such enzyme cascades are widely found in signaling networks and metabolic pathways in

living cells and can be used to produce predator-prey like nanomotors systems44

The

30

enzymes being used in the present study are horseradish peroxidase and glucose oxidase

Both enzymes are extremely stable4546

Glucose is the substrate for glucose oxidase

which is widely available and highly stable47

Horseradish peroxidase acts on hydrogen

peroxide It is involved in a cascade reaction with glucose oxidase which produces

hydrogen peroxide by converting glucose into gluconic acid (Figure 3 1)48

Figure 3 1 Concept of particle aggregation due to cascade reactions of immobilized

enzymes

In this experiment glucose oxidase (GOx) and horseradish peroxidase (HRP) were

immobilized on polystyrene carboxylate beads of different sizes using EDCNHS

coupling method Glucose and 3355-Tetramethylbenzidine (TMB) were dissolved

together in water and this solution was used as the substrate solution All the components

were present in a single system in DI water Aggregation of particles and precipitation of

oxidized TMB from the solution was expected to be observed when all these components

were present in such a system

Properties of horseradish peroxidase have been discussed in the previous chapter (Section

211 Horseradish peroxidase)

GOxGOx HRPHRP

Glucose + O2Gluconic acid + H2O2

TMB TMB oxidizedBlue precipitate

H2O

Aggregation

31

311 Glucose oxidase

Figure 3 2 Glucose oxidase

Glucose oxidase (GOx) belongs to the group of oxidoreductases and is also called

glucose aerodehydrogenase49

It is obtained from fungi and has been identified as an

antimicrobial enzyme50

GOx binds to β-D-glucopyranose and catalyzes the oxidation

reaction to from gluconolactone while simultaneously reducing oxygen to hydrogen

peroxide51

GOx has high substrate affinity and specificity high stability and high

turnover number which makes it a suitable biocatalyst for industrial applications52

Figure 3 3 Redox reaction of glucose catalyzed by glucose oxidase53

32

GOx is a glycoprotein with molecular weight of 155 kDa and consists of two identical

polypeptide subunits linked together by a disulfide bond The optimum pH for GOx is

55 though it is stable in a broad pH range of 4 to 7 and can be stored for a long period of

time51

The functional parameters for glucose oxidase include Km of 151 mM and kcat of

337 s-1

at temperature of 20 ordmC and pH of 754

Some of the inhibitors of GOx are 2-

Deoxy-D-glucose adenine nucleotides and NaNO3

32 Experimental

321 Materials and methods

Uniform carboxyl modified polystyrene microspheres 05 microm in diameter were obtained

from Polysciences Inc The size of the purchased microspheres was verified by FESEM

Peroxidase Type I from horseradish glucose oxidase Type II-S from Aspergillus niger

N-(3-dimethyl-aminopropyl)-Nrsquo-ethylcarbodiimide (EDC) and Hydrogen peroxide

solution 50 wt in water were purchased from Sigma-Aldrich N-hydroxysuccinimide

(NHS) was purchased from Fluka D-Glucose (99) was purchased from VWR

International Secure-Seal imaging spacers (9mm diameter) for glass slides were obtained

from Sigma Aldrich All chemicals were of reagent grade and used without further

purification Nanopure deionized water was used for all experiments and preparation of

solutions All measurements were made at room temperature (25 oC) unless mentioned

otherwise The UV-Visible measurements were performed using Agilent 8453A diode-

array UV-Visible Spectrophotometer with ChemStation UV-Vis Software Optical

33

microscopy experiments were performed using Axiovert 200 MAT microscope by Carl

Zeiss Diffusion coefficient measurements were obtained using Nanosight LM10

322 Optical microscopy experiments

The enzyme substrate system was observed under optical microscope to check for

aggregation and precipitation due to cascade reactions Several such systems were

studied Each system in general consisted of GOx and HRP immobilized on different

polystyrene particles The substrate solutions consisted of 01 M glucose and different

concentrations of TMB solution The systems were studied in the presence of all the

constituents and in the absence of glucose GOxHRP and TMB Systems consisting of

different sized HRP immobilized particles in substrate solution of 003 hydrogen

peroxide and TMB were also studied Videos were recorded over a period of 24 hours to

observe changes in the system Each experiment was performed atleast thrice to assess

reproducibility of results

323 Diffusion coefficient measurements using Nanosight LM10

Diffusion coefficient of GOx and HRP immobilized particles were measured using

Nanosight LM10 nanoparticle tracking instrument with Nanoparticle Tracking Analysis

(NTA) 20 software Diffusion coefficients were measured for different systems namely

GOx particles in 01 M glucose and oxygen both GOx and HRP functionalized particles

in 01 M glucose and oxygen along with controls for the individual systems Videos were

34

recorded for 30 seconds at the rate of 30 frames per second Each experiment was carried

out in triplicate to assess reproducibility

33 Results

331 Optical microscopy

According to the enzyme cascade model precipitation of TMB was possible only upon

the interaction of GOx and HRP immobilized particles in presence of appropriate

substrate solution Hence in the first set of experiments precipitation of TMB was used

as an indication of the type of interaction between GOx and HRP immobilized particles

Fan-shaped structures were observed when both HRP and GOx immobilized particles

were present in a substrate solution consisting of glucose and TMB These structures

started forming as a single strand and then branched out and intertwined to form the fan-

shaped structures (Figure 3 4)

Table 3 1 Observations for formation of fan-shaped structures in enzyme cascade

system in presence of different components

GOx HRP Glucose TMB Observation

Yes Yes Yes Yes

Fan-shaped structures observed particles associated with

the fan-shaped structures

No Yes Yes Yes No fan-shaped structures

Yes No Yes Yes No fan-shaped structures

Yes Yes No Yes No fan-shaped structures

Yes Yes Yes No No fan-shaped structures

35

Fan-shaped structures can also be observed in the system consisting of only HRP

immobilized particles in substrate solution of hydrogen peroxide and TMB The size of

the fan-shaped structures depends on the size of the enzyme immobilized particles Free

enzymes show extremely tiny structures which are difficult to view under the optical

microscope

Figure 3 4 Formation of fan-shaped structures when both HRP and GOx

immobilized particles were present in a substrate solution consisting of glucose and

TMB A In the initial stages single strands form on the surface of liquid (5Ox) B

Single strands branch out and intertwine to form fan-shaped structures (50x) C Many

such structures form and settle down after 20 minutes (20x)

A

C

B

36

Experiments were performed in the absence of TMB that is the system consisted of GOx

and HRP immobilized particles in glucose substrate solution In the absence of TMB the

particles were expected to move closer to each other without any precipitation due to the

cascade reaction However there was no conclusive aggregation of particles observed in

the system

Free Enzyme 005 μm HRP - immobilized particle

10 μm HRP - immobilized particle 05 μm HRP - immobilized particle

Figure 3 5 Size dependence of fan-shaped structures on the size of HRP -

immobilized particles (20x)

37

Table 3 2 Observations for particle aggregation in enzyme cascade system in presence

and absence of TMB

Glucose GOx HRP TMB Observation

Present Present Present Present Aggregation and precipitate

Present Present Present Absent No conclusive aggregation

Present Absent Present Present No aggregation or ppt

Absent Present Present Present No aggregation or ppt

Present Present Absent Present No aggregation or ppt

332 Diffusion coefficient measurements

Diffusion coefficients were measured for GOx immobilized particles of 05 μm diameter

in substrate solution of 01 M glucose and oxygen Diffusion coefficients of particles

were also measured for system consisting of GOx and HRP functionalized particles in

glucose and oxygen Each experiment was done in triplicate to assess reproducibility

Nanosight visualization chamber being a closed chamber oxygen was purged into all the

solutions before starting the experiments to ensure sufficient supply of oxygen for

catalytic reaction of GOx

38

Table 3 3 Diffusion coefficients for 05 μm GOx immobilized particles in 01 M

glucose purged with O2

Mean

GOx

particles in

01 M

glucose

Control - GOx

particles in DI

water

Control - GOx

particles in 01 M

glucose without

oxygen purging

Control -

particles in 01

M Glucose

Diffusion

coefficient

(x10-10

cm2s)

13316 12888 13827 13702

Standard

deviation 6307 4926 4424 7468

Table 3 4 Diffusion coefficients for GOx and HRP immobilized particles in 01 M

glucose and purged with O2

Mean

GOx- HRP-

functionalized

particles in 01 M

glucose

Control ndash Gox-

HRP- functionalized

particles in DI water

Control - particles

in 01 M glucose

Diffusion coefficient

(x10-10

cm2s)

13005 14434 13702

Standard deviation 5263 6622 7468

No significant increase was observed in diffusion coefficient of enzyme immobilized

particles in any of the systems

39

34 Discussion

Formation of fan-shaped structures due to patterned deposition of oxidized TMB

indicates some kind of interaction between GOx and HRP immobilized particles Particle

diameter dependence of the size of structures can be explained with a simple model in

Figure 3 6 A larger particle will have a higher surface area as compared to a smaller

particle Hence more enzyme is immobilized on the larger particle As a result higher

quantity of TMB is oxidized and precipitated by the larger particle as compared to the

smaller particle This leads to thicker strands of fan-shaped structures eventually scaling

up the whole system to give bigger fan-shaped structures

The exact reason for the formation of such fan-shaped structures which are dependent on

particle size could not be determined It is hypothesized that hydrogen peroxide andor

Figure 3 6 Model for size dependence of fan-shaped structures on the particle

diameter of enzyme immobilized particles Larger the particle more surface area is

available for precipitation of TMB which eventually leads to bigger fan-shaped

structures

Larger particle Smaller particle

40

TMB gradient in the substrate solution might be responsible for such structures If there

is higher concentration of H2O2 on one side of the particle and assuming an even

concentration distribution of TMB there will be selective precipitation in the direction of

H2O2 When several particles come in the vicinity of higher concentration of H2O2 the

precipitation lines will converge to give fan-shaped structures (Figure 3 7)

Figure 3 7 Hypothesis for formation of fan-shaped structures

In the system consisting of GOx immobilized particles HRP immobilized particles

glucose and TMB it was observed that the fan-shaped structures formed at the liquid-air

interface and eventually settled down to the bottom of the glass slide This makes sense

as oxygen is necessary for the catalytic reaction of conversion of glucose to gluconic acid

and hydrogen peroxide which takes place on the surface of GOx immobilized particle As

a result H2O2 is primarily formed at the liquid-air interface and is then used by HRP for

precipitation of TMB

Following the observations that precipitation of TMB occurs at the surface GOx and

HRP immobilized particles are also expected to interact at the surface in the absence of

TMB Due the cascade reaction it was expected that HRP immobilized particles will be

41

attracted to GOx immobilized particles in appropriate substrate solution Experiments

were carried out to visualize inter-particle interactions at the liquid-air interface

However liquid-air interface not being a stable system to image the precise interaction

of GOx and HRP immobilized particles could not be determined Significant external

disturbances were involved at the interface To enable the cascade reaction in the bulk of

the liquid oxygen was purged into the reaction system before starting the experiment

However this did not lead to significant reaction in the bulk of the liquid and the reaction

prominently took place at the liquid-air interface where the oxygen concentration was

higher

In order to observe the interaction of GOx and HRP immobilized particles better imaging

techniques need to be used to observe diffusion of particles in absence of external

disturbances at the liquid-air interface Large quantity of oxygen needs to be purged into

the system and retained to make the reaction more pronounced in the bulk of the substrate

solution Higher concentration of oxygen in water may also be achieved by using oxygen

enriched water which is commercially available55

Alternatively a different enzyme cascade system may be used which does not require a

gaseous substrate56

For example glycogen synthase synthesizes glycogen from glucose-

1-phosphate and glycogen phosphorylase breaks down glycogen to glucose-1-

phosphate57

This system does not require a gaseous substrate and might be able to

overcome the deficiencies of GOx and HRP enzyme cascade system

42

Chapter 4

Silver-titania nanomotors

41 Introduction

Bimetallic nanowires made of platinum and gold have been developed by WPaxton

etal1 The rods move autonomously in a solution of hydrogen peroxide In the present

study silver-titania (Ag-TiO2) particles are being synthesized and investigated for motion

in presence of hydrogen peroxide and UV light With proper control of conditions and

UV light trigger it can be possible to control the speed and direction of motion of these

nanoparticles

Hydrogen peroxide is produced in the human body by phagocytes as an immune response

to foreign particles and pathogens5859

Also silver nanoparticles have been shown to be

biocompatible and capable of interacting with cells in human body without disrupting

cellular function60

Similarly it has been demonstrated that titania is compatible with

different cells DNA and proteins in human body and does not cause toxic effects Hence

silver-titania nanomotors which swim in hydrogen peroxide may open interesting

avenues in applications like targeted drug delivery in those parts of the body where

hydrogen peroxide is present

43

42 Experimental

421 Materials and methods

Silver nitrate (999995) was purchased from Alfa Aesar Gum arabic was purchased

from MP Biomedicals Hydrogen peroxide solution 50 wt in water and ascorbic acid

were purchased from Sigma-Aldrich Nanopure deionized water was used for all

experiments and preparation of all solutions Secure-Seal imaging spacers (9 mm

diameter) were obtained from Sigma Aldrich All experiments were carried out at room

temperature of 25ordmC unless otherwise mentioned Ag-TiO2 particles were synthesized by

evaporation of titania in Semicore electron beam evaporator Particles were characterized

using field-emission scanning electron microscopy Optical microscopy experiments

were performed using Axiovert 200 MAT microscope by Carl Zeiss Particles were

tracked using Physvis particle tracking software (Figure 4 1) in which the position

information of a particle on screen is stored on a frame by frame basis The data is stored

in a dat file as X and Y co-ordinates of particles tracked on the screen This information

can be converted to diffusion coefficients of the particles by further manipulation using

MathWorks-MATLABreg software

44

Figure 4 1 Physvis particle tracking software

422 Synthesis of silver colloidal particles

Silver particles were synthesized by a nucleationgrowth type reaction developed by

Goya and Matijevic61

and modified by Velikov62

Silver nitrate was used as precursor

metal salt for synthesis of silver particles Solution A was 75 ml of 50 mM AgNO3 and

035 by weight gum arabic Solution B was 75 mL of 100 mM 1-ascorbic acid and

035 by weight gum arabic Solution B was rapidly added to solution A in a 250 mL

reaction flask The reaction flask was covered and particles were stirred for 48 hrs After

48 hours particles were allowed to settle for 2-3 hours in refrigerator and supernatant

was separated from the particles Particles were then rinsed with DI water and centrifuged

thrice to obtain pure silver particles Zeta potential of these particles was reported to be

45

around -50 to -60 mV and the size was around 1μm The size of the particles can be

varied by varying the amounts of ascorbic acid and silver nitrate Using molar ratio of 31

of ascorbic acid silver nitrate can give particles of 200 nm diameter whereas ratio of

151 can give particles of upto 5 μm diameter

423 Preparation of silver-titania (Ag-TiO2) particles

A monolayer of silver particles was prepared according to the procedure by Goldenberg

et al63

Glass slides were placed on the bottom of a petridish Water was put into the

petridish so that the entire slide was immersed in water A thin layer of hexane was then

added on top of water in the petridish A suspension of silver particles in ethanol (5

ww) was slowly introduced to the water-hexane interface in the dish After the

monolayer was formed the glass slide was lifted up through the monolayer with tweezers

The silver particles-covered glass slides were left at room temperature to dry overnight

Following drying a thin film of titania was evaporated on the glass slides in Semicore

evaporator (Figure 2 7) The spheres were partially coated with a 10 nm film of titania

Ag-TiO2 particles were removed from the glass slide by gentle sonication FE-SEM was

used to characterize the particles (Figure 4 2) The thin titania layer was not easily

distinguishable due to roughness of the particles

46

Figure 4 2 FESEM image of Ag-TiO2 particles

424 Optical microscopy experiments

Imaging spacers were placed on a glass slide Suspension of Ag-TiO2 particles was

placed on the glass slide and the particles were allowed to settle Movement of particles

was observed in DI water and videos were recorded at 50x magnification of optical

microscope for 30 sec 05 H2O2 was added to the particle suspension on the slide

Videos were recorded at 50x ranging from 30 sec to 10 min to study the movement of

silver titania particles in hydrogen peroxide solution Particles in both DI water and

hydrogen peroxide were subjected to UV light and videos were recorded to study particle

dynamics in presence of UV radiation Particle tracking was performed using Physvis

particle tracking software Atleast 30 particles in every system were tracked for 10

seconds each Particle tracking data was obtained in terms of X and Y coordinates of

particles on screen This data was manipulated using MathWorks-Matlab software and

graphs of mean square displacement of particles versus time were plotted Diffusion

coefficients of particles were calculated from these graphs

47

43 Results and Discussion

Ag-TiO2 particles demonstrated autonomous motion in the presence of H2O2 No UV

light was required for this motion Motion of the particles was accelerated by addition of

fresh H2O2 to the system When subjected to UV radiation the particles displayed

momentary acceleration for 4-5 seconds

Figure 4 3 Mechanism of motion of Ag-TiO2 nanomotors

In the presence of H2O2 silver part of the Ag-TiO2 particle converts H2O2 to H2O and O2

This may lead to propulsion of the Ag-TiO2 particle due to bubble propulsion andor self-

diffusiophoresis In the presence of UV light additional reaction takes place on the

titania part of Ag-TiO2 particle which increases the velocity of the Ag-TiO2 particles

(Figure 4 4) This UV triggered photoactivity of titania is due to hole-electron separation

caused by photons having energy equal to or greater than the band gap of titania646566

Most of the holes-electrons recombine but a small fraction move to the surface and react

with the available redox species like water and oxygen to form protons (H+) superoxide

(O2-) and hydroxyl (OH) radicals

6566 This may lead to propulsion by diffusiophoresis

as well as electrophoresis

48

Figure 4 4 Silver-titania motors move faster when subjected to UV radiation

In addition to linear motion rotational motion is also observed in several particles and

particle aggregates Aggregation of particles can lead to a net circular propulsive force

which can rotate the aggregate as shown in Figure 4 5

Figure 4 5 Rotation due to particle aggregation of Ag-TiO2 particles

Particle tracking by Physvis software revealed a significant increase in diffusion

coefficient of Ag-TiO2 particles in hydrogen peroxide solution as compared to DI water

49

The Ag-TiO2 particles have a diffusion coefficient of 1491 μm2s in hydrogen peroxide

versus only 180 μm2s in DI water Thus the Ag-TiO2 particles move around more than

10 times their body length per second in hydrogen peroxide (Figure 4 6) When

subjected to UV light the particles experience a significant acceleration in hydrogen

peroxide which lasts only for a short duration of time (4-5 sec) after which the particles

return to their original velocity The diffusion coefficient of the particles during this UV

light trigger is found to be around 5093 μm2s UV light trigger does not have any visible

effect on the Ag-TiO2 particles in DI water

Table 4 1 Translational diffusion coefficients of Ag-TiO2 particles in hydrogen

peroxide and water

Mean 05 H2O2 solution DI water

Diffusion coefficient of Ag-TiO2 (μm2s)

without UV light

1491 180

Diffusion coefficient of Ag-TiO2 (μm2s)

in the presence of UV light

5093 113

50

Figure 4 6 Mean square displacement curves of Ag-TiO2 Janus particles in hydrogen

peroxide and DI water

When higher concentration (~30 vv) of Ag-TiO2 particles in hydrogen peroxide is

subjected to UV radiation the particles exhibit pattern formation (Figure 4 7)

Aggregation of Ag-TiO2 Janus particles is observed when UV light is turned on and the

particles disaggregated when UV light is switched off This can be due to predator-prey

kind of interaction between silver and titania half of the Janus particles in the presence of

UV light Oxygen and water that are produced at the silver end by decomposition of

hydrogen peroxide are used up by titania when subjected to UV radiation This can lead

to attraction between titania and silver parts of neighboring Janus particles causing

aggregation

y = 20373x

y = 59994x

y = 71848x

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10 12

Mean

sq

uare

dis

pla

cem

en

t (μ

m2s

)

Time (sec)

Mean square displacement vs Time for Ag-TiO2

particles

05 Hydrogen peroxide and UV05 Hydrogen peroxide

DI Water

51

A UV OFF B UV ON

C UV OFF

Figure 4 7 Higher concentrations of Ag-TiO2 particles showing aggregation when UV

is turned on

52

Chapter 5

Summary and Conclusion

In summary no significant enhancement in diffusion was observed in case of enzyme

immobilized Janus particles in substrate solution Presence of a linker between enzyme

and particle did not increase the diffusion coefficient of particles Alternative enzyme

immobilization methods need to be tested like thiolation of enzyme and subsequent

binding to gold surface of a Janus particle35

or expressing the enzyme with polyhistidine

or avidin tag67

This can result in higher enzyme activity upon immobilization and may

lead to enhanced diffusion of Janus particles

Predator-prey system of GOx and HRP immobilized particles showed some promising

results Aggregation of particles and formation of fan-shaped structures by TMB

oxidation were observed under optical microscope when GOx and HRP immobilized

particles glucose and TMB were present in the system The size of the fan-shaped

structures was dependent on the size of enzyme immobilized particles This can be

indicative of some kind of interaction between the GOx and HRP immobilized particles

No conclusive aggregation of GOx and HRP immobilized particles was observed in

absence of TMB Better imaging techniques are required to study the interaction of GOx

and HRP immobilized particles at the unstable liquid-air interface where majority of the

reaction takes place Other enzyme cascade systems not requiring gaseous reactants like

glycogen synthase and glycogen phosphorylase should be tested for similar activity

53

Ag-TiO2 particles were found to exhibit autonomous motion in the presence of hydrogen

peroxide solution The motion was momentarily accelerated upon exposure to UV

radiation The particles showed a diffusion coefficient of 1491 μm2s in hydrogen

peroxide versus only 180 μm2s in DI water Upon subjecting particles to UV radiation in

presence of H2O2 diffusion coefficient increased to 5093 μm2s for a period of 4-5

seconds In addition to linear motion rotating motion could also be observed in some

particle aggregates Further development of these aggregate structures can lead to

formation of rotors at nanoscale Higher concentration of silver-titania particles in

hydrogen peroxide displayed aggregation in presence of UV light and disaggregated

when UV light was switched off This may be attributed to the predator-prey kind of

interaction between silver and titania where oxygen and water that are produced by

silver by decomposition of hydrogen peroxide are used up by titania when subjected to

UV radiation This promises to be an interesting system and needs to be further

investigated to explore its properties

54

References

1 Paxton WF Sundararajan S Mallouk TE Sen A Angew Chem Int Ed Engl

2006 45 5420-5429

2 Golestanian R Liverpool TB Adjari A Phys Rev Lett 2005 94 220801

3 httpwwwsilvermedicineorgh2o2html

4 Rami Pedahzur Ovadia Lev Badri Fattal Hillel Shuval Water Science and Technology

1995 31(5-6) 123-129

5 W Went F American Scientist 1968 56

6 Adriano Cavalcanti Tad Hogg Bijan Shirinzadeh IEEE MHS 2006 International

Symposium on Micro-NanoMechatronics and Human Science 2006 226-232

7 EM Purcell American Journal of Physics 1977 45 3-11

8 Golestanian R Phys Rev Lett 2010 105 018103(1)-018103(4)

9 S S Dukhin Z R Ulberg G L Dvornichenko B V Deryagin Russian Chemical

Bulletin 1983 31(8) 1535-1544

10 Dukhin AS Goetz PJ Ultrasound for characterizing colloids Particle sizing zeta

potential rheology Elsevier 2002 59

11 HJKeh HJTu Trends in Colloid and Interface Science XIV Progress in Colloid and

Polymer Science 2000 115 174-180

12 D Zinemanas A Nir International Journal of Multiphase Flow 1995 21(5) 787-800

55

13 Stephen Ebbens Jonathan Howse Soft matter 2010 6 726-739

14 J Vicario R Eelkema W R Browne A Meetsma R M La Crois B L Feringa

Chem Commun 2005 31 3936

15 Davide Pantarotto Wesley R Browne Ben L Feringa Chem Commun 2008 1533 -

1535

16 Lisa A Cameron Matthew J Footer Alexander van Oudenaarden Julie A Theriot

PNAS 1999 96(9) 4908-4913

17 Ali Najafi Ramin Golestanian Phys Rev E 2004 69 062901

18 Jinseok Kim Jungyul Park Sungwook Yang Jeongeun Baek Byungkyu Kim Sang

Ho Lee Eui-Sung Yoon Kukjin Chun Sukho Park Lab Chip 2007 7 1504 - 1508

19 Kurt M Dubowski Clinical Chemistry 2008 5411 1919ndash1920

20 Spector DA Yang Q Wade JB Am J Physiol Renal Physiol 2007 292(1) 467-474

21 Rhee SG Chang TS Bae YS Lee SR Kang SW J Am Soc Nephrol 2003 14(8-3)

211-215

22 Sakaue T Kapral R Mikhailov A S Eur Phys J B 2010 75 381ndash387

23 Muddana H S Sengupta S Mallouk T E Sen A Butler P J J Am Chem Soc

2010 132 (7) 2110ndash2111

24 Baker CJ Deahl K Domek J Orlandi EW Arch Biochem Biophys 2000 382(2) 232-

237

25 V Vojinovic RH Carvalho F Lemos JMS Cabral LP Fonseca BS Ferreira

Biochemical Engineering Journal 2007 35 126-135

56

26

OV Lebedeva NN Ugarova Russian Chemical Bulletin 1996 45(1) 18-25

27 Nigel C Veitch Phytochemistry 2004 65(3) 249-259

28 Kenneth H Petersen Journal of Immunological Methods 2009 340(1) 86-89

29 httpwwwbrenda-enzymesorgphpresult_flatphp4ecno=11117

30 Goldenberg L M Wagner J Stumpe J Paulke B-R Goumlrnitz E Langmuir 2002 18

5627-5629

31 httpwwwgoldbiocompdf3761-Protocol201pdf

32 Av Rovisco Pais Lisboa Portugal T amp D Technology West Yorkshire UK Journal

of Molecular Catalysis B Enzymatic 2004 28(2-3) 129-135

33 httpwwwphysicsnyuedugrierlabmethodsnode11html

34 Yoon-Mee Lee1 O-Yul Kwon Yeo-Joon Yoon Keungarp Ryu Biotechnology Letters

2006 28 39ndash43

35 JC Pyun SD Kim JW Chung Analytical Biochemistry 2005 347(2) 227-233

36 George H Pimbley Jr Mathematical Biosciences 1974 20(1-2) 27-51

37 Andy Fenton Sarah E Perkins Parasitology 2010 137 1027-1038

38 Mead James G Brownell Robert L Mammal Species of the World A Taxonomic and

Geographic Reference (3rd ed) Johns Hopkins University Press 2005 2 2142

57

39

Krause KH Schweiz Med Wochenschr 2000 130(4) 97-100

40 Irina Kareva Faina Berezovskaya Carlos Castillo-Chavez Journal of Biological

Dynamics 2010 4(4)315 - 327

41 Delaittre G Reynhout I Cornelissen J Nolte R Chemistry ndash A European Journal

2009 15 12600ndash12603

42 C Buller DH Johnson RC Tschumper Investigative Ophthalmology amp Visual Science

1990 31 2156-2163

43 Robert M Mazo Brownian Motion Fluctuations Dynamics and Applications Oxford

Science Publications 2009 57

44 Vivek K Mutalik KV Venkatesh Theoretical Biology and Medical Modelling 2005 2

19

45 Satoshi Nakamura Kunimasa Koga Biochemical and Biophysical Research

Communications 1977 78(2) 806-810

46 Krishnananda Chattopadhyay Shyamalava Mazumdar Biochemistry 2000 39 (1)

263ndash270

47 Satoshi Nakamura Yasuyuki Ogura J Biochem 1968 63 (3) 308-316

48 van Dongen SF Nallani M Cornelissen JJ Nolte RJ van Hest JC Chemistry 2009

15(5) 1107-1114

49 Ronald Bentley Methods in Enzymology 1955 1 340-345

50 Vartiainen J Raumlttouml M Paulussen S Packaging Technology and Science 2005

18 243ndash251

58

51 GA Sherbeny AA Shindia YMMM Sheriff International Journal of Agriculture

and Biology 2005 7

52 D Rando G-W Kohring F Giffhorn Appl Microbiol Biotechnol 1997 48

53 Worthington enzyme manual Worthington Biochemical Corporation

httpworthington-biochemcomgopdefaulthtml

54 httpwwwbrenda-enzymesorgphpresult_flatphp4ecno=11117

55 httpwwwoxygenphwatercom

56 P Boon Chock Sue Goo Rhee and Earl R Stadtman Annu Rev Biochem 1980 49

813-841

57 Sybil Golden P A Wals Joseph Katz Analytical Biochemistry 1977 77(2) 436-445

58 R L Baehner S K Murrmann J Davis R B Johnston Jr J Clin Invest 1975 56(3)

571ndash576

59 I Bejarano M P Terroacuten S D Paredes C Barriga A B Rodriacuteguez J A Pariente

Molecular and Cellular Biochemistry 2007 296(1-2) 77-84

60 Michael C Moulton Laura K Braydich-Stolle Mallikarjuna N Nadagouda Samantha

Kunzelman Saber M Hussain Rajender S Varma Nanoscale 2010 2 763-770

61 Goya DV Matijevic E Colloids and Surfaces A 1999 146 139

62 Velikov KP Zegers GE van Blaanderen A Langmuir 2003 19 1384

63 Goldenberg L M Wagner J Stumpe J Paulke B-R Goumlrnitz E Langmuir 2002 18

5627-5629

59

64

Banerjee S Gopal J Muraleedharan P Tyagi A K Raj B Curr Sci 2006

901378ndash1383

65 Bogomolov V N Kudinov E K Firsov Y A Sov Phys Solid State 1968 35 555

66 Kudo A Miseki Y Chem Soc Rev 2009 38 253ndash278

67 Gaj T Meyer SC Ghosh I Protein expression and Purification 2007 56 54-61

6

motion Diffusiophoretic motion of particles is caused due to concentration gradients of

solutes in an electrolytenonelectrolyte solution9 Electrophoresis is the motion of a

particle relative to a fluid under the influence of an electric field10

This occurs as the

particle carries an electric surface charge which is acted upon by an electrostatic

coulombic force of the external electric field Osmophoresis is the motion of a

vesiclefluid particle encapsulated in a semi-permeable membrane towards lower

concentration gradient when it is placed in a solution containing solute concentration

gradient1112

Thus considering the numerous mechanisms of motion known to work at

the nanoscale several suitable options are available for the development of artificial

nanomotors

12 Existing nanomotors Mechanisms and uses

Several nanomotors have been developed in the past decade or so A general principle of

working of these nanomotors is that they function by catalytically decomposing the

dissolved fuel to generate asymmetry at their surface or at their interfacial region thereby

causing propulsion13

The self-electrophoretic bimetallic nanowires developed by

WPaxton and A Sen etal consisted of a 2 μm long rod with one tip made up of Au and

the other tip made up of Pt1 These motors were synthesized by electrodeposition in an

alumina template The rods moved autonomously in a solution of hydrogen peroxide

with platinum end forward Pt oxidizes H2O2 to produce H+ and O2 and Au utilizes H

+ to

reduce H2O2 to water This generates an electron flow through the rod and a proton flow

outside the rod resulting in an electric field around the rod in the solution This electric

7

field in solution acts on the unbalanced charges within the nanorodrsquos double layer This

moves the nanorod with respect to the surrounding fuel due to the ion flux that is

generated

Bubble propulsion has been predominantly used as mechanism of motion in the motors

developed by Feringa etal His group developed a micromotor by attaching catalase to a

microparticle and placing the particle in a solution containing hydrogen peroxide This

system allowed the microparticle to move by producing oxygen bubbles from the

decomposition of hydrogen peroxide14

A similar motor was later created by Feringarsquos

group which consisted of glucose oxidase and catalase immobilized on a carbon

nanotube in a substrate solution of hydrogen peroxide Glucose oxidase converted

glucose to H2O2 which was then utilized by catalase to produce water and oxygen

thereby propelling the carbon nanotube15

Another interesting example of a self-propelled motor is the polymerization driven motor

developed by Cameron et al16

It consists of polystyrene beads immobilized with ActA

protein which catalyzes actin polymerization leading to the formation of an actin

filament which resembles the flagella of bacteria This actin polymerization propels the

beads at the speed of 001-015 μms

A theoretical model has been proposed by Nafaji and Golestanian etal which consists

of three spheres linked by rigid rods in one dimension17

The length of the rods can

switch between two specific values Due to the nonreciprocal motion of the rod this

8

model can swim in low Reynolds number systems An actual model based on

nonreciprocal motion has been developed by Kim etal in which heart cells have been

linked to an asymmetric polydimethylsiloxane robot As the heart cells beat the

asymmetric structure of the device resembles a walking movement18

Most of the nanomicromotors mentioned above are either metallic or work in

environments not compatible with human body Enzymes being an integral part of the

body can solve this problem by operating in suitable substrates already present in the

body fluids The nonreciprocal conformation changes of the enzyme as well as

diffusiophoresis and electrophoresis can conceivably be used to generate motion in case

of enzyme driven nanomotors such motors will be discussed in detail in the following

chapter

9

Chapter 2

Immobilized enzymes on Janus particles as catalytic motors

21 Introduction

Enzymes are widely found in biological systems and act on a large variety of substrates

This can make enzymes an ideal choice for powering nanomotors which need to be

biocompatible Several fluids found in the body can act as substrates to the enzymes For

example dissolved glucose which is abundantly found in the body19

is a susbtrate for

enzyme glucose oxidase Urea which is found in the urinary tract20

is a substrate for

urease Hydrogen peroxide produced by phagocytic cells in the body21

is a substrate for

horseradish peroxidase Having motors driven by enzymes can open possibilities of

targeted drug delivery to these parts of the body containing such substrates

Enzymes have already been used to generate motion of carbon nanotubes by mechanism

of bubble propulsion by Feringa etal15

Two other possible mechanisms which may be

able to power motion of nanoparticles using enzymes are asymmetrical decomposition of

substrates into products leading to self-diffusiophoresis (Figure 2 1) and nonreciprocal

changes in enzyme conformation leading to propulsion (Figure 2 2) As seen in Figure 2

1 enzymes which are immobilized on a Janus particle can breakdown the available

substrate in the solution into products causing asymmetric distribution of solutes in the

surrounding fluid This can lead to movement of particle towards the region of lower

10

solute concentration by principle of self-diffusiophoresis Figure 2-2 shows the possible

nonreciprocal changes in the conformation of a theoretical protein which can lead to

motion of the protein22

These nonreciprocal changes in conformation are triggered by the

attachment of substrate molecule and release of product molecules from the enzymersquos

active site

Figure 2 1 Enzyme functionalized Janus particle moves upon catalyzing asymmetrical

decomposition of substrate into product

11

Figure 2 2 Schematic showing idealized nonreciprocal conformation changes in a

theoretical enzyme22

Enhanced diffusion of a single enzyme molecule by catalysis in a substrate solution has

been demonstrated23

The diffusion coefficient of a single urease molecule was measured

using fluorescence correlation spectroscopy and it was observed that the diffusion of the

urease molecule increases by 16-28 by varying urea concentrations from 0001 M to 1

M Addition of enzyme inhibitors to the system curbed the enhanced diffusion This

enhanced diffusion has been attributed to the force generated by self-electrophoresis by

the enzyme in substrate solution

12

Based on the above facts it might be possible to propel a nanoparticle by asymmetric

distribution of enzymes on its surface thereby leading to self-propulsion of the particles

in substrate solution (Figure 2 1) This propulsion may be due to self-

electrophoresisself-diffusiophoresis andor due to nonreciprocal changes in enzyme

conformations Here we aim to investigate a system consisting of polystyrene particles

half coated with enzyme introduced in a suitable substrate solution The enzyme can be

directly linked to the polystyrene particle or it can be linked through a long spacer

molecule (linker) to provide more degrees of freedom to the enzyme

Several factors need to be considered for choosing enzyme for powering motion at the

nanoscale Some of the important factors are the ratio of kcatKm which denotes the

catalytic efficiency of the enzyme possible substrates stability of enzymessubstrates

ease of immobilization relationship with other enzymes in cascade reactions and

commercial availability of the enzymes The enzyme being used in the present study is

horseradish peroxidase It acts on a molecule of hydrogen peroxide and reduces it while

simultaneously oxidizing another substrate molecule In the absence of alternate

oxidizable substrate HRP can oxidize water and oxygen back to hydrogen peroxide24

thus giving an equilibrium reaction

13

211 Horseradish peroxidase

Figure 2 3 Horseradish peroxidase

Horseradish peroxidase belongs to the group of peroxidases which are a group of heme-

containing oxidoreductases that act on peroxide as hydrogen donor25

It is a redox

enzyme which contains a protoheme a complex of trivalent iron with 1358-

tetramethyl-24-divinylporphin-67-dipropionic acid as a prosthetic group It is a 44 kDa

protein which includes 33900 Da polypeptide chain bound with eight oligosaccharide

chains The carbohydrate chains improve the stability of the enzyme by protecting it

against radicals formed in the course of the reaction it catalyzes26

Horseradish

peroxidase is found in horseradish and is used widely in bioanalytical applications such

as ELISA and Western blots It decomposes hydrogen peroxide into water and oxygen

while simultaneously oxidizing another substrate27

The oxidizable substrates can be

chromogenic substrates such as 22rsquo55rsquo-tetramethyl-benzidine (TMB) and 33-

diaminobenzidine (DMB) or chemoluminescent substrates like SuperSignal and ECL

luminal28

14

Figure 2 4 Reaction catalyzed by horseradish peroxidase

The functional parameters for horseradish peroxidase include Km of 015 mM and kcat of

790 s-1

at an optimum temperature of 20 ordmC and an optimum pH of 6429

Some of the

inhibitors of HRP are 110-phenanthroline aminotriazole and bromine

22 Experimental

221 Materials and methods

Uniform carboxyl modified polystyrene microspheres 05 microm in diameter were obtained

from Polysciences Inc The size of the purchased microspheres was verified by Field

Emission Scanning Electron Microscopy (FESEM) Peroxidase Type I from

Horseradish N-(3-dimethyl-aminopropyl)-Nrsquo-ethylcarbodiimide (EDC) and hydrogen

peroxide solution 50 wt in water were purchased from Sigma-Aldrich N-

hydroxysuccinimide (NHS) ge 97 was purchased from Fluka All chemicals were of

reagent grade and used without further purification Nanopure deionized water was used

for all experiments and preparation of solutions All measurements were made at room

15

temperature (25 oC) unless mentioned otherwise The UV-Visible spectroscopy

measurements were performed using Agilent 8453A diode-array UV-Visible

Spectrophotometer with ChemStation UV-Vis Software Diffusion coefficient

measurements were performed using Nanosight LM10

222 Synthesis of Janus particles

A monolayer of particles was prepared according to the procedure (Figure 2 5) by

Goldenberg L et al30

Glass slides were placed on the bottom of a petridish Water was

put into the petridish so that the entire slide was immersed in water A thin layer of

hexane was added on top of water in the petridish A suspension of microspheres in

ethanol (5 ww) was introduced to the water-hexane interface After the monolayer was

formed the glass slide was lifted up through the monolayer with tweezers The

microsphere-covered glass slides were left to dry overnight at room temperature

Following drying a thin film of silica was evaporated on the glass slides in Semicore

evaporator The spheres were partially coated with 30 nm of silica Particles were

removed from the glass slide by gentle sonication The polystyrene-silica (PS-SiO2)

Janus particles thus obtained were washed in deionized water several times before

further immobilization steps were performed FESEM was used to confirm synthesis of

Janus particles

16

Figure 2 5 Preparation of PS-SiO2 Janus particles by evaporation

223 Immobilization of enzymes using EDCNHS coupling method

1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide and N-hydroxysulfosuccinimide

(EDCNHS) coupling procedure was followed for immobilizing horseradish peroxidase

type I on 05 μm PS-SiO2 Janus particles (Figure 2 6) In this process EDC reacts with a

carboxyl groups on the Janus particles to form an amine-reactive O-acylisourea

intermediate If this intermediate does not encounter an amine it can hydrolyze and

regenerate the carboxyl group In the presence of NHS EDC can be used to convert

carboxyl groups to amine-reactive sulfo-NHS esters which are much more stable and do

not readily hydrolyze to regenerate carboxyl groups In the current experiment

immobilization was performed in the presence as well as absence of a long spacer

17

molecule (linker) between Janus particles and enzymes The chemical 6-aminohexanoic

acid was used as the linker (Figure 2 7) HRP immobilized PS-SiO2 Janus particles were

stored in deionized water until required for further experiments

Figure 2 6 EDCNHS coupling method for immobilization of enzyme on carboxylated

polystyrene particles

Figure 2 7 Immobilization of enzyme on Janus particles with and without 6-

aminohexanoic acid linker

18

224 Enzyme assay

Assay for activity of HRP was performed using a spectrophotometric method which

involved using 4-aminoantipyrene as hydrogen donor3132

The reaction rate was

determined by measuring increase in absorbance at 510 nm resulting from the

decomposition of hydrogen peroxide One unit represents the decomposition of 1 μM of

hydrogen peroxide per minute at 25 degC and pH 70 under the specified conditions

The spectrophotometer was set to the wavelength of 510 nm and temperature of 25 degC

Phenol4-aminoantipyrene solution (14 mL) and 00017 M H2O2 solution (15 mL) were

pipetted into each cuvette The cuvette was incubated for 3-4 mins at 25degC and blank was

established A 01 mL solution of diluted enzyme was added to the above mixture and

absorbance was recorded for 4-5 mins Standard graphs of absorbance versus time were

drawn for known concentration of the free enzyme and compared to that of the

immobilized enzyme on the Janus particles The estimate of the amount of enzyme

present on the Janus particles was deduced from the difference between the quantity of

the free enzyme previously added for immobilization and the amount that was measured

to be in the supernatant after some of the enzyme became attached to the Janus particles

The experiment was carried out in triplicate to assess reproducibility

19

The formula used to calculate the activity of enzyme is

where ∆Amin denotes the change in absorbance per minute

225 Diffusion coefficient measurements using Nanosight LM10

Diffusion coefficients for HRP immobilized particles were measured using Nanosight

LM 10 nanoparticle tracking instrument with Nanoparticle Tracking Analysis (NTA) 20

software in the presence of 003 H2O2 solution as the substrate and also in the presence

of only deionized water The videos were recorded for 30 seconds at the rate of 30 frames

per second Each experiment was carried out in triplicate to assess reproducibility

Particles

visualization

zone

Microscope with

LM 10 unit

LM 10 unit

Figure 2 8 Nanosight LM 10 instrumentation

20

Nanosight LM 10 (Figure 2 8) generates videos of population of moving nanoparticles in

a liquid when illuminated by laser light The particles which pass through the laser beam

path are seen by the instrument as small points of light undergoing Brownian motion

(Figure 2 9a)

The videos are further analyzed by NTA (Nanoparticle tracking analysis) software

(Figure 2 9b) which identifies and tracks each particle independently on a frame-by-

frame basis The software also calculates and subtracts any bulk flow in the system The

mean square displacement is calculated for each particle as long as it is visible If the

particle blinks that is if it disappears momentarily and then reappears on the screen the

software will consider the same particle as two separate ones All analysis data is

exported to the excel files in terms of diffusion coefficients of the tracked particles

B A

Figure 2 9 NTA video analysis (a) Particles as they are seen on the screen during

experimentation in Nanosight LM10 (b) NTA screen as the particles are being analyzed for

their diffusion coefficients

21

23 Results

231 Enzyme assay

The activity of free HRP enzyme reported by Sigma Aldrich is 150 unitsmg However

the activity of HRP upon analysis was found to be 52158 unitsmg which is much lower

than the reported value The activity of HRP was found to be further lowered to 2133

unitsmg when it was immobilized on the PS-SiO2 Janus particles in the presence of a

linker The following formula is used to calculate enzyme activity

where ΔAmin is the change in absorbance per minute

22

Figure 2 10 Graph for the assay of solution of free enzyme containing 05mgL of HRP

ΔAmin = 01716 Unitsmg of free enzyme = 52158

During EDCNHS coupling 04 mg enzyme was added to 1 ml of 025 vv suspension

of Janus particles Thus initial concentration of enzyme in the mixture was 04 mgml

Assuming retained activity of the enzyme in supernatant solution the amount of enzyme

in supernatant was calculated to be 03868 mgml from the assay Amount of enzyme

functionalized on the Janus particles was calculated as the difference between enzyme

previously added for immobilization (04 mgml) and the enzyme present in supernatant

(03868 mgml) and was found to be 00132 mgml Enzyme assay was performed on the

enzyme immobilized Janus particles to estimate the activity of immobilized enzyme

(Figure 2 11) and was found to be 2133 unitsmg

y = 0002x + 00516

0

01

02

03

04

05

06

07

08

0 50 100 150 200 250 300 350 400

Ab

sorb

an

ce

51

0 n

m

Time (sec)

Assay of free enzyme HRP

23

Figure 2 11 Graph for the assay of of immobilized enzyme on 05 μm Janus particles

(~025 vv) ΔAmin = 01853 Unitsmg of free enzyme = 2133

232 Diffusion coefficient measurements

No significant increase was observed in diffusion coefficient of HRP-immobilized Janus

particles in the presence of H2O2 as the substrate Similar results were obtained for

diffusion coefficient measurement with and without linker moieties bridging the enzymes

and particles(Table 2 1 Table 2 2) Thus the linker did not seem to assist in increasing

the particle diffusion by improving or retaining the activity of the enzyme

y = 00015x + 00953

0

01

02

03

04

05

06

07

0 50 100 150 200 250 300 350 400

Ab

sorb

an

ce

510 n

m

Time (sec)

Assay of HRP immobilized 05 μm Janus particles

24

Table 2 1 Diffusion coefficient measurements of HRP immobilized Janus particles

without linker

HRP immobilized

Janus particles in

003 H2O2

Control - Janus

particles in

003 H2O2

Control - HRP

immobilized Janus

particles in DI water

Diffusion coefficient

(10-10

cm2s)

1359416 1051777 1308675

Standard deviation 4534801 4122075 4918597

Table 2 2 Diffusion coefficient measurements of HRP immobilized Janus particles in

the presence of linker

HRP immobilized

Janus particles in

003 H2O2

Control - Janus

particles in

003 H2O2

Control - HRP

immobilized Janus

particles in DI water

Diffusion coefficient

(10-10

cm2s)

13838 1339829 1407891

Standard deviation 5644692 5649364 5797218

25

Figure 2 12 Diffusion coefficient measurements of PS-SiO2 Janus particles without and

with 6-aminohexanoic acid linker

24 Discussion

To compare the diffusion coefficients observed during the experiments theoretical

diffusion coefficient was calculated using following equation33

r

kTD altraslation

6 Equation 21

where r is the hydrodynamic radius of particle Dtranslational is translational diffusion

coefficient of the particle T is temperature of the system η is the viscosity of fluid

through which the particles are moving and k is the Boltzmann constant From FE-SEM

images the size of the particles used was determined to be 450 nm For 450 nm diameter

0

20

40

60

80

100

120

140

160

Without linker With linker

Dif

fusio

n c

oeff

icie

nt

(10

-10 c

m2s

)

HRP immobilized 05 μm Janus particles - Diffusion coefficient measurements

HRP immobilized Janus particles in 003 H2O2

Control - Janus particles in 003 H2O2

Control - HRP immobilized Janus particles in DI water

26

particle Dtranslational is equal to 109 μm2s which is comparable to the diffusion

coefficient measured for the enzyme immobilized particles

HRP was found to be active on the Janus particles however the activity of the enzyme

was reduced upon immobilization Lower enzyme activity can be due to the coupling

method used which in this case was EDCNHS covalent binding However EDCNHS

coupling method has been shown to significantly retain the enzyme activity when the

enzyme is immobilized on carboxylated multi-walled carbon nanotubes under appropriate

conditions34

Hence EDCNHS binding method by itself is not believed to be

responsible for reduction of enzyme activity upon immobilization

Direct binding of enzyme to the Janus particle may lead to reduction in the enzyme

activity because of space constraints for the enzyme and difficulty for the passage of

substrate to the active site of the enzyme due to stearic hindrance Hence a linker was

inserted between the enzyme and Janus particle during enzyme immobilization However

inserting a linker between the enzyme and the particle did not lead to enhanced diffusion

of Janus particles or increase in enzyme activity of immobilized enzyme

Lack of enhanced diffusion can be firstly attributed to the reduction in enzyme activity

upon immobilization Enzyme activity may be retained by modifying the conditions

during EDCNHS coupling or changing the binding method altogether The degree of

immobilization of HRP on carboxylated Janus particles can possibly be affected by

several factors such as temperature the concentration of the coupling reagent EDC

27

concentration HRP concentration in solution and incubation time34

Different

combinations of these factors can be tested to find optimum conditions for

immobilization of HRP Alternate binding method can involve thiolation of HRP by the

covalent conjugation of mercaptopropionic acid and then the thiolated HRP can then be

attached on the gold surface of a polystyrene-gold Janus particle35

Thus further

modifications can be made to the system in order to retain enzyme activity on the

immobilized particles and thereby power nanomotors using enzymes

28

Chapter 3

Predator-prey system of enzyme immobilized particles

31 Introduction

Predator-prey systems are widely found in nature and play a major role in biological

processes3637

Predator-prey systems include all the interactions in which one organism

pursues andor consumes another organism One familiar predator-prey system at the

macroscale is that of a tiger chasing and consuming a deer Another not so familiar one

includes Baleen whales which eat millions of microscopic planktons at once38

At the

microscale such kind of interactions can be observed in the case of phagocytes in the

body which are capable of engulfing foreign microorganisms and killing them with a

combination of microbicidal systems39

In the case of cancer the tumor is the prey and

the mature myeloid cells in the body are the predator However the tumor here has a

defense mechanism against the predator in which the tumor produces cytokines which

inhibits the maturation of the myeloid cells and thereby prevents predation40

One similarity that can be observed in all these systems is that the prey possesses some

attribute which is desired by the predator which makes the predator chase the prey A

similar relationship is seen in case of enzymes which are involved in cascade reactions41

In cascade reactions product synthesized by one enzyme is the reactant required by

another enzyme to carry out its own catalytic reaction As such there is a predator-prey

29

kind of dependence between the two enzymes which may cause one enzyme to chase the

other enzyme when placed in suitable substrate solutions However the mechanism

hypothesized for such a system is totally different than the mechanism involved in

biological systems In biological systems such as bacterial chemotaxis or phagocytes

chasing foreign particles in human body complex biochemical reactions are responsible

for the predator-prey behavior42

In the proposed enzyme cascade system there are no

complex biochemical reactions involved The principle hypothesized for predator-prey

behavior of enzyme cascade reactions can be explained as follows Motion of synthetic

motors at nanoscale involves two components translational diffusion and rotational

Brownian diffusion At very short time scales of less than 1Dr where Dr (s-1

) is

rotational Brownian diffusion coefficient the motion of particles is unidirectional For

longer time scales of greater than 1Dr the direction of motion is random giving

poweredactive diffusion43

If the chemical homogeneity is broken that is when there is

higher concentration of a substrate present in one direction of the enzyme the enzyme

might acquire a subtle bias which may lead to a spatial aggregation of the enzymes based

on the substrate concentration gradient The bias can be a result of the relationship

between the speed of enzyme diffusion and substrate concentration where higher

substrate concentration lead to higher enzyme diffusion As a result in an enzyme

cascade system one enzyme will migrate towards higher concentration of substrate

which is present near the other enzyme thus giving rise to a predator-prey behavior

Such enzyme cascades are widely found in signaling networks and metabolic pathways in

living cells and can be used to produce predator-prey like nanomotors systems44

The

30

enzymes being used in the present study are horseradish peroxidase and glucose oxidase

Both enzymes are extremely stable4546

Glucose is the substrate for glucose oxidase

which is widely available and highly stable47

Horseradish peroxidase acts on hydrogen

peroxide It is involved in a cascade reaction with glucose oxidase which produces

hydrogen peroxide by converting glucose into gluconic acid (Figure 3 1)48

Figure 3 1 Concept of particle aggregation due to cascade reactions of immobilized

enzymes

In this experiment glucose oxidase (GOx) and horseradish peroxidase (HRP) were

immobilized on polystyrene carboxylate beads of different sizes using EDCNHS

coupling method Glucose and 3355-Tetramethylbenzidine (TMB) were dissolved

together in water and this solution was used as the substrate solution All the components

were present in a single system in DI water Aggregation of particles and precipitation of

oxidized TMB from the solution was expected to be observed when all these components

were present in such a system

Properties of horseradish peroxidase have been discussed in the previous chapter (Section

211 Horseradish peroxidase)

GOxGOx HRPHRP

Glucose + O2Gluconic acid + H2O2

TMB TMB oxidizedBlue precipitate

H2O

Aggregation

31

311 Glucose oxidase

Figure 3 2 Glucose oxidase

Glucose oxidase (GOx) belongs to the group of oxidoreductases and is also called

glucose aerodehydrogenase49

It is obtained from fungi and has been identified as an

antimicrobial enzyme50

GOx binds to β-D-glucopyranose and catalyzes the oxidation

reaction to from gluconolactone while simultaneously reducing oxygen to hydrogen

peroxide51

GOx has high substrate affinity and specificity high stability and high

turnover number which makes it a suitable biocatalyst for industrial applications52

Figure 3 3 Redox reaction of glucose catalyzed by glucose oxidase53

32

GOx is a glycoprotein with molecular weight of 155 kDa and consists of two identical

polypeptide subunits linked together by a disulfide bond The optimum pH for GOx is

55 though it is stable in a broad pH range of 4 to 7 and can be stored for a long period of

time51

The functional parameters for glucose oxidase include Km of 151 mM and kcat of

337 s-1

at temperature of 20 ordmC and pH of 754

Some of the inhibitors of GOx are 2-

Deoxy-D-glucose adenine nucleotides and NaNO3

32 Experimental

321 Materials and methods

Uniform carboxyl modified polystyrene microspheres 05 microm in diameter were obtained

from Polysciences Inc The size of the purchased microspheres was verified by FESEM

Peroxidase Type I from horseradish glucose oxidase Type II-S from Aspergillus niger

N-(3-dimethyl-aminopropyl)-Nrsquo-ethylcarbodiimide (EDC) and Hydrogen peroxide

solution 50 wt in water were purchased from Sigma-Aldrich N-hydroxysuccinimide

(NHS) was purchased from Fluka D-Glucose (99) was purchased from VWR

International Secure-Seal imaging spacers (9mm diameter) for glass slides were obtained

from Sigma Aldrich All chemicals were of reagent grade and used without further

purification Nanopure deionized water was used for all experiments and preparation of

solutions All measurements were made at room temperature (25 oC) unless mentioned

otherwise The UV-Visible measurements were performed using Agilent 8453A diode-

array UV-Visible Spectrophotometer with ChemStation UV-Vis Software Optical

33

microscopy experiments were performed using Axiovert 200 MAT microscope by Carl

Zeiss Diffusion coefficient measurements were obtained using Nanosight LM10

322 Optical microscopy experiments

The enzyme substrate system was observed under optical microscope to check for

aggregation and precipitation due to cascade reactions Several such systems were

studied Each system in general consisted of GOx and HRP immobilized on different

polystyrene particles The substrate solutions consisted of 01 M glucose and different

concentrations of TMB solution The systems were studied in the presence of all the

constituents and in the absence of glucose GOxHRP and TMB Systems consisting of

different sized HRP immobilized particles in substrate solution of 003 hydrogen

peroxide and TMB were also studied Videos were recorded over a period of 24 hours to

observe changes in the system Each experiment was performed atleast thrice to assess

reproducibility of results

323 Diffusion coefficient measurements using Nanosight LM10

Diffusion coefficient of GOx and HRP immobilized particles were measured using

Nanosight LM10 nanoparticle tracking instrument with Nanoparticle Tracking Analysis

(NTA) 20 software Diffusion coefficients were measured for different systems namely

GOx particles in 01 M glucose and oxygen both GOx and HRP functionalized particles

in 01 M glucose and oxygen along with controls for the individual systems Videos were

34

recorded for 30 seconds at the rate of 30 frames per second Each experiment was carried

out in triplicate to assess reproducibility

33 Results

331 Optical microscopy

According to the enzyme cascade model precipitation of TMB was possible only upon

the interaction of GOx and HRP immobilized particles in presence of appropriate

substrate solution Hence in the first set of experiments precipitation of TMB was used

as an indication of the type of interaction between GOx and HRP immobilized particles

Fan-shaped structures were observed when both HRP and GOx immobilized particles

were present in a substrate solution consisting of glucose and TMB These structures

started forming as a single strand and then branched out and intertwined to form the fan-

shaped structures (Figure 3 4)

Table 3 1 Observations for formation of fan-shaped structures in enzyme cascade

system in presence of different components

GOx HRP Glucose TMB Observation

Yes Yes Yes Yes

Fan-shaped structures observed particles associated with

the fan-shaped structures

No Yes Yes Yes No fan-shaped structures

Yes No Yes Yes No fan-shaped structures

Yes Yes No Yes No fan-shaped structures

Yes Yes Yes No No fan-shaped structures

35

Fan-shaped structures can also be observed in the system consisting of only HRP

immobilized particles in substrate solution of hydrogen peroxide and TMB The size of

the fan-shaped structures depends on the size of the enzyme immobilized particles Free

enzymes show extremely tiny structures which are difficult to view under the optical

microscope

Figure 3 4 Formation of fan-shaped structures when both HRP and GOx

immobilized particles were present in a substrate solution consisting of glucose and

TMB A In the initial stages single strands form on the surface of liquid (5Ox) B

Single strands branch out and intertwine to form fan-shaped structures (50x) C Many

such structures form and settle down after 20 minutes (20x)

A

C

B

36

Experiments were performed in the absence of TMB that is the system consisted of GOx

and HRP immobilized particles in glucose substrate solution In the absence of TMB the

particles were expected to move closer to each other without any precipitation due to the

cascade reaction However there was no conclusive aggregation of particles observed in

the system

Free Enzyme 005 μm HRP - immobilized particle

10 μm HRP - immobilized particle 05 μm HRP - immobilized particle

Figure 3 5 Size dependence of fan-shaped structures on the size of HRP -

immobilized particles (20x)

37

Table 3 2 Observations for particle aggregation in enzyme cascade system in presence

and absence of TMB

Glucose GOx HRP TMB Observation

Present Present Present Present Aggregation and precipitate

Present Present Present Absent No conclusive aggregation

Present Absent Present Present No aggregation or ppt

Absent Present Present Present No aggregation or ppt

Present Present Absent Present No aggregation or ppt

332 Diffusion coefficient measurements

Diffusion coefficients were measured for GOx immobilized particles of 05 μm diameter

in substrate solution of 01 M glucose and oxygen Diffusion coefficients of particles

were also measured for system consisting of GOx and HRP functionalized particles in

glucose and oxygen Each experiment was done in triplicate to assess reproducibility

Nanosight visualization chamber being a closed chamber oxygen was purged into all the

solutions before starting the experiments to ensure sufficient supply of oxygen for

catalytic reaction of GOx

38

Table 3 3 Diffusion coefficients for 05 μm GOx immobilized particles in 01 M

glucose purged with O2

Mean

GOx

particles in

01 M

glucose

Control - GOx

particles in DI

water

Control - GOx

particles in 01 M

glucose without

oxygen purging

Control -

particles in 01

M Glucose

Diffusion

coefficient

(x10-10

cm2s)

13316 12888 13827 13702

Standard

deviation 6307 4926 4424 7468

Table 3 4 Diffusion coefficients for GOx and HRP immobilized particles in 01 M

glucose and purged with O2

Mean

GOx- HRP-

functionalized

particles in 01 M

glucose

Control ndash Gox-

HRP- functionalized

particles in DI water

Control - particles

in 01 M glucose

Diffusion coefficient

(x10-10

cm2s)

13005 14434 13702

Standard deviation 5263 6622 7468

No significant increase was observed in diffusion coefficient of enzyme immobilized

particles in any of the systems

39

34 Discussion

Formation of fan-shaped structures due to patterned deposition of oxidized TMB

indicates some kind of interaction between GOx and HRP immobilized particles Particle

diameter dependence of the size of structures can be explained with a simple model in

Figure 3 6 A larger particle will have a higher surface area as compared to a smaller

particle Hence more enzyme is immobilized on the larger particle As a result higher

quantity of TMB is oxidized and precipitated by the larger particle as compared to the

smaller particle This leads to thicker strands of fan-shaped structures eventually scaling

up the whole system to give bigger fan-shaped structures

The exact reason for the formation of such fan-shaped structures which are dependent on

particle size could not be determined It is hypothesized that hydrogen peroxide andor

Figure 3 6 Model for size dependence of fan-shaped structures on the particle

diameter of enzyme immobilized particles Larger the particle more surface area is

available for precipitation of TMB which eventually leads to bigger fan-shaped

structures

Larger particle Smaller particle

40

TMB gradient in the substrate solution might be responsible for such structures If there

is higher concentration of H2O2 on one side of the particle and assuming an even

concentration distribution of TMB there will be selective precipitation in the direction of

H2O2 When several particles come in the vicinity of higher concentration of H2O2 the

precipitation lines will converge to give fan-shaped structures (Figure 3 7)

Figure 3 7 Hypothesis for formation of fan-shaped structures

In the system consisting of GOx immobilized particles HRP immobilized particles

glucose and TMB it was observed that the fan-shaped structures formed at the liquid-air

interface and eventually settled down to the bottom of the glass slide This makes sense

as oxygen is necessary for the catalytic reaction of conversion of glucose to gluconic acid

and hydrogen peroxide which takes place on the surface of GOx immobilized particle As

a result H2O2 is primarily formed at the liquid-air interface and is then used by HRP for

precipitation of TMB

Following the observations that precipitation of TMB occurs at the surface GOx and

HRP immobilized particles are also expected to interact at the surface in the absence of

TMB Due the cascade reaction it was expected that HRP immobilized particles will be

41

attracted to GOx immobilized particles in appropriate substrate solution Experiments

were carried out to visualize inter-particle interactions at the liquid-air interface

However liquid-air interface not being a stable system to image the precise interaction

of GOx and HRP immobilized particles could not be determined Significant external

disturbances were involved at the interface To enable the cascade reaction in the bulk of

the liquid oxygen was purged into the reaction system before starting the experiment

However this did not lead to significant reaction in the bulk of the liquid and the reaction

prominently took place at the liquid-air interface where the oxygen concentration was

higher

In order to observe the interaction of GOx and HRP immobilized particles better imaging

techniques need to be used to observe diffusion of particles in absence of external

disturbances at the liquid-air interface Large quantity of oxygen needs to be purged into

the system and retained to make the reaction more pronounced in the bulk of the substrate

solution Higher concentration of oxygen in water may also be achieved by using oxygen

enriched water which is commercially available55

Alternatively a different enzyme cascade system may be used which does not require a

gaseous substrate56

For example glycogen synthase synthesizes glycogen from glucose-

1-phosphate and glycogen phosphorylase breaks down glycogen to glucose-1-

phosphate57

This system does not require a gaseous substrate and might be able to

overcome the deficiencies of GOx and HRP enzyme cascade system

42

Chapter 4

Silver-titania nanomotors

41 Introduction

Bimetallic nanowires made of platinum and gold have been developed by WPaxton

etal1 The rods move autonomously in a solution of hydrogen peroxide In the present

study silver-titania (Ag-TiO2) particles are being synthesized and investigated for motion

in presence of hydrogen peroxide and UV light With proper control of conditions and

UV light trigger it can be possible to control the speed and direction of motion of these

nanoparticles

Hydrogen peroxide is produced in the human body by phagocytes as an immune response

to foreign particles and pathogens5859

Also silver nanoparticles have been shown to be

biocompatible and capable of interacting with cells in human body without disrupting

cellular function60

Similarly it has been demonstrated that titania is compatible with

different cells DNA and proteins in human body and does not cause toxic effects Hence

silver-titania nanomotors which swim in hydrogen peroxide may open interesting

avenues in applications like targeted drug delivery in those parts of the body where

hydrogen peroxide is present

43

42 Experimental

421 Materials and methods

Silver nitrate (999995) was purchased from Alfa Aesar Gum arabic was purchased

from MP Biomedicals Hydrogen peroxide solution 50 wt in water and ascorbic acid

were purchased from Sigma-Aldrich Nanopure deionized water was used for all

experiments and preparation of all solutions Secure-Seal imaging spacers (9 mm

diameter) were obtained from Sigma Aldrich All experiments were carried out at room

temperature of 25ordmC unless otherwise mentioned Ag-TiO2 particles were synthesized by

evaporation of titania in Semicore electron beam evaporator Particles were characterized

using field-emission scanning electron microscopy Optical microscopy experiments

were performed using Axiovert 200 MAT microscope by Carl Zeiss Particles were

tracked using Physvis particle tracking software (Figure 4 1) in which the position

information of a particle on screen is stored on a frame by frame basis The data is stored

in a dat file as X and Y co-ordinates of particles tracked on the screen This information

can be converted to diffusion coefficients of the particles by further manipulation using

MathWorks-MATLABreg software

44

Figure 4 1 Physvis particle tracking software

422 Synthesis of silver colloidal particles

Silver particles were synthesized by a nucleationgrowth type reaction developed by

Goya and Matijevic61

and modified by Velikov62

Silver nitrate was used as precursor

metal salt for synthesis of silver particles Solution A was 75 ml of 50 mM AgNO3 and

035 by weight gum arabic Solution B was 75 mL of 100 mM 1-ascorbic acid and

035 by weight gum arabic Solution B was rapidly added to solution A in a 250 mL

reaction flask The reaction flask was covered and particles were stirred for 48 hrs After

48 hours particles were allowed to settle for 2-3 hours in refrigerator and supernatant

was separated from the particles Particles were then rinsed with DI water and centrifuged

thrice to obtain pure silver particles Zeta potential of these particles was reported to be

45

around -50 to -60 mV and the size was around 1μm The size of the particles can be

varied by varying the amounts of ascorbic acid and silver nitrate Using molar ratio of 31

of ascorbic acid silver nitrate can give particles of 200 nm diameter whereas ratio of

151 can give particles of upto 5 μm diameter

423 Preparation of silver-titania (Ag-TiO2) particles

A monolayer of silver particles was prepared according to the procedure by Goldenberg

et al63

Glass slides were placed on the bottom of a petridish Water was put into the

petridish so that the entire slide was immersed in water A thin layer of hexane was then

added on top of water in the petridish A suspension of silver particles in ethanol (5

ww) was slowly introduced to the water-hexane interface in the dish After the

monolayer was formed the glass slide was lifted up through the monolayer with tweezers

The silver particles-covered glass slides were left at room temperature to dry overnight

Following drying a thin film of titania was evaporated on the glass slides in Semicore

evaporator (Figure 2 7) The spheres were partially coated with a 10 nm film of titania

Ag-TiO2 particles were removed from the glass slide by gentle sonication FE-SEM was

used to characterize the particles (Figure 4 2) The thin titania layer was not easily

distinguishable due to roughness of the particles

46

Figure 4 2 FESEM image of Ag-TiO2 particles

424 Optical microscopy experiments

Imaging spacers were placed on a glass slide Suspension of Ag-TiO2 particles was

placed on the glass slide and the particles were allowed to settle Movement of particles

was observed in DI water and videos were recorded at 50x magnification of optical

microscope for 30 sec 05 H2O2 was added to the particle suspension on the slide

Videos were recorded at 50x ranging from 30 sec to 10 min to study the movement of

silver titania particles in hydrogen peroxide solution Particles in both DI water and

hydrogen peroxide were subjected to UV light and videos were recorded to study particle

dynamics in presence of UV radiation Particle tracking was performed using Physvis

particle tracking software Atleast 30 particles in every system were tracked for 10

seconds each Particle tracking data was obtained in terms of X and Y coordinates of

particles on screen This data was manipulated using MathWorks-Matlab software and

graphs of mean square displacement of particles versus time were plotted Diffusion

coefficients of particles were calculated from these graphs

47

43 Results and Discussion

Ag-TiO2 particles demonstrated autonomous motion in the presence of H2O2 No UV

light was required for this motion Motion of the particles was accelerated by addition of

fresh H2O2 to the system When subjected to UV radiation the particles displayed

momentary acceleration for 4-5 seconds

Figure 4 3 Mechanism of motion of Ag-TiO2 nanomotors

In the presence of H2O2 silver part of the Ag-TiO2 particle converts H2O2 to H2O and O2

This may lead to propulsion of the Ag-TiO2 particle due to bubble propulsion andor self-

diffusiophoresis In the presence of UV light additional reaction takes place on the

titania part of Ag-TiO2 particle which increases the velocity of the Ag-TiO2 particles

(Figure 4 4) This UV triggered photoactivity of titania is due to hole-electron separation

caused by photons having energy equal to or greater than the band gap of titania646566

Most of the holes-electrons recombine but a small fraction move to the surface and react

with the available redox species like water and oxygen to form protons (H+) superoxide

(O2-) and hydroxyl (OH) radicals

6566 This may lead to propulsion by diffusiophoresis

as well as electrophoresis

48

Figure 4 4 Silver-titania motors move faster when subjected to UV radiation

In addition to linear motion rotational motion is also observed in several particles and

particle aggregates Aggregation of particles can lead to a net circular propulsive force

which can rotate the aggregate as shown in Figure 4 5

Figure 4 5 Rotation due to particle aggregation of Ag-TiO2 particles

Particle tracking by Physvis software revealed a significant increase in diffusion

coefficient of Ag-TiO2 particles in hydrogen peroxide solution as compared to DI water

49

The Ag-TiO2 particles have a diffusion coefficient of 1491 μm2s in hydrogen peroxide

versus only 180 μm2s in DI water Thus the Ag-TiO2 particles move around more than

10 times their body length per second in hydrogen peroxide (Figure 4 6) When

subjected to UV light the particles experience a significant acceleration in hydrogen

peroxide which lasts only for a short duration of time (4-5 sec) after which the particles

return to their original velocity The diffusion coefficient of the particles during this UV

light trigger is found to be around 5093 μm2s UV light trigger does not have any visible

effect on the Ag-TiO2 particles in DI water

Table 4 1 Translational diffusion coefficients of Ag-TiO2 particles in hydrogen

peroxide and water

Mean 05 H2O2 solution DI water

Diffusion coefficient of Ag-TiO2 (μm2s)

without UV light

1491 180

Diffusion coefficient of Ag-TiO2 (μm2s)

in the presence of UV light

5093 113

50

Figure 4 6 Mean square displacement curves of Ag-TiO2 Janus particles in hydrogen

peroxide and DI water

When higher concentration (~30 vv) of Ag-TiO2 particles in hydrogen peroxide is

subjected to UV radiation the particles exhibit pattern formation (Figure 4 7)

Aggregation of Ag-TiO2 Janus particles is observed when UV light is turned on and the

particles disaggregated when UV light is switched off This can be due to predator-prey

kind of interaction between silver and titania half of the Janus particles in the presence of

UV light Oxygen and water that are produced at the silver end by decomposition of

hydrogen peroxide are used up by titania when subjected to UV radiation This can lead

to attraction between titania and silver parts of neighboring Janus particles causing

aggregation

y = 20373x

y = 59994x

y = 71848x

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10 12

Mean

sq

uare

dis

pla

cem

en

t (μ

m2s

)

Time (sec)

Mean square displacement vs Time for Ag-TiO2

particles

05 Hydrogen peroxide and UV05 Hydrogen peroxide

DI Water

51

A UV OFF B UV ON

C UV OFF

Figure 4 7 Higher concentrations of Ag-TiO2 particles showing aggregation when UV

is turned on

52

Chapter 5

Summary and Conclusion

In summary no significant enhancement in diffusion was observed in case of enzyme

immobilized Janus particles in substrate solution Presence of a linker between enzyme

and particle did not increase the diffusion coefficient of particles Alternative enzyme

immobilization methods need to be tested like thiolation of enzyme and subsequent

binding to gold surface of a Janus particle35

or expressing the enzyme with polyhistidine

or avidin tag67

This can result in higher enzyme activity upon immobilization and may

lead to enhanced diffusion of Janus particles

Predator-prey system of GOx and HRP immobilized particles showed some promising

results Aggregation of particles and formation of fan-shaped structures by TMB

oxidation were observed under optical microscope when GOx and HRP immobilized

particles glucose and TMB were present in the system The size of the fan-shaped

structures was dependent on the size of enzyme immobilized particles This can be

indicative of some kind of interaction between the GOx and HRP immobilized particles

No conclusive aggregation of GOx and HRP immobilized particles was observed in

absence of TMB Better imaging techniques are required to study the interaction of GOx

and HRP immobilized particles at the unstable liquid-air interface where majority of the

reaction takes place Other enzyme cascade systems not requiring gaseous reactants like

glycogen synthase and glycogen phosphorylase should be tested for similar activity

53

Ag-TiO2 particles were found to exhibit autonomous motion in the presence of hydrogen

peroxide solution The motion was momentarily accelerated upon exposure to UV

radiation The particles showed a diffusion coefficient of 1491 μm2s in hydrogen

peroxide versus only 180 μm2s in DI water Upon subjecting particles to UV radiation in

presence of H2O2 diffusion coefficient increased to 5093 μm2s for a period of 4-5

seconds In addition to linear motion rotating motion could also be observed in some

particle aggregates Further development of these aggregate structures can lead to

formation of rotors at nanoscale Higher concentration of silver-titania particles in

hydrogen peroxide displayed aggregation in presence of UV light and disaggregated

when UV light was switched off This may be attributed to the predator-prey kind of

interaction between silver and titania where oxygen and water that are produced by

silver by decomposition of hydrogen peroxide are used up by titania when subjected to

UV radiation This promises to be an interesting system and needs to be further

investigated to explore its properties

54

References

1 Paxton WF Sundararajan S Mallouk TE Sen A Angew Chem Int Ed Engl

2006 45 5420-5429

2 Golestanian R Liverpool TB Adjari A Phys Rev Lett 2005 94 220801

3 httpwwwsilvermedicineorgh2o2html

4 Rami Pedahzur Ovadia Lev Badri Fattal Hillel Shuval Water Science and Technology

1995 31(5-6) 123-129

5 W Went F American Scientist 1968 56

6 Adriano Cavalcanti Tad Hogg Bijan Shirinzadeh IEEE MHS 2006 International

Symposium on Micro-NanoMechatronics and Human Science 2006 226-232

7 EM Purcell American Journal of Physics 1977 45 3-11

8 Golestanian R Phys Rev Lett 2010 105 018103(1)-018103(4)

9 S S Dukhin Z R Ulberg G L Dvornichenko B V Deryagin Russian Chemical

Bulletin 1983 31(8) 1535-1544

10 Dukhin AS Goetz PJ Ultrasound for characterizing colloids Particle sizing zeta

potential rheology Elsevier 2002 59

11 HJKeh HJTu Trends in Colloid and Interface Science XIV Progress in Colloid and

Polymer Science 2000 115 174-180

12 D Zinemanas A Nir International Journal of Multiphase Flow 1995 21(5) 787-800

55

13 Stephen Ebbens Jonathan Howse Soft matter 2010 6 726-739

14 J Vicario R Eelkema W R Browne A Meetsma R M La Crois B L Feringa

Chem Commun 2005 31 3936

15 Davide Pantarotto Wesley R Browne Ben L Feringa Chem Commun 2008 1533 -

1535

16 Lisa A Cameron Matthew J Footer Alexander van Oudenaarden Julie A Theriot

PNAS 1999 96(9) 4908-4913

17 Ali Najafi Ramin Golestanian Phys Rev E 2004 69 062901

18 Jinseok Kim Jungyul Park Sungwook Yang Jeongeun Baek Byungkyu Kim Sang

Ho Lee Eui-Sung Yoon Kukjin Chun Sukho Park Lab Chip 2007 7 1504 - 1508

19 Kurt M Dubowski Clinical Chemistry 2008 5411 1919ndash1920

20 Spector DA Yang Q Wade JB Am J Physiol Renal Physiol 2007 292(1) 467-474

21 Rhee SG Chang TS Bae YS Lee SR Kang SW J Am Soc Nephrol 2003 14(8-3)

211-215

22 Sakaue T Kapral R Mikhailov A S Eur Phys J B 2010 75 381ndash387

23 Muddana H S Sengupta S Mallouk T E Sen A Butler P J J Am Chem Soc

2010 132 (7) 2110ndash2111

24 Baker CJ Deahl K Domek J Orlandi EW Arch Biochem Biophys 2000 382(2) 232-

237

25 V Vojinovic RH Carvalho F Lemos JMS Cabral LP Fonseca BS Ferreira

Biochemical Engineering Journal 2007 35 126-135

56

26

OV Lebedeva NN Ugarova Russian Chemical Bulletin 1996 45(1) 18-25

27 Nigel C Veitch Phytochemistry 2004 65(3) 249-259

28 Kenneth H Petersen Journal of Immunological Methods 2009 340(1) 86-89

29 httpwwwbrenda-enzymesorgphpresult_flatphp4ecno=11117

30 Goldenberg L M Wagner J Stumpe J Paulke B-R Goumlrnitz E Langmuir 2002 18

5627-5629

31 httpwwwgoldbiocompdf3761-Protocol201pdf

32 Av Rovisco Pais Lisboa Portugal T amp D Technology West Yorkshire UK Journal

of Molecular Catalysis B Enzymatic 2004 28(2-3) 129-135

33 httpwwwphysicsnyuedugrierlabmethodsnode11html

34 Yoon-Mee Lee1 O-Yul Kwon Yeo-Joon Yoon Keungarp Ryu Biotechnology Letters

2006 28 39ndash43

35 JC Pyun SD Kim JW Chung Analytical Biochemistry 2005 347(2) 227-233

36 George H Pimbley Jr Mathematical Biosciences 1974 20(1-2) 27-51

37 Andy Fenton Sarah E Perkins Parasitology 2010 137 1027-1038

38 Mead James G Brownell Robert L Mammal Species of the World A Taxonomic and

Geographic Reference (3rd ed) Johns Hopkins University Press 2005 2 2142

57

39

Krause KH Schweiz Med Wochenschr 2000 130(4) 97-100

40 Irina Kareva Faina Berezovskaya Carlos Castillo-Chavez Journal of Biological

Dynamics 2010 4(4)315 - 327

41 Delaittre G Reynhout I Cornelissen J Nolte R Chemistry ndash A European Journal

2009 15 12600ndash12603

42 C Buller DH Johnson RC Tschumper Investigative Ophthalmology amp Visual Science

1990 31 2156-2163

43 Robert M Mazo Brownian Motion Fluctuations Dynamics and Applications Oxford

Science Publications 2009 57

44 Vivek K Mutalik KV Venkatesh Theoretical Biology and Medical Modelling 2005 2

19

45 Satoshi Nakamura Kunimasa Koga Biochemical and Biophysical Research

Communications 1977 78(2) 806-810

46 Krishnananda Chattopadhyay Shyamalava Mazumdar Biochemistry 2000 39 (1)

263ndash270

47 Satoshi Nakamura Yasuyuki Ogura J Biochem 1968 63 (3) 308-316

48 van Dongen SF Nallani M Cornelissen JJ Nolte RJ van Hest JC Chemistry 2009

15(5) 1107-1114

49 Ronald Bentley Methods in Enzymology 1955 1 340-345

50 Vartiainen J Raumlttouml M Paulussen S Packaging Technology and Science 2005

18 243ndash251

58

51 GA Sherbeny AA Shindia YMMM Sheriff International Journal of Agriculture

and Biology 2005 7

52 D Rando G-W Kohring F Giffhorn Appl Microbiol Biotechnol 1997 48

53 Worthington enzyme manual Worthington Biochemical Corporation

httpworthington-biochemcomgopdefaulthtml

54 httpwwwbrenda-enzymesorgphpresult_flatphp4ecno=11117

55 httpwwwoxygenphwatercom

56 P Boon Chock Sue Goo Rhee and Earl R Stadtman Annu Rev Biochem 1980 49

813-841

57 Sybil Golden P A Wals Joseph Katz Analytical Biochemistry 1977 77(2) 436-445

58 R L Baehner S K Murrmann J Davis R B Johnston Jr J Clin Invest 1975 56(3)

571ndash576

59 I Bejarano M P Terroacuten S D Paredes C Barriga A B Rodriacuteguez J A Pariente

Molecular and Cellular Biochemistry 2007 296(1-2) 77-84

60 Michael C Moulton Laura K Braydich-Stolle Mallikarjuna N Nadagouda Samantha

Kunzelman Saber M Hussain Rajender S Varma Nanoscale 2010 2 763-770

61 Goya DV Matijevic E Colloids and Surfaces A 1999 146 139

62 Velikov KP Zegers GE van Blaanderen A Langmuir 2003 19 1384

63 Goldenberg L M Wagner J Stumpe J Paulke B-R Goumlrnitz E Langmuir 2002 18

5627-5629

59

64

Banerjee S Gopal J Muraleedharan P Tyagi A K Raj B Curr Sci 2006

901378ndash1383

65 Bogomolov V N Kudinov E K Firsov Y A Sov Phys Solid State 1968 35 555

66 Kudo A Miseki Y Chem Soc Rev 2009 38 253ndash278

67 Gaj T Meyer SC Ghosh I Protein expression and Purification 2007 56 54-61

7

field in solution acts on the unbalanced charges within the nanorodrsquos double layer This

moves the nanorod with respect to the surrounding fuel due to the ion flux that is

generated

Bubble propulsion has been predominantly used as mechanism of motion in the motors

developed by Feringa etal His group developed a micromotor by attaching catalase to a

microparticle and placing the particle in a solution containing hydrogen peroxide This

system allowed the microparticle to move by producing oxygen bubbles from the

decomposition of hydrogen peroxide14

A similar motor was later created by Feringarsquos

group which consisted of glucose oxidase and catalase immobilized on a carbon

nanotube in a substrate solution of hydrogen peroxide Glucose oxidase converted

glucose to H2O2 which was then utilized by catalase to produce water and oxygen

thereby propelling the carbon nanotube15

Another interesting example of a self-propelled motor is the polymerization driven motor

developed by Cameron et al16

It consists of polystyrene beads immobilized with ActA

protein which catalyzes actin polymerization leading to the formation of an actin

filament which resembles the flagella of bacteria This actin polymerization propels the

beads at the speed of 001-015 μms

A theoretical model has been proposed by Nafaji and Golestanian etal which consists

of three spheres linked by rigid rods in one dimension17

The length of the rods can

switch between two specific values Due to the nonreciprocal motion of the rod this

8

model can swim in low Reynolds number systems An actual model based on

nonreciprocal motion has been developed by Kim etal in which heart cells have been

linked to an asymmetric polydimethylsiloxane robot As the heart cells beat the

asymmetric structure of the device resembles a walking movement18

Most of the nanomicromotors mentioned above are either metallic or work in

environments not compatible with human body Enzymes being an integral part of the

body can solve this problem by operating in suitable substrates already present in the

body fluids The nonreciprocal conformation changes of the enzyme as well as

diffusiophoresis and electrophoresis can conceivably be used to generate motion in case

of enzyme driven nanomotors such motors will be discussed in detail in the following

chapter

9

Chapter 2

Immobilized enzymes on Janus particles as catalytic motors

21 Introduction

Enzymes are widely found in biological systems and act on a large variety of substrates

This can make enzymes an ideal choice for powering nanomotors which need to be

biocompatible Several fluids found in the body can act as substrates to the enzymes For

example dissolved glucose which is abundantly found in the body19

is a susbtrate for

enzyme glucose oxidase Urea which is found in the urinary tract20

is a substrate for

urease Hydrogen peroxide produced by phagocytic cells in the body21

is a substrate for

horseradish peroxidase Having motors driven by enzymes can open possibilities of

targeted drug delivery to these parts of the body containing such substrates

Enzymes have already been used to generate motion of carbon nanotubes by mechanism

of bubble propulsion by Feringa etal15

Two other possible mechanisms which may be

able to power motion of nanoparticles using enzymes are asymmetrical decomposition of

substrates into products leading to self-diffusiophoresis (Figure 2 1) and nonreciprocal

changes in enzyme conformation leading to propulsion (Figure 2 2) As seen in Figure 2

1 enzymes which are immobilized on a Janus particle can breakdown the available

substrate in the solution into products causing asymmetric distribution of solutes in the

surrounding fluid This can lead to movement of particle towards the region of lower

10

solute concentration by principle of self-diffusiophoresis Figure 2-2 shows the possible

nonreciprocal changes in the conformation of a theoretical protein which can lead to

motion of the protein22

These nonreciprocal changes in conformation are triggered by the

attachment of substrate molecule and release of product molecules from the enzymersquos

active site

Figure 2 1 Enzyme functionalized Janus particle moves upon catalyzing asymmetrical

decomposition of substrate into product

11

Figure 2 2 Schematic showing idealized nonreciprocal conformation changes in a

theoretical enzyme22

Enhanced diffusion of a single enzyme molecule by catalysis in a substrate solution has

been demonstrated23

The diffusion coefficient of a single urease molecule was measured

using fluorescence correlation spectroscopy and it was observed that the diffusion of the

urease molecule increases by 16-28 by varying urea concentrations from 0001 M to 1

M Addition of enzyme inhibitors to the system curbed the enhanced diffusion This

enhanced diffusion has been attributed to the force generated by self-electrophoresis by

the enzyme in substrate solution

12

Based on the above facts it might be possible to propel a nanoparticle by asymmetric

distribution of enzymes on its surface thereby leading to self-propulsion of the particles

in substrate solution (Figure 2 1) This propulsion may be due to self-

electrophoresisself-diffusiophoresis andor due to nonreciprocal changes in enzyme

conformations Here we aim to investigate a system consisting of polystyrene particles

half coated with enzyme introduced in a suitable substrate solution The enzyme can be

directly linked to the polystyrene particle or it can be linked through a long spacer

molecule (linker) to provide more degrees of freedom to the enzyme

Several factors need to be considered for choosing enzyme for powering motion at the

nanoscale Some of the important factors are the ratio of kcatKm which denotes the

catalytic efficiency of the enzyme possible substrates stability of enzymessubstrates

ease of immobilization relationship with other enzymes in cascade reactions and

commercial availability of the enzymes The enzyme being used in the present study is

horseradish peroxidase It acts on a molecule of hydrogen peroxide and reduces it while

simultaneously oxidizing another substrate molecule In the absence of alternate

oxidizable substrate HRP can oxidize water and oxygen back to hydrogen peroxide24

thus giving an equilibrium reaction

13

211 Horseradish peroxidase

Figure 2 3 Horseradish peroxidase

Horseradish peroxidase belongs to the group of peroxidases which are a group of heme-

containing oxidoreductases that act on peroxide as hydrogen donor25

It is a redox

enzyme which contains a protoheme a complex of trivalent iron with 1358-

tetramethyl-24-divinylporphin-67-dipropionic acid as a prosthetic group It is a 44 kDa

protein which includes 33900 Da polypeptide chain bound with eight oligosaccharide

chains The carbohydrate chains improve the stability of the enzyme by protecting it

against radicals formed in the course of the reaction it catalyzes26

Horseradish

peroxidase is found in horseradish and is used widely in bioanalytical applications such

as ELISA and Western blots It decomposes hydrogen peroxide into water and oxygen

while simultaneously oxidizing another substrate27

The oxidizable substrates can be

chromogenic substrates such as 22rsquo55rsquo-tetramethyl-benzidine (TMB) and 33-

diaminobenzidine (DMB) or chemoluminescent substrates like SuperSignal and ECL

luminal28

14

Figure 2 4 Reaction catalyzed by horseradish peroxidase

The functional parameters for horseradish peroxidase include Km of 015 mM and kcat of

790 s-1

at an optimum temperature of 20 ordmC and an optimum pH of 6429

Some of the

inhibitors of HRP are 110-phenanthroline aminotriazole and bromine

22 Experimental

221 Materials and methods

Uniform carboxyl modified polystyrene microspheres 05 microm in diameter were obtained

from Polysciences Inc The size of the purchased microspheres was verified by Field

Emission Scanning Electron Microscopy (FESEM) Peroxidase Type I from

Horseradish N-(3-dimethyl-aminopropyl)-Nrsquo-ethylcarbodiimide (EDC) and hydrogen

peroxide solution 50 wt in water were purchased from Sigma-Aldrich N-

hydroxysuccinimide (NHS) ge 97 was purchased from Fluka All chemicals were of

reagent grade and used without further purification Nanopure deionized water was used

for all experiments and preparation of solutions All measurements were made at room

15

temperature (25 oC) unless mentioned otherwise The UV-Visible spectroscopy

measurements were performed using Agilent 8453A diode-array UV-Visible

Spectrophotometer with ChemStation UV-Vis Software Diffusion coefficient

measurements were performed using Nanosight LM10

222 Synthesis of Janus particles

A monolayer of particles was prepared according to the procedure (Figure 2 5) by

Goldenberg L et al30

Glass slides were placed on the bottom of a petridish Water was

put into the petridish so that the entire slide was immersed in water A thin layer of

hexane was added on top of water in the petridish A suspension of microspheres in

ethanol (5 ww) was introduced to the water-hexane interface After the monolayer was

formed the glass slide was lifted up through the monolayer with tweezers The

microsphere-covered glass slides were left to dry overnight at room temperature

Following drying a thin film of silica was evaporated on the glass slides in Semicore

evaporator The spheres were partially coated with 30 nm of silica Particles were

removed from the glass slide by gentle sonication The polystyrene-silica (PS-SiO2)

Janus particles thus obtained were washed in deionized water several times before

further immobilization steps were performed FESEM was used to confirm synthesis of

Janus particles

16

Figure 2 5 Preparation of PS-SiO2 Janus particles by evaporation

223 Immobilization of enzymes using EDCNHS coupling method

1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide and N-hydroxysulfosuccinimide

(EDCNHS) coupling procedure was followed for immobilizing horseradish peroxidase

type I on 05 μm PS-SiO2 Janus particles (Figure 2 6) In this process EDC reacts with a

carboxyl groups on the Janus particles to form an amine-reactive O-acylisourea

intermediate If this intermediate does not encounter an amine it can hydrolyze and

regenerate the carboxyl group In the presence of NHS EDC can be used to convert

carboxyl groups to amine-reactive sulfo-NHS esters which are much more stable and do

not readily hydrolyze to regenerate carboxyl groups In the current experiment

immobilization was performed in the presence as well as absence of a long spacer

17

molecule (linker) between Janus particles and enzymes The chemical 6-aminohexanoic

acid was used as the linker (Figure 2 7) HRP immobilized PS-SiO2 Janus particles were

stored in deionized water until required for further experiments

Figure 2 6 EDCNHS coupling method for immobilization of enzyme on carboxylated

polystyrene particles

Figure 2 7 Immobilization of enzyme on Janus particles with and without 6-

aminohexanoic acid linker

18

224 Enzyme assay

Assay for activity of HRP was performed using a spectrophotometric method which

involved using 4-aminoantipyrene as hydrogen donor3132

The reaction rate was

determined by measuring increase in absorbance at 510 nm resulting from the

decomposition of hydrogen peroxide One unit represents the decomposition of 1 μM of

hydrogen peroxide per minute at 25 degC and pH 70 under the specified conditions

The spectrophotometer was set to the wavelength of 510 nm and temperature of 25 degC

Phenol4-aminoantipyrene solution (14 mL) and 00017 M H2O2 solution (15 mL) were

pipetted into each cuvette The cuvette was incubated for 3-4 mins at 25degC and blank was

established A 01 mL solution of diluted enzyme was added to the above mixture and

absorbance was recorded for 4-5 mins Standard graphs of absorbance versus time were

drawn for known concentration of the free enzyme and compared to that of the

immobilized enzyme on the Janus particles The estimate of the amount of enzyme

present on the Janus particles was deduced from the difference between the quantity of

the free enzyme previously added for immobilization and the amount that was measured

to be in the supernatant after some of the enzyme became attached to the Janus particles

The experiment was carried out in triplicate to assess reproducibility

19

The formula used to calculate the activity of enzyme is

where ∆Amin denotes the change in absorbance per minute

225 Diffusion coefficient measurements using Nanosight LM10

Diffusion coefficients for HRP immobilized particles were measured using Nanosight

LM 10 nanoparticle tracking instrument with Nanoparticle Tracking Analysis (NTA) 20

software in the presence of 003 H2O2 solution as the substrate and also in the presence

of only deionized water The videos were recorded for 30 seconds at the rate of 30 frames

per second Each experiment was carried out in triplicate to assess reproducibility

Particles

visualization

zone

Microscope with

LM 10 unit

LM 10 unit

Figure 2 8 Nanosight LM 10 instrumentation

20

Nanosight LM 10 (Figure 2 8) generates videos of population of moving nanoparticles in

a liquid when illuminated by laser light The particles which pass through the laser beam

path are seen by the instrument as small points of light undergoing Brownian motion

(Figure 2 9a)

The videos are further analyzed by NTA (Nanoparticle tracking analysis) software

(Figure 2 9b) which identifies and tracks each particle independently on a frame-by-

frame basis The software also calculates and subtracts any bulk flow in the system The

mean square displacement is calculated for each particle as long as it is visible If the

particle blinks that is if it disappears momentarily and then reappears on the screen the

software will consider the same particle as two separate ones All analysis data is

exported to the excel files in terms of diffusion coefficients of the tracked particles

B A

Figure 2 9 NTA video analysis (a) Particles as they are seen on the screen during

experimentation in Nanosight LM10 (b) NTA screen as the particles are being analyzed for

their diffusion coefficients

21

23 Results

231 Enzyme assay

The activity of free HRP enzyme reported by Sigma Aldrich is 150 unitsmg However

the activity of HRP upon analysis was found to be 52158 unitsmg which is much lower

than the reported value The activity of HRP was found to be further lowered to 2133

unitsmg when it was immobilized on the PS-SiO2 Janus particles in the presence of a

linker The following formula is used to calculate enzyme activity

where ΔAmin is the change in absorbance per minute

22

Figure 2 10 Graph for the assay of solution of free enzyme containing 05mgL of HRP

ΔAmin = 01716 Unitsmg of free enzyme = 52158

During EDCNHS coupling 04 mg enzyme was added to 1 ml of 025 vv suspension

of Janus particles Thus initial concentration of enzyme in the mixture was 04 mgml

Assuming retained activity of the enzyme in supernatant solution the amount of enzyme

in supernatant was calculated to be 03868 mgml from the assay Amount of enzyme

functionalized on the Janus particles was calculated as the difference between enzyme

previously added for immobilization (04 mgml) and the enzyme present in supernatant

(03868 mgml) and was found to be 00132 mgml Enzyme assay was performed on the

enzyme immobilized Janus particles to estimate the activity of immobilized enzyme

(Figure 2 11) and was found to be 2133 unitsmg

y = 0002x + 00516

0

01

02

03

04

05

06

07

08

0 50 100 150 200 250 300 350 400

Ab

sorb

an

ce

51

0 n

m

Time (sec)

Assay of free enzyme HRP

23

Figure 2 11 Graph for the assay of of immobilized enzyme on 05 μm Janus particles

(~025 vv) ΔAmin = 01853 Unitsmg of free enzyme = 2133

232 Diffusion coefficient measurements

No significant increase was observed in diffusion coefficient of HRP-immobilized Janus

particles in the presence of H2O2 as the substrate Similar results were obtained for

diffusion coefficient measurement with and without linker moieties bridging the enzymes

and particles(Table 2 1 Table 2 2) Thus the linker did not seem to assist in increasing

the particle diffusion by improving or retaining the activity of the enzyme

y = 00015x + 00953

0

01

02

03

04

05

06

07

0 50 100 150 200 250 300 350 400

Ab

sorb

an

ce

510 n

m

Time (sec)

Assay of HRP immobilized 05 μm Janus particles

24

Table 2 1 Diffusion coefficient measurements of HRP immobilized Janus particles

without linker

HRP immobilized

Janus particles in

003 H2O2

Control - Janus

particles in

003 H2O2

Control - HRP

immobilized Janus

particles in DI water

Diffusion coefficient

(10-10

cm2s)

1359416 1051777 1308675

Standard deviation 4534801 4122075 4918597

Table 2 2 Diffusion coefficient measurements of HRP immobilized Janus particles in

the presence of linker

HRP immobilized

Janus particles in

003 H2O2

Control - Janus

particles in

003 H2O2

Control - HRP

immobilized Janus

particles in DI water

Diffusion coefficient

(10-10

cm2s)

13838 1339829 1407891

Standard deviation 5644692 5649364 5797218

25

Figure 2 12 Diffusion coefficient measurements of PS-SiO2 Janus particles without and

with 6-aminohexanoic acid linker

24 Discussion

To compare the diffusion coefficients observed during the experiments theoretical

diffusion coefficient was calculated using following equation33

r

kTD altraslation

6 Equation 21

where r is the hydrodynamic radius of particle Dtranslational is translational diffusion

coefficient of the particle T is temperature of the system η is the viscosity of fluid

through which the particles are moving and k is the Boltzmann constant From FE-SEM

images the size of the particles used was determined to be 450 nm For 450 nm diameter

0

20

40

60

80

100

120

140

160

Without linker With linker

Dif

fusio

n c

oeff

icie

nt

(10

-10 c

m2s

)

HRP immobilized 05 μm Janus particles - Diffusion coefficient measurements

HRP immobilized Janus particles in 003 H2O2

Control - Janus particles in 003 H2O2

Control - HRP immobilized Janus particles in DI water

26

particle Dtranslational is equal to 109 μm2s which is comparable to the diffusion

coefficient measured for the enzyme immobilized particles

HRP was found to be active on the Janus particles however the activity of the enzyme

was reduced upon immobilization Lower enzyme activity can be due to the coupling

method used which in this case was EDCNHS covalent binding However EDCNHS

coupling method has been shown to significantly retain the enzyme activity when the

enzyme is immobilized on carboxylated multi-walled carbon nanotubes under appropriate

conditions34

Hence EDCNHS binding method by itself is not believed to be

responsible for reduction of enzyme activity upon immobilization

Direct binding of enzyme to the Janus particle may lead to reduction in the enzyme

activity because of space constraints for the enzyme and difficulty for the passage of

substrate to the active site of the enzyme due to stearic hindrance Hence a linker was

inserted between the enzyme and Janus particle during enzyme immobilization However

inserting a linker between the enzyme and the particle did not lead to enhanced diffusion

of Janus particles or increase in enzyme activity of immobilized enzyme

Lack of enhanced diffusion can be firstly attributed to the reduction in enzyme activity

upon immobilization Enzyme activity may be retained by modifying the conditions

during EDCNHS coupling or changing the binding method altogether The degree of

immobilization of HRP on carboxylated Janus particles can possibly be affected by

several factors such as temperature the concentration of the coupling reagent EDC

27

concentration HRP concentration in solution and incubation time34

Different

combinations of these factors can be tested to find optimum conditions for

immobilization of HRP Alternate binding method can involve thiolation of HRP by the

covalent conjugation of mercaptopropionic acid and then the thiolated HRP can then be

attached on the gold surface of a polystyrene-gold Janus particle35

Thus further

modifications can be made to the system in order to retain enzyme activity on the

immobilized particles and thereby power nanomotors using enzymes

28

Chapter 3

Predator-prey system of enzyme immobilized particles

31 Introduction

Predator-prey systems are widely found in nature and play a major role in biological

processes3637

Predator-prey systems include all the interactions in which one organism

pursues andor consumes another organism One familiar predator-prey system at the

macroscale is that of a tiger chasing and consuming a deer Another not so familiar one

includes Baleen whales which eat millions of microscopic planktons at once38

At the

microscale such kind of interactions can be observed in the case of phagocytes in the

body which are capable of engulfing foreign microorganisms and killing them with a

combination of microbicidal systems39

In the case of cancer the tumor is the prey and

the mature myeloid cells in the body are the predator However the tumor here has a

defense mechanism against the predator in which the tumor produces cytokines which

inhibits the maturation of the myeloid cells and thereby prevents predation40

One similarity that can be observed in all these systems is that the prey possesses some

attribute which is desired by the predator which makes the predator chase the prey A

similar relationship is seen in case of enzymes which are involved in cascade reactions41

In cascade reactions product synthesized by one enzyme is the reactant required by

another enzyme to carry out its own catalytic reaction As such there is a predator-prey

29

kind of dependence between the two enzymes which may cause one enzyme to chase the

other enzyme when placed in suitable substrate solutions However the mechanism

hypothesized for such a system is totally different than the mechanism involved in

biological systems In biological systems such as bacterial chemotaxis or phagocytes

chasing foreign particles in human body complex biochemical reactions are responsible

for the predator-prey behavior42

In the proposed enzyme cascade system there are no

complex biochemical reactions involved The principle hypothesized for predator-prey

behavior of enzyme cascade reactions can be explained as follows Motion of synthetic

motors at nanoscale involves two components translational diffusion and rotational

Brownian diffusion At very short time scales of less than 1Dr where Dr (s-1

) is

rotational Brownian diffusion coefficient the motion of particles is unidirectional For

longer time scales of greater than 1Dr the direction of motion is random giving

poweredactive diffusion43

If the chemical homogeneity is broken that is when there is

higher concentration of a substrate present in one direction of the enzyme the enzyme

might acquire a subtle bias which may lead to a spatial aggregation of the enzymes based

on the substrate concentration gradient The bias can be a result of the relationship

between the speed of enzyme diffusion and substrate concentration where higher

substrate concentration lead to higher enzyme diffusion As a result in an enzyme

cascade system one enzyme will migrate towards higher concentration of substrate

which is present near the other enzyme thus giving rise to a predator-prey behavior

Such enzyme cascades are widely found in signaling networks and metabolic pathways in

living cells and can be used to produce predator-prey like nanomotors systems44

The

30

enzymes being used in the present study are horseradish peroxidase and glucose oxidase

Both enzymes are extremely stable4546

Glucose is the substrate for glucose oxidase

which is widely available and highly stable47

Horseradish peroxidase acts on hydrogen

peroxide It is involved in a cascade reaction with glucose oxidase which produces

hydrogen peroxide by converting glucose into gluconic acid (Figure 3 1)48

Figure 3 1 Concept of particle aggregation due to cascade reactions of immobilized

enzymes

In this experiment glucose oxidase (GOx) and horseradish peroxidase (HRP) were

immobilized on polystyrene carboxylate beads of different sizes using EDCNHS

coupling method Glucose and 3355-Tetramethylbenzidine (TMB) were dissolved

together in water and this solution was used as the substrate solution All the components

were present in a single system in DI water Aggregation of particles and precipitation of

oxidized TMB from the solution was expected to be observed when all these components

were present in such a system

Properties of horseradish peroxidase have been discussed in the previous chapter (Section

211 Horseradish peroxidase)

GOxGOx HRPHRP

Glucose + O2Gluconic acid + H2O2

TMB TMB oxidizedBlue precipitate

H2O

Aggregation

31

311 Glucose oxidase

Figure 3 2 Glucose oxidase

Glucose oxidase (GOx) belongs to the group of oxidoreductases and is also called

glucose aerodehydrogenase49

It is obtained from fungi and has been identified as an

antimicrobial enzyme50

GOx binds to β-D-glucopyranose and catalyzes the oxidation

reaction to from gluconolactone while simultaneously reducing oxygen to hydrogen

peroxide51

GOx has high substrate affinity and specificity high stability and high

turnover number which makes it a suitable biocatalyst for industrial applications52

Figure 3 3 Redox reaction of glucose catalyzed by glucose oxidase53

32

GOx is a glycoprotein with molecular weight of 155 kDa and consists of two identical

polypeptide subunits linked together by a disulfide bond The optimum pH for GOx is

55 though it is stable in a broad pH range of 4 to 7 and can be stored for a long period of

time51

The functional parameters for glucose oxidase include Km of 151 mM and kcat of

337 s-1

at temperature of 20 ordmC and pH of 754

Some of the inhibitors of GOx are 2-

Deoxy-D-glucose adenine nucleotides and NaNO3

32 Experimental

321 Materials and methods

Uniform carboxyl modified polystyrene microspheres 05 microm in diameter were obtained

from Polysciences Inc The size of the purchased microspheres was verified by FESEM

Peroxidase Type I from horseradish glucose oxidase Type II-S from Aspergillus niger

N-(3-dimethyl-aminopropyl)-Nrsquo-ethylcarbodiimide (EDC) and Hydrogen peroxide

solution 50 wt in water were purchased from Sigma-Aldrich N-hydroxysuccinimide

(NHS) was purchased from Fluka D-Glucose (99) was purchased from VWR

International Secure-Seal imaging spacers (9mm diameter) for glass slides were obtained

from Sigma Aldrich All chemicals were of reagent grade and used without further

purification Nanopure deionized water was used for all experiments and preparation of

solutions All measurements were made at room temperature (25 oC) unless mentioned

otherwise The UV-Visible measurements were performed using Agilent 8453A diode-

array UV-Visible Spectrophotometer with ChemStation UV-Vis Software Optical

33

microscopy experiments were performed using Axiovert 200 MAT microscope by Carl

Zeiss Diffusion coefficient measurements were obtained using Nanosight LM10

322 Optical microscopy experiments

The enzyme substrate system was observed under optical microscope to check for

aggregation and precipitation due to cascade reactions Several such systems were

studied Each system in general consisted of GOx and HRP immobilized on different

polystyrene particles The substrate solutions consisted of 01 M glucose and different

concentrations of TMB solution The systems were studied in the presence of all the

constituents and in the absence of glucose GOxHRP and TMB Systems consisting of

different sized HRP immobilized particles in substrate solution of 003 hydrogen

peroxide and TMB were also studied Videos were recorded over a period of 24 hours to

observe changes in the system Each experiment was performed atleast thrice to assess

reproducibility of results

323 Diffusion coefficient measurements using Nanosight LM10

Diffusion coefficient of GOx and HRP immobilized particles were measured using

Nanosight LM10 nanoparticle tracking instrument with Nanoparticle Tracking Analysis

(NTA) 20 software Diffusion coefficients were measured for different systems namely

GOx particles in 01 M glucose and oxygen both GOx and HRP functionalized particles

in 01 M glucose and oxygen along with controls for the individual systems Videos were

34

recorded for 30 seconds at the rate of 30 frames per second Each experiment was carried

out in triplicate to assess reproducibility

33 Results

331 Optical microscopy

According to the enzyme cascade model precipitation of TMB was possible only upon

the interaction of GOx and HRP immobilized particles in presence of appropriate

substrate solution Hence in the first set of experiments precipitation of TMB was used

as an indication of the type of interaction between GOx and HRP immobilized particles

Fan-shaped structures were observed when both HRP and GOx immobilized particles

were present in a substrate solution consisting of glucose and TMB These structures

started forming as a single strand and then branched out and intertwined to form the fan-

shaped structures (Figure 3 4)

Table 3 1 Observations for formation of fan-shaped structures in enzyme cascade

system in presence of different components

GOx HRP Glucose TMB Observation

Yes Yes Yes Yes

Fan-shaped structures observed particles associated with

the fan-shaped structures

No Yes Yes Yes No fan-shaped structures

Yes No Yes Yes No fan-shaped structures

Yes Yes No Yes No fan-shaped structures

Yes Yes Yes No No fan-shaped structures

35

Fan-shaped structures can also be observed in the system consisting of only HRP

immobilized particles in substrate solution of hydrogen peroxide and TMB The size of

the fan-shaped structures depends on the size of the enzyme immobilized particles Free

enzymes show extremely tiny structures which are difficult to view under the optical

microscope

Figure 3 4 Formation of fan-shaped structures when both HRP and GOx

immobilized particles were present in a substrate solution consisting of glucose and

TMB A In the initial stages single strands form on the surface of liquid (5Ox) B

Single strands branch out and intertwine to form fan-shaped structures (50x) C Many

such structures form and settle down after 20 minutes (20x)

A

C

B

36

Experiments were performed in the absence of TMB that is the system consisted of GOx

and HRP immobilized particles in glucose substrate solution In the absence of TMB the

particles were expected to move closer to each other without any precipitation due to the

cascade reaction However there was no conclusive aggregation of particles observed in

the system

Free Enzyme 005 μm HRP - immobilized particle

10 μm HRP - immobilized particle 05 μm HRP - immobilized particle

Figure 3 5 Size dependence of fan-shaped structures on the size of HRP -

immobilized particles (20x)

37

Table 3 2 Observations for particle aggregation in enzyme cascade system in presence

and absence of TMB

Glucose GOx HRP TMB Observation

Present Present Present Present Aggregation and precipitate

Present Present Present Absent No conclusive aggregation

Present Absent Present Present No aggregation or ppt

Absent Present Present Present No aggregation or ppt

Present Present Absent Present No aggregation or ppt

332 Diffusion coefficient measurements

Diffusion coefficients were measured for GOx immobilized particles of 05 μm diameter

in substrate solution of 01 M glucose and oxygen Diffusion coefficients of particles

were also measured for system consisting of GOx and HRP functionalized particles in

glucose and oxygen Each experiment was done in triplicate to assess reproducibility

Nanosight visualization chamber being a closed chamber oxygen was purged into all the

solutions before starting the experiments to ensure sufficient supply of oxygen for

catalytic reaction of GOx

38

Table 3 3 Diffusion coefficients for 05 μm GOx immobilized particles in 01 M

glucose purged with O2

Mean

GOx

particles in

01 M

glucose

Control - GOx

particles in DI

water

Control - GOx

particles in 01 M

glucose without

oxygen purging

Control -

particles in 01

M Glucose

Diffusion

coefficient

(x10-10

cm2s)

13316 12888 13827 13702

Standard

deviation 6307 4926 4424 7468

Table 3 4 Diffusion coefficients for GOx and HRP immobilized particles in 01 M

glucose and purged with O2

Mean

GOx- HRP-

functionalized

particles in 01 M

glucose

Control ndash Gox-

HRP- functionalized

particles in DI water

Control - particles

in 01 M glucose

Diffusion coefficient

(x10-10

cm2s)

13005 14434 13702

Standard deviation 5263 6622 7468

No significant increase was observed in diffusion coefficient of enzyme immobilized

particles in any of the systems

39

34 Discussion

Formation of fan-shaped structures due to patterned deposition of oxidized TMB

indicates some kind of interaction between GOx and HRP immobilized particles Particle

diameter dependence of the size of structures can be explained with a simple model in

Figure 3 6 A larger particle will have a higher surface area as compared to a smaller

particle Hence more enzyme is immobilized on the larger particle As a result higher

quantity of TMB is oxidized and precipitated by the larger particle as compared to the

smaller particle This leads to thicker strands of fan-shaped structures eventually scaling

up the whole system to give bigger fan-shaped structures

The exact reason for the formation of such fan-shaped structures which are dependent on

particle size could not be determined It is hypothesized that hydrogen peroxide andor

Figure 3 6 Model for size dependence of fan-shaped structures on the particle

diameter of enzyme immobilized particles Larger the particle more surface area is

available for precipitation of TMB which eventually leads to bigger fan-shaped

structures

Larger particle Smaller particle

40

TMB gradient in the substrate solution might be responsible for such structures If there

is higher concentration of H2O2 on one side of the particle and assuming an even

concentration distribution of TMB there will be selective precipitation in the direction of

H2O2 When several particles come in the vicinity of higher concentration of H2O2 the

precipitation lines will converge to give fan-shaped structures (Figure 3 7)

Figure 3 7 Hypothesis for formation of fan-shaped structures

In the system consisting of GOx immobilized particles HRP immobilized particles

glucose and TMB it was observed that the fan-shaped structures formed at the liquid-air

interface and eventually settled down to the bottom of the glass slide This makes sense

as oxygen is necessary for the catalytic reaction of conversion of glucose to gluconic acid

and hydrogen peroxide which takes place on the surface of GOx immobilized particle As

a result H2O2 is primarily formed at the liquid-air interface and is then used by HRP for

precipitation of TMB

Following the observations that precipitation of TMB occurs at the surface GOx and

HRP immobilized particles are also expected to interact at the surface in the absence of

TMB Due the cascade reaction it was expected that HRP immobilized particles will be

41

attracted to GOx immobilized particles in appropriate substrate solution Experiments

were carried out to visualize inter-particle interactions at the liquid-air interface

However liquid-air interface not being a stable system to image the precise interaction

of GOx and HRP immobilized particles could not be determined Significant external

disturbances were involved at the interface To enable the cascade reaction in the bulk of

the liquid oxygen was purged into the reaction system before starting the experiment

However this did not lead to significant reaction in the bulk of the liquid and the reaction

prominently took place at the liquid-air interface where the oxygen concentration was

higher

In order to observe the interaction of GOx and HRP immobilized particles better imaging

techniques need to be used to observe diffusion of particles in absence of external

disturbances at the liquid-air interface Large quantity of oxygen needs to be purged into

the system and retained to make the reaction more pronounced in the bulk of the substrate

solution Higher concentration of oxygen in water may also be achieved by using oxygen

enriched water which is commercially available55

Alternatively a different enzyme cascade system may be used which does not require a

gaseous substrate56

For example glycogen synthase synthesizes glycogen from glucose-

1-phosphate and glycogen phosphorylase breaks down glycogen to glucose-1-

phosphate57

This system does not require a gaseous substrate and might be able to

overcome the deficiencies of GOx and HRP enzyme cascade system

42

Chapter 4

Silver-titania nanomotors

41 Introduction

Bimetallic nanowires made of platinum and gold have been developed by WPaxton

etal1 The rods move autonomously in a solution of hydrogen peroxide In the present

study silver-titania (Ag-TiO2) particles are being synthesized and investigated for motion

in presence of hydrogen peroxide and UV light With proper control of conditions and

UV light trigger it can be possible to control the speed and direction of motion of these

nanoparticles

Hydrogen peroxide is produced in the human body by phagocytes as an immune response

to foreign particles and pathogens5859

Also silver nanoparticles have been shown to be

biocompatible and capable of interacting with cells in human body without disrupting

cellular function60

Similarly it has been demonstrated that titania is compatible with

different cells DNA and proteins in human body and does not cause toxic effects Hence

silver-titania nanomotors which swim in hydrogen peroxide may open interesting

avenues in applications like targeted drug delivery in those parts of the body where

hydrogen peroxide is present

43

42 Experimental

421 Materials and methods

Silver nitrate (999995) was purchased from Alfa Aesar Gum arabic was purchased

from MP Biomedicals Hydrogen peroxide solution 50 wt in water and ascorbic acid

were purchased from Sigma-Aldrich Nanopure deionized water was used for all

experiments and preparation of all solutions Secure-Seal imaging spacers (9 mm

diameter) were obtained from Sigma Aldrich All experiments were carried out at room

temperature of 25ordmC unless otherwise mentioned Ag-TiO2 particles were synthesized by

evaporation of titania in Semicore electron beam evaporator Particles were characterized

using field-emission scanning electron microscopy Optical microscopy experiments

were performed using Axiovert 200 MAT microscope by Carl Zeiss Particles were

tracked using Physvis particle tracking software (Figure 4 1) in which the position

information of a particle on screen is stored on a frame by frame basis The data is stored

in a dat file as X and Y co-ordinates of particles tracked on the screen This information

can be converted to diffusion coefficients of the particles by further manipulation using

MathWorks-MATLABreg software

44

Figure 4 1 Physvis particle tracking software

422 Synthesis of silver colloidal particles

Silver particles were synthesized by a nucleationgrowth type reaction developed by

Goya and Matijevic61

and modified by Velikov62

Silver nitrate was used as precursor

metal salt for synthesis of silver particles Solution A was 75 ml of 50 mM AgNO3 and

035 by weight gum arabic Solution B was 75 mL of 100 mM 1-ascorbic acid and

035 by weight gum arabic Solution B was rapidly added to solution A in a 250 mL

reaction flask The reaction flask was covered and particles were stirred for 48 hrs After

48 hours particles were allowed to settle for 2-3 hours in refrigerator and supernatant

was separated from the particles Particles were then rinsed with DI water and centrifuged

thrice to obtain pure silver particles Zeta potential of these particles was reported to be

45

around -50 to -60 mV and the size was around 1μm The size of the particles can be

varied by varying the amounts of ascorbic acid and silver nitrate Using molar ratio of 31

of ascorbic acid silver nitrate can give particles of 200 nm diameter whereas ratio of

151 can give particles of upto 5 μm diameter

423 Preparation of silver-titania (Ag-TiO2) particles

A monolayer of silver particles was prepared according to the procedure by Goldenberg

et al63

Glass slides were placed on the bottom of a petridish Water was put into the

petridish so that the entire slide was immersed in water A thin layer of hexane was then

added on top of water in the petridish A suspension of silver particles in ethanol (5

ww) was slowly introduced to the water-hexane interface in the dish After the

monolayer was formed the glass slide was lifted up through the monolayer with tweezers

The silver particles-covered glass slides were left at room temperature to dry overnight

Following drying a thin film of titania was evaporated on the glass slides in Semicore

evaporator (Figure 2 7) The spheres were partially coated with a 10 nm film of titania

Ag-TiO2 particles were removed from the glass slide by gentle sonication FE-SEM was

used to characterize the particles (Figure 4 2) The thin titania layer was not easily

distinguishable due to roughness of the particles

46

Figure 4 2 FESEM image of Ag-TiO2 particles

424 Optical microscopy experiments

Imaging spacers were placed on a glass slide Suspension of Ag-TiO2 particles was

placed on the glass slide and the particles were allowed to settle Movement of particles

was observed in DI water and videos were recorded at 50x magnification of optical

microscope for 30 sec 05 H2O2 was added to the particle suspension on the slide

Videos were recorded at 50x ranging from 30 sec to 10 min to study the movement of

silver titania particles in hydrogen peroxide solution Particles in both DI water and

hydrogen peroxide were subjected to UV light and videos were recorded to study particle

dynamics in presence of UV radiation Particle tracking was performed using Physvis

particle tracking software Atleast 30 particles in every system were tracked for 10

seconds each Particle tracking data was obtained in terms of X and Y coordinates of

particles on screen This data was manipulated using MathWorks-Matlab software and

graphs of mean square displacement of particles versus time were plotted Diffusion

coefficients of particles were calculated from these graphs

47

43 Results and Discussion

Ag-TiO2 particles demonstrated autonomous motion in the presence of H2O2 No UV

light was required for this motion Motion of the particles was accelerated by addition of

fresh H2O2 to the system When subjected to UV radiation the particles displayed

momentary acceleration for 4-5 seconds

Figure 4 3 Mechanism of motion of Ag-TiO2 nanomotors

In the presence of H2O2 silver part of the Ag-TiO2 particle converts H2O2 to H2O and O2

This may lead to propulsion of the Ag-TiO2 particle due to bubble propulsion andor self-

diffusiophoresis In the presence of UV light additional reaction takes place on the

titania part of Ag-TiO2 particle which increases the velocity of the Ag-TiO2 particles

(Figure 4 4) This UV triggered photoactivity of titania is due to hole-electron separation

caused by photons having energy equal to or greater than the band gap of titania646566

Most of the holes-electrons recombine but a small fraction move to the surface and react

with the available redox species like water and oxygen to form protons (H+) superoxide

(O2-) and hydroxyl (OH) radicals

6566 This may lead to propulsion by diffusiophoresis

as well as electrophoresis

48

Figure 4 4 Silver-titania motors move faster when subjected to UV radiation

In addition to linear motion rotational motion is also observed in several particles and

particle aggregates Aggregation of particles can lead to a net circular propulsive force

which can rotate the aggregate as shown in Figure 4 5

Figure 4 5 Rotation due to particle aggregation of Ag-TiO2 particles

Particle tracking by Physvis software revealed a significant increase in diffusion

coefficient of Ag-TiO2 particles in hydrogen peroxide solution as compared to DI water

49

The Ag-TiO2 particles have a diffusion coefficient of 1491 μm2s in hydrogen peroxide

versus only 180 μm2s in DI water Thus the Ag-TiO2 particles move around more than

10 times their body length per second in hydrogen peroxide (Figure 4 6) When

subjected to UV light the particles experience a significant acceleration in hydrogen

peroxide which lasts only for a short duration of time (4-5 sec) after which the particles

return to their original velocity The diffusion coefficient of the particles during this UV

light trigger is found to be around 5093 μm2s UV light trigger does not have any visible

effect on the Ag-TiO2 particles in DI water

Table 4 1 Translational diffusion coefficients of Ag-TiO2 particles in hydrogen

peroxide and water

Mean 05 H2O2 solution DI water

Diffusion coefficient of Ag-TiO2 (μm2s)

without UV light

1491 180

Diffusion coefficient of Ag-TiO2 (μm2s)

in the presence of UV light

5093 113

50

Figure 4 6 Mean square displacement curves of Ag-TiO2 Janus particles in hydrogen

peroxide and DI water

When higher concentration (~30 vv) of Ag-TiO2 particles in hydrogen peroxide is

subjected to UV radiation the particles exhibit pattern formation (Figure 4 7)

Aggregation of Ag-TiO2 Janus particles is observed when UV light is turned on and the

particles disaggregated when UV light is switched off This can be due to predator-prey

kind of interaction between silver and titania half of the Janus particles in the presence of

UV light Oxygen and water that are produced at the silver end by decomposition of

hydrogen peroxide are used up by titania when subjected to UV radiation This can lead

to attraction between titania and silver parts of neighboring Janus particles causing

aggregation

y = 20373x

y = 59994x

y = 71848x

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10 12

Mean

sq

uare

dis

pla

cem

en

t (μ

m2s

)

Time (sec)

Mean square displacement vs Time for Ag-TiO2

particles

05 Hydrogen peroxide and UV05 Hydrogen peroxide

DI Water

51

A UV OFF B UV ON

C UV OFF

Figure 4 7 Higher concentrations of Ag-TiO2 particles showing aggregation when UV

is turned on

52

Chapter 5

Summary and Conclusion

In summary no significant enhancement in diffusion was observed in case of enzyme

immobilized Janus particles in substrate solution Presence of a linker between enzyme

and particle did not increase the diffusion coefficient of particles Alternative enzyme

immobilization methods need to be tested like thiolation of enzyme and subsequent

binding to gold surface of a Janus particle35

or expressing the enzyme with polyhistidine

or avidin tag67

This can result in higher enzyme activity upon immobilization and may

lead to enhanced diffusion of Janus particles

Predator-prey system of GOx and HRP immobilized particles showed some promising

results Aggregation of particles and formation of fan-shaped structures by TMB

oxidation were observed under optical microscope when GOx and HRP immobilized

particles glucose and TMB were present in the system The size of the fan-shaped

structures was dependent on the size of enzyme immobilized particles This can be

indicative of some kind of interaction between the GOx and HRP immobilized particles

No conclusive aggregation of GOx and HRP immobilized particles was observed in

absence of TMB Better imaging techniques are required to study the interaction of GOx

and HRP immobilized particles at the unstable liquid-air interface where majority of the

reaction takes place Other enzyme cascade systems not requiring gaseous reactants like

glycogen synthase and glycogen phosphorylase should be tested for similar activity

53

Ag-TiO2 particles were found to exhibit autonomous motion in the presence of hydrogen

peroxide solution The motion was momentarily accelerated upon exposure to UV

radiation The particles showed a diffusion coefficient of 1491 μm2s in hydrogen

peroxide versus only 180 μm2s in DI water Upon subjecting particles to UV radiation in

presence of H2O2 diffusion coefficient increased to 5093 μm2s for a period of 4-5

seconds In addition to linear motion rotating motion could also be observed in some

particle aggregates Further development of these aggregate structures can lead to

formation of rotors at nanoscale Higher concentration of silver-titania particles in

hydrogen peroxide displayed aggregation in presence of UV light and disaggregated

when UV light was switched off This may be attributed to the predator-prey kind of

interaction between silver and titania where oxygen and water that are produced by

silver by decomposition of hydrogen peroxide are used up by titania when subjected to

UV radiation This promises to be an interesting system and needs to be further

investigated to explore its properties

54

References

1 Paxton WF Sundararajan S Mallouk TE Sen A Angew Chem Int Ed Engl

2006 45 5420-5429

2 Golestanian R Liverpool TB Adjari A Phys Rev Lett 2005 94 220801

3 httpwwwsilvermedicineorgh2o2html

4 Rami Pedahzur Ovadia Lev Badri Fattal Hillel Shuval Water Science and Technology

1995 31(5-6) 123-129

5 W Went F American Scientist 1968 56

6 Adriano Cavalcanti Tad Hogg Bijan Shirinzadeh IEEE MHS 2006 International

Symposium on Micro-NanoMechatronics and Human Science 2006 226-232

7 EM Purcell American Journal of Physics 1977 45 3-11

8 Golestanian R Phys Rev Lett 2010 105 018103(1)-018103(4)

9 S S Dukhin Z R Ulberg G L Dvornichenko B V Deryagin Russian Chemical

Bulletin 1983 31(8) 1535-1544

10 Dukhin AS Goetz PJ Ultrasound for characterizing colloids Particle sizing zeta

potential rheology Elsevier 2002 59

11 HJKeh HJTu Trends in Colloid and Interface Science XIV Progress in Colloid and

Polymer Science 2000 115 174-180

12 D Zinemanas A Nir International Journal of Multiphase Flow 1995 21(5) 787-800

55

13 Stephen Ebbens Jonathan Howse Soft matter 2010 6 726-739

14 J Vicario R Eelkema W R Browne A Meetsma R M La Crois B L Feringa

Chem Commun 2005 31 3936

15 Davide Pantarotto Wesley R Browne Ben L Feringa Chem Commun 2008 1533 -

1535

16 Lisa A Cameron Matthew J Footer Alexander van Oudenaarden Julie A Theriot

PNAS 1999 96(9) 4908-4913

17 Ali Najafi Ramin Golestanian Phys Rev E 2004 69 062901

18 Jinseok Kim Jungyul Park Sungwook Yang Jeongeun Baek Byungkyu Kim Sang

Ho Lee Eui-Sung Yoon Kukjin Chun Sukho Park Lab Chip 2007 7 1504 - 1508

19 Kurt M Dubowski Clinical Chemistry 2008 5411 1919ndash1920

20 Spector DA Yang Q Wade JB Am J Physiol Renal Physiol 2007 292(1) 467-474

21 Rhee SG Chang TS Bae YS Lee SR Kang SW J Am Soc Nephrol 2003 14(8-3)

211-215

22 Sakaue T Kapral R Mikhailov A S Eur Phys J B 2010 75 381ndash387

23 Muddana H S Sengupta S Mallouk T E Sen A Butler P J J Am Chem Soc

2010 132 (7) 2110ndash2111

24 Baker CJ Deahl K Domek J Orlandi EW Arch Biochem Biophys 2000 382(2) 232-

237

25 V Vojinovic RH Carvalho F Lemos JMS Cabral LP Fonseca BS Ferreira

Biochemical Engineering Journal 2007 35 126-135

56

26

OV Lebedeva NN Ugarova Russian Chemical Bulletin 1996 45(1) 18-25

27 Nigel C Veitch Phytochemistry 2004 65(3) 249-259

28 Kenneth H Petersen Journal of Immunological Methods 2009 340(1) 86-89

29 httpwwwbrenda-enzymesorgphpresult_flatphp4ecno=11117

30 Goldenberg L M Wagner J Stumpe J Paulke B-R Goumlrnitz E Langmuir 2002 18

5627-5629

31 httpwwwgoldbiocompdf3761-Protocol201pdf

32 Av Rovisco Pais Lisboa Portugal T amp D Technology West Yorkshire UK Journal

of Molecular Catalysis B Enzymatic 2004 28(2-3) 129-135

33 httpwwwphysicsnyuedugrierlabmethodsnode11html

34 Yoon-Mee Lee1 O-Yul Kwon Yeo-Joon Yoon Keungarp Ryu Biotechnology Letters

2006 28 39ndash43

35 JC Pyun SD Kim JW Chung Analytical Biochemistry 2005 347(2) 227-233

36 George H Pimbley Jr Mathematical Biosciences 1974 20(1-2) 27-51

37 Andy Fenton Sarah E Perkins Parasitology 2010 137 1027-1038

38 Mead James G Brownell Robert L Mammal Species of the World A Taxonomic and

Geographic Reference (3rd ed) Johns Hopkins University Press 2005 2 2142

57

39

Krause KH Schweiz Med Wochenschr 2000 130(4) 97-100

40 Irina Kareva Faina Berezovskaya Carlos Castillo-Chavez Journal of Biological

Dynamics 2010 4(4)315 - 327

41 Delaittre G Reynhout I Cornelissen J Nolte R Chemistry ndash A European Journal

2009 15 12600ndash12603

42 C Buller DH Johnson RC Tschumper Investigative Ophthalmology amp Visual Science

1990 31 2156-2163

43 Robert M Mazo Brownian Motion Fluctuations Dynamics and Applications Oxford

Science Publications 2009 57

44 Vivek K Mutalik KV Venkatesh Theoretical Biology and Medical Modelling 2005 2

19

45 Satoshi Nakamura Kunimasa Koga Biochemical and Biophysical Research

Communications 1977 78(2) 806-810

46 Krishnananda Chattopadhyay Shyamalava Mazumdar Biochemistry 2000 39 (1)

263ndash270

47 Satoshi Nakamura Yasuyuki Ogura J Biochem 1968 63 (3) 308-316

48 van Dongen SF Nallani M Cornelissen JJ Nolte RJ van Hest JC Chemistry 2009

15(5) 1107-1114

49 Ronald Bentley Methods in Enzymology 1955 1 340-345

50 Vartiainen J Raumlttouml M Paulussen S Packaging Technology and Science 2005

18 243ndash251

58

51 GA Sherbeny AA Shindia YMMM Sheriff International Journal of Agriculture

and Biology 2005 7

52 D Rando G-W Kohring F Giffhorn Appl Microbiol Biotechnol 1997 48

53 Worthington enzyme manual Worthington Biochemical Corporation

httpworthington-biochemcomgopdefaulthtml

54 httpwwwbrenda-enzymesorgphpresult_flatphp4ecno=11117

55 httpwwwoxygenphwatercom

56 P Boon Chock Sue Goo Rhee and Earl R Stadtman Annu Rev Biochem 1980 49

813-841

57 Sybil Golden P A Wals Joseph Katz Analytical Biochemistry 1977 77(2) 436-445

58 R L Baehner S K Murrmann J Davis R B Johnston Jr J Clin Invest 1975 56(3)

571ndash576

59 I Bejarano M P Terroacuten S D Paredes C Barriga A B Rodriacuteguez J A Pariente

Molecular and Cellular Biochemistry 2007 296(1-2) 77-84

60 Michael C Moulton Laura K Braydich-Stolle Mallikarjuna N Nadagouda Samantha

Kunzelman Saber M Hussain Rajender S Varma Nanoscale 2010 2 763-770

61 Goya DV Matijevic E Colloids and Surfaces A 1999 146 139

62 Velikov KP Zegers GE van Blaanderen A Langmuir 2003 19 1384

63 Goldenberg L M Wagner J Stumpe J Paulke B-R Goumlrnitz E Langmuir 2002 18

5627-5629

59

64

Banerjee S Gopal J Muraleedharan P Tyagi A K Raj B Curr Sci 2006

901378ndash1383

65 Bogomolov V N Kudinov E K Firsov Y A Sov Phys Solid State 1968 35 555

66 Kudo A Miseki Y Chem Soc Rev 2009 38 253ndash278

67 Gaj T Meyer SC Ghosh I Protein expression and Purification 2007 56 54-61

8

model can swim in low Reynolds number systems An actual model based on

nonreciprocal motion has been developed by Kim etal in which heart cells have been

linked to an asymmetric polydimethylsiloxane robot As the heart cells beat the

asymmetric structure of the device resembles a walking movement18

Most of the nanomicromotors mentioned above are either metallic or work in

environments not compatible with human body Enzymes being an integral part of the

body can solve this problem by operating in suitable substrates already present in the

body fluids The nonreciprocal conformation changes of the enzyme as well as

diffusiophoresis and electrophoresis can conceivably be used to generate motion in case

of enzyme driven nanomotors such motors will be discussed in detail in the following

chapter

9

Chapter 2

Immobilized enzymes on Janus particles as catalytic motors

21 Introduction

Enzymes are widely found in biological systems and act on a large variety of substrates

This can make enzymes an ideal choice for powering nanomotors which need to be

biocompatible Several fluids found in the body can act as substrates to the enzymes For

example dissolved glucose which is abundantly found in the body19

is a susbtrate for

enzyme glucose oxidase Urea which is found in the urinary tract20

is a substrate for

urease Hydrogen peroxide produced by phagocytic cells in the body21

is a substrate for

horseradish peroxidase Having motors driven by enzymes can open possibilities of

targeted drug delivery to these parts of the body containing such substrates

Enzymes have already been used to generate motion of carbon nanotubes by mechanism

of bubble propulsion by Feringa etal15

Two other possible mechanisms which may be

able to power motion of nanoparticles using enzymes are asymmetrical decomposition of

substrates into products leading to self-diffusiophoresis (Figure 2 1) and nonreciprocal

changes in enzyme conformation leading to propulsion (Figure 2 2) As seen in Figure 2

1 enzymes which are immobilized on a Janus particle can breakdown the available

substrate in the solution into products causing asymmetric distribution of solutes in the

surrounding fluid This can lead to movement of particle towards the region of lower

10

solute concentration by principle of self-diffusiophoresis Figure 2-2 shows the possible

nonreciprocal changes in the conformation of a theoretical protein which can lead to

motion of the protein22

These nonreciprocal changes in conformation are triggered by the

attachment of substrate molecule and release of product molecules from the enzymersquos

active site

Figure 2 1 Enzyme functionalized Janus particle moves upon catalyzing asymmetrical

decomposition of substrate into product

11

Figure 2 2 Schematic showing idealized nonreciprocal conformation changes in a

theoretical enzyme22

Enhanced diffusion of a single enzyme molecule by catalysis in a substrate solution has

been demonstrated23

The diffusion coefficient of a single urease molecule was measured

using fluorescence correlation spectroscopy and it was observed that the diffusion of the

urease molecule increases by 16-28 by varying urea concentrations from 0001 M to 1

M Addition of enzyme inhibitors to the system curbed the enhanced diffusion This

enhanced diffusion has been attributed to the force generated by self-electrophoresis by

the enzyme in substrate solution

12

Based on the above facts it might be possible to propel a nanoparticle by asymmetric

distribution of enzymes on its surface thereby leading to self-propulsion of the particles

in substrate solution (Figure 2 1) This propulsion may be due to self-

electrophoresisself-diffusiophoresis andor due to nonreciprocal changes in enzyme

conformations Here we aim to investigate a system consisting of polystyrene particles

half coated with enzyme introduced in a suitable substrate solution The enzyme can be

directly linked to the polystyrene particle or it can be linked through a long spacer

molecule (linker) to provide more degrees of freedom to the enzyme

Several factors need to be considered for choosing enzyme for powering motion at the

nanoscale Some of the important factors are the ratio of kcatKm which denotes the

catalytic efficiency of the enzyme possible substrates stability of enzymessubstrates

ease of immobilization relationship with other enzymes in cascade reactions and

commercial availability of the enzymes The enzyme being used in the present study is

horseradish peroxidase It acts on a molecule of hydrogen peroxide and reduces it while

simultaneously oxidizing another substrate molecule In the absence of alternate

oxidizable substrate HRP can oxidize water and oxygen back to hydrogen peroxide24

thus giving an equilibrium reaction

13

211 Horseradish peroxidase

Figure 2 3 Horseradish peroxidase

Horseradish peroxidase belongs to the group of peroxidases which are a group of heme-

containing oxidoreductases that act on peroxide as hydrogen donor25

It is a redox

enzyme which contains a protoheme a complex of trivalent iron with 1358-

tetramethyl-24-divinylporphin-67-dipropionic acid as a prosthetic group It is a 44 kDa

protein which includes 33900 Da polypeptide chain bound with eight oligosaccharide

chains The carbohydrate chains improve the stability of the enzyme by protecting it

against radicals formed in the course of the reaction it catalyzes26

Horseradish

peroxidase is found in horseradish and is used widely in bioanalytical applications such

as ELISA and Western blots It decomposes hydrogen peroxide into water and oxygen

while simultaneously oxidizing another substrate27

The oxidizable substrates can be

chromogenic substrates such as 22rsquo55rsquo-tetramethyl-benzidine (TMB) and 33-

diaminobenzidine (DMB) or chemoluminescent substrates like SuperSignal and ECL

luminal28

14

Figure 2 4 Reaction catalyzed by horseradish peroxidase

The functional parameters for horseradish peroxidase include Km of 015 mM and kcat of

790 s-1

at an optimum temperature of 20 ordmC and an optimum pH of 6429

Some of the

inhibitors of HRP are 110-phenanthroline aminotriazole and bromine

22 Experimental

221 Materials and methods

Uniform carboxyl modified polystyrene microspheres 05 microm in diameter were obtained

from Polysciences Inc The size of the purchased microspheres was verified by Field

Emission Scanning Electron Microscopy (FESEM) Peroxidase Type I from

Horseradish N-(3-dimethyl-aminopropyl)-Nrsquo-ethylcarbodiimide (EDC) and hydrogen

peroxide solution 50 wt in water were purchased from Sigma-Aldrich N-

hydroxysuccinimide (NHS) ge 97 was purchased from Fluka All chemicals were of

reagent grade and used without further purification Nanopure deionized water was used

for all experiments and preparation of solutions All measurements were made at room

15

temperature (25 oC) unless mentioned otherwise The UV-Visible spectroscopy

measurements were performed using Agilent 8453A diode-array UV-Visible

Spectrophotometer with ChemStation UV-Vis Software Diffusion coefficient

measurements were performed using Nanosight LM10

222 Synthesis of Janus particles

A monolayer of particles was prepared according to the procedure (Figure 2 5) by

Goldenberg L et al30

Glass slides were placed on the bottom of a petridish Water was

put into the petridish so that the entire slide was immersed in water A thin layer of

hexane was added on top of water in the petridish A suspension of microspheres in

ethanol (5 ww) was introduced to the water-hexane interface After the monolayer was

formed the glass slide was lifted up through the monolayer with tweezers The

microsphere-covered glass slides were left to dry overnight at room temperature

Following drying a thin film of silica was evaporated on the glass slides in Semicore

evaporator The spheres were partially coated with 30 nm of silica Particles were

removed from the glass slide by gentle sonication The polystyrene-silica (PS-SiO2)

Janus particles thus obtained were washed in deionized water several times before

further immobilization steps were performed FESEM was used to confirm synthesis of

Janus particles

16

Figure 2 5 Preparation of PS-SiO2 Janus particles by evaporation

223 Immobilization of enzymes using EDCNHS coupling method

1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide and N-hydroxysulfosuccinimide

(EDCNHS) coupling procedure was followed for immobilizing horseradish peroxidase

type I on 05 μm PS-SiO2 Janus particles (Figure 2 6) In this process EDC reacts with a

carboxyl groups on the Janus particles to form an amine-reactive O-acylisourea

intermediate If this intermediate does not encounter an amine it can hydrolyze and

regenerate the carboxyl group In the presence of NHS EDC can be used to convert

carboxyl groups to amine-reactive sulfo-NHS esters which are much more stable and do

not readily hydrolyze to regenerate carboxyl groups In the current experiment

immobilization was performed in the presence as well as absence of a long spacer

17

molecule (linker) between Janus particles and enzymes The chemical 6-aminohexanoic

acid was used as the linker (Figure 2 7) HRP immobilized PS-SiO2 Janus particles were

stored in deionized water until required for further experiments

Figure 2 6 EDCNHS coupling method for immobilization of enzyme on carboxylated

polystyrene particles

Figure 2 7 Immobilization of enzyme on Janus particles with and without 6-

aminohexanoic acid linker

18

224 Enzyme assay

Assay for activity of HRP was performed using a spectrophotometric method which

involved using 4-aminoantipyrene as hydrogen donor3132

The reaction rate was

determined by measuring increase in absorbance at 510 nm resulting from the

decomposition of hydrogen peroxide One unit represents the decomposition of 1 μM of

hydrogen peroxide per minute at 25 degC and pH 70 under the specified conditions

The spectrophotometer was set to the wavelength of 510 nm and temperature of 25 degC

Phenol4-aminoantipyrene solution (14 mL) and 00017 M H2O2 solution (15 mL) were

pipetted into each cuvette The cuvette was incubated for 3-4 mins at 25degC and blank was

established A 01 mL solution of diluted enzyme was added to the above mixture and

absorbance was recorded for 4-5 mins Standard graphs of absorbance versus time were

drawn for known concentration of the free enzyme and compared to that of the

immobilized enzyme on the Janus particles The estimate of the amount of enzyme

present on the Janus particles was deduced from the difference between the quantity of

the free enzyme previously added for immobilization and the amount that was measured

to be in the supernatant after some of the enzyme became attached to the Janus particles

The experiment was carried out in triplicate to assess reproducibility

19

The formula used to calculate the activity of enzyme is

where ∆Amin denotes the change in absorbance per minute

225 Diffusion coefficient measurements using Nanosight LM10

Diffusion coefficients for HRP immobilized particles were measured using Nanosight

LM 10 nanoparticle tracking instrument with Nanoparticle Tracking Analysis (NTA) 20

software in the presence of 003 H2O2 solution as the substrate and also in the presence

of only deionized water The videos were recorded for 30 seconds at the rate of 30 frames

per second Each experiment was carried out in triplicate to assess reproducibility

Particles

visualization

zone

Microscope with

LM 10 unit

LM 10 unit

Figure 2 8 Nanosight LM 10 instrumentation

20

Nanosight LM 10 (Figure 2 8) generates videos of population of moving nanoparticles in

a liquid when illuminated by laser light The particles which pass through the laser beam

path are seen by the instrument as small points of light undergoing Brownian motion

(Figure 2 9a)

The videos are further analyzed by NTA (Nanoparticle tracking analysis) software

(Figure 2 9b) which identifies and tracks each particle independently on a frame-by-

frame basis The software also calculates and subtracts any bulk flow in the system The

mean square displacement is calculated for each particle as long as it is visible If the

particle blinks that is if it disappears momentarily and then reappears on the screen the

software will consider the same particle as two separate ones All analysis data is

exported to the excel files in terms of diffusion coefficients of the tracked particles

B A

Figure 2 9 NTA video analysis (a) Particles as they are seen on the screen during

experimentation in Nanosight LM10 (b) NTA screen as the particles are being analyzed for

their diffusion coefficients

21

23 Results

231 Enzyme assay

The activity of free HRP enzyme reported by Sigma Aldrich is 150 unitsmg However

the activity of HRP upon analysis was found to be 52158 unitsmg which is much lower

than the reported value The activity of HRP was found to be further lowered to 2133

unitsmg when it was immobilized on the PS-SiO2 Janus particles in the presence of a

linker The following formula is used to calculate enzyme activity

where ΔAmin is the change in absorbance per minute

22

Figure 2 10 Graph for the assay of solution of free enzyme containing 05mgL of HRP

ΔAmin = 01716 Unitsmg of free enzyme = 52158

During EDCNHS coupling 04 mg enzyme was added to 1 ml of 025 vv suspension

of Janus particles Thus initial concentration of enzyme in the mixture was 04 mgml

Assuming retained activity of the enzyme in supernatant solution the amount of enzyme

in supernatant was calculated to be 03868 mgml from the assay Amount of enzyme

functionalized on the Janus particles was calculated as the difference between enzyme

previously added for immobilization (04 mgml) and the enzyme present in supernatant

(03868 mgml) and was found to be 00132 mgml Enzyme assay was performed on the

enzyme immobilized Janus particles to estimate the activity of immobilized enzyme

(Figure 2 11) and was found to be 2133 unitsmg

y = 0002x + 00516

0

01

02

03

04

05

06

07

08

0 50 100 150 200 250 300 350 400

Ab

sorb

an

ce

51

0 n

m

Time (sec)

Assay of free enzyme HRP

23

Figure 2 11 Graph for the assay of of immobilized enzyme on 05 μm Janus particles

(~025 vv) ΔAmin = 01853 Unitsmg of free enzyme = 2133

232 Diffusion coefficient measurements

No significant increase was observed in diffusion coefficient of HRP-immobilized Janus

particles in the presence of H2O2 as the substrate Similar results were obtained for

diffusion coefficient measurement with and without linker moieties bridging the enzymes

and particles(Table 2 1 Table 2 2) Thus the linker did not seem to assist in increasing

the particle diffusion by improving or retaining the activity of the enzyme

y = 00015x + 00953

0

01

02

03

04

05

06

07

0 50 100 150 200 250 300 350 400

Ab

sorb

an

ce

510 n

m

Time (sec)

Assay of HRP immobilized 05 μm Janus particles

24

Table 2 1 Diffusion coefficient measurements of HRP immobilized Janus particles

without linker

HRP immobilized

Janus particles in

003 H2O2

Control - Janus

particles in

003 H2O2

Control - HRP

immobilized Janus

particles in DI water

Diffusion coefficient

(10-10

cm2s)

1359416 1051777 1308675

Standard deviation 4534801 4122075 4918597

Table 2 2 Diffusion coefficient measurements of HRP immobilized Janus particles in

the presence of linker

HRP immobilized

Janus particles in

003 H2O2

Control - Janus

particles in

003 H2O2

Control - HRP

immobilized Janus

particles in DI water

Diffusion coefficient

(10-10

cm2s)

13838 1339829 1407891

Standard deviation 5644692 5649364 5797218

25

Figure 2 12 Diffusion coefficient measurements of PS-SiO2 Janus particles without and

with 6-aminohexanoic acid linker

24 Discussion

To compare the diffusion coefficients observed during the experiments theoretical

diffusion coefficient was calculated using following equation33

r

kTD altraslation

6 Equation 21

where r is the hydrodynamic radius of particle Dtranslational is translational diffusion

coefficient of the particle T is temperature of the system η is the viscosity of fluid

through which the particles are moving and k is the Boltzmann constant From FE-SEM

images the size of the particles used was determined to be 450 nm For 450 nm diameter

0

20

40

60

80

100

120

140

160

Without linker With linker

Dif

fusio

n c

oeff

icie

nt

(10

-10 c

m2s

)

HRP immobilized 05 μm Janus particles - Diffusion coefficient measurements

HRP immobilized Janus particles in 003 H2O2

Control - Janus particles in 003 H2O2

Control - HRP immobilized Janus particles in DI water

26

particle Dtranslational is equal to 109 μm2s which is comparable to the diffusion

coefficient measured for the enzyme immobilized particles

HRP was found to be active on the Janus particles however the activity of the enzyme

was reduced upon immobilization Lower enzyme activity can be due to the coupling

method used which in this case was EDCNHS covalent binding However EDCNHS

coupling method has been shown to significantly retain the enzyme activity when the

enzyme is immobilized on carboxylated multi-walled carbon nanotubes under appropriate

conditions34

Hence EDCNHS binding method by itself is not believed to be

responsible for reduction of enzyme activity upon immobilization

Direct binding of enzyme to the Janus particle may lead to reduction in the enzyme

activity because of space constraints for the enzyme and difficulty for the passage of

substrate to the active site of the enzyme due to stearic hindrance Hence a linker was

inserted between the enzyme and Janus particle during enzyme immobilization However

inserting a linker between the enzyme and the particle did not lead to enhanced diffusion

of Janus particles or increase in enzyme activity of immobilized enzyme

Lack of enhanced diffusion can be firstly attributed to the reduction in enzyme activity

upon immobilization Enzyme activity may be retained by modifying the conditions

during EDCNHS coupling or changing the binding method altogether The degree of

immobilization of HRP on carboxylated Janus particles can possibly be affected by

several factors such as temperature the concentration of the coupling reagent EDC

27

concentration HRP concentration in solution and incubation time34

Different

combinations of these factors can be tested to find optimum conditions for

immobilization of HRP Alternate binding method can involve thiolation of HRP by the

covalent conjugation of mercaptopropionic acid and then the thiolated HRP can then be

attached on the gold surface of a polystyrene-gold Janus particle35

Thus further

modifications can be made to the system in order to retain enzyme activity on the

immobilized particles and thereby power nanomotors using enzymes

28

Chapter 3

Predator-prey system of enzyme immobilized particles

31 Introduction

Predator-prey systems are widely found in nature and play a major role in biological

processes3637

Predator-prey systems include all the interactions in which one organism

pursues andor consumes another organism One familiar predator-prey system at the

macroscale is that of a tiger chasing and consuming a deer Another not so familiar one

includes Baleen whales which eat millions of microscopic planktons at once38

At the

microscale such kind of interactions can be observed in the case of phagocytes in the

body which are capable of engulfing foreign microorganisms and killing them with a

combination of microbicidal systems39

In the case of cancer the tumor is the prey and

the mature myeloid cells in the body are the predator However the tumor here has a

defense mechanism against the predator in which the tumor produces cytokines which

inhibits the maturation of the myeloid cells and thereby prevents predation40

One similarity that can be observed in all these systems is that the prey possesses some

attribute which is desired by the predator which makes the predator chase the prey A

similar relationship is seen in case of enzymes which are involved in cascade reactions41

In cascade reactions product synthesized by one enzyme is the reactant required by

another enzyme to carry out its own catalytic reaction As such there is a predator-prey

29

kind of dependence between the two enzymes which may cause one enzyme to chase the

other enzyme when placed in suitable substrate solutions However the mechanism

hypothesized for such a system is totally different than the mechanism involved in

biological systems In biological systems such as bacterial chemotaxis or phagocytes

chasing foreign particles in human body complex biochemical reactions are responsible

for the predator-prey behavior42

In the proposed enzyme cascade system there are no

complex biochemical reactions involved The principle hypothesized for predator-prey

behavior of enzyme cascade reactions can be explained as follows Motion of synthetic

motors at nanoscale involves two components translational diffusion and rotational

Brownian diffusion At very short time scales of less than 1Dr where Dr (s-1

) is

rotational Brownian diffusion coefficient the motion of particles is unidirectional For

longer time scales of greater than 1Dr the direction of motion is random giving

poweredactive diffusion43

If the chemical homogeneity is broken that is when there is

higher concentration of a substrate present in one direction of the enzyme the enzyme

might acquire a subtle bias which may lead to a spatial aggregation of the enzymes based

on the substrate concentration gradient The bias can be a result of the relationship

between the speed of enzyme diffusion and substrate concentration where higher

substrate concentration lead to higher enzyme diffusion As a result in an enzyme

cascade system one enzyme will migrate towards higher concentration of substrate

which is present near the other enzyme thus giving rise to a predator-prey behavior

Such enzyme cascades are widely found in signaling networks and metabolic pathways in

living cells and can be used to produce predator-prey like nanomotors systems44

The

30

enzymes being used in the present study are horseradish peroxidase and glucose oxidase

Both enzymes are extremely stable4546

Glucose is the substrate for glucose oxidase

which is widely available and highly stable47

Horseradish peroxidase acts on hydrogen

peroxide It is involved in a cascade reaction with glucose oxidase which produces

hydrogen peroxide by converting glucose into gluconic acid (Figure 3 1)48

Figure 3 1 Concept of particle aggregation due to cascade reactions of immobilized

enzymes

In this experiment glucose oxidase (GOx) and horseradish peroxidase (HRP) were

immobilized on polystyrene carboxylate beads of different sizes using EDCNHS

coupling method Glucose and 3355-Tetramethylbenzidine (TMB) were dissolved

together in water and this solution was used as the substrate solution All the components

were present in a single system in DI water Aggregation of particles and precipitation of

oxidized TMB from the solution was expected to be observed when all these components

were present in such a system

Properties of horseradish peroxidase have been discussed in the previous chapter (Section

211 Horseradish peroxidase)

GOxGOx HRPHRP

Glucose + O2Gluconic acid + H2O2

TMB TMB oxidizedBlue precipitate

H2O

Aggregation

31

311 Glucose oxidase

Figure 3 2 Glucose oxidase

Glucose oxidase (GOx) belongs to the group of oxidoreductases and is also called

glucose aerodehydrogenase49

It is obtained from fungi and has been identified as an

antimicrobial enzyme50

GOx binds to β-D-glucopyranose and catalyzes the oxidation

reaction to from gluconolactone while simultaneously reducing oxygen to hydrogen

peroxide51

GOx has high substrate affinity and specificity high stability and high

turnover number which makes it a suitable biocatalyst for industrial applications52

Figure 3 3 Redox reaction of glucose catalyzed by glucose oxidase53

32

GOx is a glycoprotein with molecular weight of 155 kDa and consists of two identical

polypeptide subunits linked together by a disulfide bond The optimum pH for GOx is

55 though it is stable in a broad pH range of 4 to 7 and can be stored for a long period of

time51

The functional parameters for glucose oxidase include Km of 151 mM and kcat of

337 s-1

at temperature of 20 ordmC and pH of 754

Some of the inhibitors of GOx are 2-

Deoxy-D-glucose adenine nucleotides and NaNO3

32 Experimental

321 Materials and methods

Uniform carboxyl modified polystyrene microspheres 05 microm in diameter were obtained

from Polysciences Inc The size of the purchased microspheres was verified by FESEM

Peroxidase Type I from horseradish glucose oxidase Type II-S from Aspergillus niger

N-(3-dimethyl-aminopropyl)-Nrsquo-ethylcarbodiimide (EDC) and Hydrogen peroxide

solution 50 wt in water were purchased from Sigma-Aldrich N-hydroxysuccinimide

(NHS) was purchased from Fluka D-Glucose (99) was purchased from VWR

International Secure-Seal imaging spacers (9mm diameter) for glass slides were obtained

from Sigma Aldrich All chemicals were of reagent grade and used without further

purification Nanopure deionized water was used for all experiments and preparation of

solutions All measurements were made at room temperature (25 oC) unless mentioned

otherwise The UV-Visible measurements were performed using Agilent 8453A diode-

array UV-Visible Spectrophotometer with ChemStation UV-Vis Software Optical

33

microscopy experiments were performed using Axiovert 200 MAT microscope by Carl

Zeiss Diffusion coefficient measurements were obtained using Nanosight LM10

322 Optical microscopy experiments

The enzyme substrate system was observed under optical microscope to check for

aggregation and precipitation due to cascade reactions Several such systems were

studied Each system in general consisted of GOx and HRP immobilized on different

polystyrene particles The substrate solutions consisted of 01 M glucose and different

concentrations of TMB solution The systems were studied in the presence of all the

constituents and in the absence of glucose GOxHRP and TMB Systems consisting of

different sized HRP immobilized particles in substrate solution of 003 hydrogen

peroxide and TMB were also studied Videos were recorded over a period of 24 hours to

observe changes in the system Each experiment was performed atleast thrice to assess

reproducibility of results

323 Diffusion coefficient measurements using Nanosight LM10

Diffusion coefficient of GOx and HRP immobilized particles were measured using

Nanosight LM10 nanoparticle tracking instrument with Nanoparticle Tracking Analysis

(NTA) 20 software Diffusion coefficients were measured for different systems namely

GOx particles in 01 M glucose and oxygen both GOx and HRP functionalized particles

in 01 M glucose and oxygen along with controls for the individual systems Videos were

34

recorded for 30 seconds at the rate of 30 frames per second Each experiment was carried

out in triplicate to assess reproducibility

33 Results

331 Optical microscopy

According to the enzyme cascade model precipitation of TMB was possible only upon

the interaction of GOx and HRP immobilized particles in presence of appropriate

substrate solution Hence in the first set of experiments precipitation of TMB was used

as an indication of the type of interaction between GOx and HRP immobilized particles

Fan-shaped structures were observed when both HRP and GOx immobilized particles

were present in a substrate solution consisting of glucose and TMB These structures

started forming as a single strand and then branched out and intertwined to form the fan-

shaped structures (Figure 3 4)

Table 3 1 Observations for formation of fan-shaped structures in enzyme cascade

system in presence of different components

GOx HRP Glucose TMB Observation

Yes Yes Yes Yes

Fan-shaped structures observed particles associated with

the fan-shaped structures

No Yes Yes Yes No fan-shaped structures

Yes No Yes Yes No fan-shaped structures

Yes Yes No Yes No fan-shaped structures

Yes Yes Yes No No fan-shaped structures

35

Fan-shaped structures can also be observed in the system consisting of only HRP

immobilized particles in substrate solution of hydrogen peroxide and TMB The size of

the fan-shaped structures depends on the size of the enzyme immobilized particles Free

enzymes show extremely tiny structures which are difficult to view under the optical

microscope

Figure 3 4 Formation of fan-shaped structures when both HRP and GOx

immobilized particles were present in a substrate solution consisting of glucose and

TMB A In the initial stages single strands form on the surface of liquid (5Ox) B

Single strands branch out and intertwine to form fan-shaped structures (50x) C Many

such structures form and settle down after 20 minutes (20x)

A

C

B

36

Experiments were performed in the absence of TMB that is the system consisted of GOx

and HRP immobilized particles in glucose substrate solution In the absence of TMB the

particles were expected to move closer to each other without any precipitation due to the

cascade reaction However there was no conclusive aggregation of particles observed in

the system

Free Enzyme 005 μm HRP - immobilized particle

10 μm HRP - immobilized particle 05 μm HRP - immobilized particle

Figure 3 5 Size dependence of fan-shaped structures on the size of HRP -

immobilized particles (20x)

37

Table 3 2 Observations for particle aggregation in enzyme cascade system in presence

and absence of TMB

Glucose GOx HRP TMB Observation

Present Present Present Present Aggregation and precipitate

Present Present Present Absent No conclusive aggregation

Present Absent Present Present No aggregation or ppt

Absent Present Present Present No aggregation or ppt

Present Present Absent Present No aggregation or ppt

332 Diffusion coefficient measurements

Diffusion coefficients were measured for GOx immobilized particles of 05 μm diameter

in substrate solution of 01 M glucose and oxygen Diffusion coefficients of particles

were also measured for system consisting of GOx and HRP functionalized particles in

glucose and oxygen Each experiment was done in triplicate to assess reproducibility

Nanosight visualization chamber being a closed chamber oxygen was purged into all the

solutions before starting the experiments to ensure sufficient supply of oxygen for

catalytic reaction of GOx

38

Table 3 3 Diffusion coefficients for 05 μm GOx immobilized particles in 01 M

glucose purged with O2

Mean

GOx

particles in

01 M

glucose

Control - GOx

particles in DI

water

Control - GOx

particles in 01 M

glucose without

oxygen purging

Control -

particles in 01

M Glucose

Diffusion

coefficient

(x10-10

cm2s)

13316 12888 13827 13702

Standard

deviation 6307 4926 4424 7468

Table 3 4 Diffusion coefficients for GOx and HRP immobilized particles in 01 M

glucose and purged with O2

Mean

GOx- HRP-

functionalized

particles in 01 M

glucose

Control ndash Gox-

HRP- functionalized

particles in DI water

Control - particles

in 01 M glucose

Diffusion coefficient

(x10-10

cm2s)

13005 14434 13702

Standard deviation 5263 6622 7468

No significant increase was observed in diffusion coefficient of enzyme immobilized

particles in any of the systems

39

34 Discussion

Formation of fan-shaped structures due to patterned deposition of oxidized TMB

indicates some kind of interaction between GOx and HRP immobilized particles Particle

diameter dependence of the size of structures can be explained with a simple model in

Figure 3 6 A larger particle will have a higher surface area as compared to a smaller

particle Hence more enzyme is immobilized on the larger particle As a result higher

quantity of TMB is oxidized and precipitated by the larger particle as compared to the

smaller particle This leads to thicker strands of fan-shaped structures eventually scaling

up the whole system to give bigger fan-shaped structures

The exact reason for the formation of such fan-shaped structures which are dependent on

particle size could not be determined It is hypothesized that hydrogen peroxide andor

Figure 3 6 Model for size dependence of fan-shaped structures on the particle

diameter of enzyme immobilized particles Larger the particle more surface area is

available for precipitation of TMB which eventually leads to bigger fan-shaped

structures

Larger particle Smaller particle

40

TMB gradient in the substrate solution might be responsible for such structures If there

is higher concentration of H2O2 on one side of the particle and assuming an even

concentration distribution of TMB there will be selective precipitation in the direction of

H2O2 When several particles come in the vicinity of higher concentration of H2O2 the

precipitation lines will converge to give fan-shaped structures (Figure 3 7)

Figure 3 7 Hypothesis for formation of fan-shaped structures

In the system consisting of GOx immobilized particles HRP immobilized particles

glucose and TMB it was observed that the fan-shaped structures formed at the liquid-air

interface and eventually settled down to the bottom of the glass slide This makes sense

as oxygen is necessary for the catalytic reaction of conversion of glucose to gluconic acid

and hydrogen peroxide which takes place on the surface of GOx immobilized particle As

a result H2O2 is primarily formed at the liquid-air interface and is then used by HRP for

precipitation of TMB

Following the observations that precipitation of TMB occurs at the surface GOx and

HRP immobilized particles are also expected to interact at the surface in the absence of

TMB Due the cascade reaction it was expected that HRP immobilized particles will be

41

attracted to GOx immobilized particles in appropriate substrate solution Experiments

were carried out to visualize inter-particle interactions at the liquid-air interface

However liquid-air interface not being a stable system to image the precise interaction

of GOx and HRP immobilized particles could not be determined Significant external

disturbances were involved at the interface To enable the cascade reaction in the bulk of

the liquid oxygen was purged into the reaction system before starting the experiment

However this did not lead to significant reaction in the bulk of the liquid and the reaction

prominently took place at the liquid-air interface where the oxygen concentration was

higher

In order to observe the interaction of GOx and HRP immobilized particles better imaging

techniques need to be used to observe diffusion of particles in absence of external

disturbances at the liquid-air interface Large quantity of oxygen needs to be purged into

the system and retained to make the reaction more pronounced in the bulk of the substrate

solution Higher concentration of oxygen in water may also be achieved by using oxygen

enriched water which is commercially available55

Alternatively a different enzyme cascade system may be used which does not require a

gaseous substrate56

For example glycogen synthase synthesizes glycogen from glucose-

1-phosphate and glycogen phosphorylase breaks down glycogen to glucose-1-

phosphate57

This system does not require a gaseous substrate and might be able to

overcome the deficiencies of GOx and HRP enzyme cascade system

42

Chapter 4

Silver-titania nanomotors

41 Introduction

Bimetallic nanowires made of platinum and gold have been developed by WPaxton

etal1 The rods move autonomously in a solution of hydrogen peroxide In the present

study silver-titania (Ag-TiO2) particles are being synthesized and investigated for motion

in presence of hydrogen peroxide and UV light With proper control of conditions and

UV light trigger it can be possible to control the speed and direction of motion of these

nanoparticles

Hydrogen peroxide is produced in the human body by phagocytes as an immune response

to foreign particles and pathogens5859

Also silver nanoparticles have been shown to be

biocompatible and capable of interacting with cells in human body without disrupting

cellular function60

Similarly it has been demonstrated that titania is compatible with

different cells DNA and proteins in human body and does not cause toxic effects Hence

silver-titania nanomotors which swim in hydrogen peroxide may open interesting

avenues in applications like targeted drug delivery in those parts of the body where

hydrogen peroxide is present

43

42 Experimental

421 Materials and methods

Silver nitrate (999995) was purchased from Alfa Aesar Gum arabic was purchased

from MP Biomedicals Hydrogen peroxide solution 50 wt in water and ascorbic acid

were purchased from Sigma-Aldrich Nanopure deionized water was used for all

experiments and preparation of all solutions Secure-Seal imaging spacers (9 mm

diameter) were obtained from Sigma Aldrich All experiments were carried out at room

temperature of 25ordmC unless otherwise mentioned Ag-TiO2 particles were synthesized by

evaporation of titania in Semicore electron beam evaporator Particles were characterized

using field-emission scanning electron microscopy Optical microscopy experiments

were performed using Axiovert 200 MAT microscope by Carl Zeiss Particles were

tracked using Physvis particle tracking software (Figure 4 1) in which the position

information of a particle on screen is stored on a frame by frame basis The data is stored

in a dat file as X and Y co-ordinates of particles tracked on the screen This information

can be converted to diffusion coefficients of the particles by further manipulation using

MathWorks-MATLABreg software

44

Figure 4 1 Physvis particle tracking software

422 Synthesis of silver colloidal particles

Silver particles were synthesized by a nucleationgrowth type reaction developed by

Goya and Matijevic61

and modified by Velikov62

Silver nitrate was used as precursor

metal salt for synthesis of silver particles Solution A was 75 ml of 50 mM AgNO3 and

035 by weight gum arabic Solution B was 75 mL of 100 mM 1-ascorbic acid and

035 by weight gum arabic Solution B was rapidly added to solution A in a 250 mL

reaction flask The reaction flask was covered and particles were stirred for 48 hrs After

48 hours particles were allowed to settle for 2-3 hours in refrigerator and supernatant

was separated from the particles Particles were then rinsed with DI water and centrifuged

thrice to obtain pure silver particles Zeta potential of these particles was reported to be

45

around -50 to -60 mV and the size was around 1μm The size of the particles can be

varied by varying the amounts of ascorbic acid and silver nitrate Using molar ratio of 31

of ascorbic acid silver nitrate can give particles of 200 nm diameter whereas ratio of

151 can give particles of upto 5 μm diameter

423 Preparation of silver-titania (Ag-TiO2) particles

A monolayer of silver particles was prepared according to the procedure by Goldenberg

et al63

Glass slides were placed on the bottom of a petridish Water was put into the

petridish so that the entire slide was immersed in water A thin layer of hexane was then

added on top of water in the petridish A suspension of silver particles in ethanol (5

ww) was slowly introduced to the water-hexane interface in the dish After the

monolayer was formed the glass slide was lifted up through the monolayer with tweezers

The silver particles-covered glass slides were left at room temperature to dry overnight

Following drying a thin film of titania was evaporated on the glass slides in Semicore

evaporator (Figure 2 7) The spheres were partially coated with a 10 nm film of titania

Ag-TiO2 particles were removed from the glass slide by gentle sonication FE-SEM was

used to characterize the particles (Figure 4 2) The thin titania layer was not easily

distinguishable due to roughness of the particles

46

Figure 4 2 FESEM image of Ag-TiO2 particles

424 Optical microscopy experiments

Imaging spacers were placed on a glass slide Suspension of Ag-TiO2 particles was

placed on the glass slide and the particles were allowed to settle Movement of particles

was observed in DI water and videos were recorded at 50x magnification of optical

microscope for 30 sec 05 H2O2 was added to the particle suspension on the slide

Videos were recorded at 50x ranging from 30 sec to 10 min to study the movement of

silver titania particles in hydrogen peroxide solution Particles in both DI water and

hydrogen peroxide were subjected to UV light and videos were recorded to study particle

dynamics in presence of UV radiation Particle tracking was performed using Physvis

particle tracking software Atleast 30 particles in every system were tracked for 10

seconds each Particle tracking data was obtained in terms of X and Y coordinates of

particles on screen This data was manipulated using MathWorks-Matlab software and

graphs of mean square displacement of particles versus time were plotted Diffusion

coefficients of particles were calculated from these graphs

47

43 Results and Discussion

Ag-TiO2 particles demonstrated autonomous motion in the presence of H2O2 No UV

light was required for this motion Motion of the particles was accelerated by addition of

fresh H2O2 to the system When subjected to UV radiation the particles displayed

momentary acceleration for 4-5 seconds

Figure 4 3 Mechanism of motion of Ag-TiO2 nanomotors

In the presence of H2O2 silver part of the Ag-TiO2 particle converts H2O2 to H2O and O2

This may lead to propulsion of the Ag-TiO2 particle due to bubble propulsion andor self-

diffusiophoresis In the presence of UV light additional reaction takes place on the

titania part of Ag-TiO2 particle which increases the velocity of the Ag-TiO2 particles

(Figure 4 4) This UV triggered photoactivity of titania is due to hole-electron separation

caused by photons having energy equal to or greater than the band gap of titania646566

Most of the holes-electrons recombine but a small fraction move to the surface and react

with the available redox species like water and oxygen to form protons (H+) superoxide

(O2-) and hydroxyl (OH) radicals

6566 This may lead to propulsion by diffusiophoresis

as well as electrophoresis

48

Figure 4 4 Silver-titania motors move faster when subjected to UV radiation

In addition to linear motion rotational motion is also observed in several particles and

particle aggregates Aggregation of particles can lead to a net circular propulsive force

which can rotate the aggregate as shown in Figure 4 5

Figure 4 5 Rotation due to particle aggregation of Ag-TiO2 particles

Particle tracking by Physvis software revealed a significant increase in diffusion

coefficient of Ag-TiO2 particles in hydrogen peroxide solution as compared to DI water

49

The Ag-TiO2 particles have a diffusion coefficient of 1491 μm2s in hydrogen peroxide

versus only 180 μm2s in DI water Thus the Ag-TiO2 particles move around more than

10 times their body length per second in hydrogen peroxide (Figure 4 6) When

subjected to UV light the particles experience a significant acceleration in hydrogen

peroxide which lasts only for a short duration of time (4-5 sec) after which the particles

return to their original velocity The diffusion coefficient of the particles during this UV

light trigger is found to be around 5093 μm2s UV light trigger does not have any visible

effect on the Ag-TiO2 particles in DI water

Table 4 1 Translational diffusion coefficients of Ag-TiO2 particles in hydrogen

peroxide and water

Mean 05 H2O2 solution DI water

Diffusion coefficient of Ag-TiO2 (μm2s)

without UV light

1491 180

Diffusion coefficient of Ag-TiO2 (μm2s)

in the presence of UV light

5093 113

50

Figure 4 6 Mean square displacement curves of Ag-TiO2 Janus particles in hydrogen

peroxide and DI water

When higher concentration (~30 vv) of Ag-TiO2 particles in hydrogen peroxide is

subjected to UV radiation the particles exhibit pattern formation (Figure 4 7)

Aggregation of Ag-TiO2 Janus particles is observed when UV light is turned on and the

particles disaggregated when UV light is switched off This can be due to predator-prey

kind of interaction between silver and titania half of the Janus particles in the presence of

UV light Oxygen and water that are produced at the silver end by decomposition of

hydrogen peroxide are used up by titania when subjected to UV radiation This can lead

to attraction between titania and silver parts of neighboring Janus particles causing

aggregation

y = 20373x

y = 59994x

y = 71848x

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10 12

Mean

sq

uare

dis

pla

cem

en

t (μ

m2s

)

Time (sec)

Mean square displacement vs Time for Ag-TiO2

particles

05 Hydrogen peroxide and UV05 Hydrogen peroxide

DI Water

51

A UV OFF B UV ON

C UV OFF

Figure 4 7 Higher concentrations of Ag-TiO2 particles showing aggregation when UV

is turned on

52

Chapter 5

Summary and Conclusion

In summary no significant enhancement in diffusion was observed in case of enzyme

immobilized Janus particles in substrate solution Presence of a linker between enzyme

and particle did not increase the diffusion coefficient of particles Alternative enzyme

immobilization methods need to be tested like thiolation of enzyme and subsequent

binding to gold surface of a Janus particle35

or expressing the enzyme with polyhistidine

or avidin tag67

This can result in higher enzyme activity upon immobilization and may

lead to enhanced diffusion of Janus particles

Predator-prey system of GOx and HRP immobilized particles showed some promising

results Aggregation of particles and formation of fan-shaped structures by TMB

oxidation were observed under optical microscope when GOx and HRP immobilized

particles glucose and TMB were present in the system The size of the fan-shaped

structures was dependent on the size of enzyme immobilized particles This can be

indicative of some kind of interaction between the GOx and HRP immobilized particles

No conclusive aggregation of GOx and HRP immobilized particles was observed in

absence of TMB Better imaging techniques are required to study the interaction of GOx

and HRP immobilized particles at the unstable liquid-air interface where majority of the

reaction takes place Other enzyme cascade systems not requiring gaseous reactants like

glycogen synthase and glycogen phosphorylase should be tested for similar activity

53

Ag-TiO2 particles were found to exhibit autonomous motion in the presence of hydrogen

peroxide solution The motion was momentarily accelerated upon exposure to UV

radiation The particles showed a diffusion coefficient of 1491 μm2s in hydrogen

peroxide versus only 180 μm2s in DI water Upon subjecting particles to UV radiation in

presence of H2O2 diffusion coefficient increased to 5093 μm2s for a period of 4-5

seconds In addition to linear motion rotating motion could also be observed in some

particle aggregates Further development of these aggregate structures can lead to

formation of rotors at nanoscale Higher concentration of silver-titania particles in

hydrogen peroxide displayed aggregation in presence of UV light and disaggregated

when UV light was switched off This may be attributed to the predator-prey kind of

interaction between silver and titania where oxygen and water that are produced by

silver by decomposition of hydrogen peroxide are used up by titania when subjected to

UV radiation This promises to be an interesting system and needs to be further

investigated to explore its properties

54

References

1 Paxton WF Sundararajan S Mallouk TE Sen A Angew Chem Int Ed Engl

2006 45 5420-5429

2 Golestanian R Liverpool TB Adjari A Phys Rev Lett 2005 94 220801

3 httpwwwsilvermedicineorgh2o2html

4 Rami Pedahzur Ovadia Lev Badri Fattal Hillel Shuval Water Science and Technology

1995 31(5-6) 123-129

5 W Went F American Scientist 1968 56

6 Adriano Cavalcanti Tad Hogg Bijan Shirinzadeh IEEE MHS 2006 International

Symposium on Micro-NanoMechatronics and Human Science 2006 226-232

7 EM Purcell American Journal of Physics 1977 45 3-11

8 Golestanian R Phys Rev Lett 2010 105 018103(1)-018103(4)

9 S S Dukhin Z R Ulberg G L Dvornichenko B V Deryagin Russian Chemical

Bulletin 1983 31(8) 1535-1544

10 Dukhin AS Goetz PJ Ultrasound for characterizing colloids Particle sizing zeta

potential rheology Elsevier 2002 59

11 HJKeh HJTu Trends in Colloid and Interface Science XIV Progress in Colloid and

Polymer Science 2000 115 174-180

12 D Zinemanas A Nir International Journal of Multiphase Flow 1995 21(5) 787-800

55

13 Stephen Ebbens Jonathan Howse Soft matter 2010 6 726-739

14 J Vicario R Eelkema W R Browne A Meetsma R M La Crois B L Feringa

Chem Commun 2005 31 3936

15 Davide Pantarotto Wesley R Browne Ben L Feringa Chem Commun 2008 1533 -

1535

16 Lisa A Cameron Matthew J Footer Alexander van Oudenaarden Julie A Theriot

PNAS 1999 96(9) 4908-4913

17 Ali Najafi Ramin Golestanian Phys Rev E 2004 69 062901

18 Jinseok Kim Jungyul Park Sungwook Yang Jeongeun Baek Byungkyu Kim Sang

Ho Lee Eui-Sung Yoon Kukjin Chun Sukho Park Lab Chip 2007 7 1504 - 1508

19 Kurt M Dubowski Clinical Chemistry 2008 5411 1919ndash1920

20 Spector DA Yang Q Wade JB Am J Physiol Renal Physiol 2007 292(1) 467-474

21 Rhee SG Chang TS Bae YS Lee SR Kang SW J Am Soc Nephrol 2003 14(8-3)

211-215

22 Sakaue T Kapral R Mikhailov A S Eur Phys J B 2010 75 381ndash387

23 Muddana H S Sengupta S Mallouk T E Sen A Butler P J J Am Chem Soc

2010 132 (7) 2110ndash2111

24 Baker CJ Deahl K Domek J Orlandi EW Arch Biochem Biophys 2000 382(2) 232-

237

25 V Vojinovic RH Carvalho F Lemos JMS Cabral LP Fonseca BS Ferreira

Biochemical Engineering Journal 2007 35 126-135

56

26

OV Lebedeva NN Ugarova Russian Chemical Bulletin 1996 45(1) 18-25

27 Nigel C Veitch Phytochemistry 2004 65(3) 249-259

28 Kenneth H Petersen Journal of Immunological Methods 2009 340(1) 86-89

29 httpwwwbrenda-enzymesorgphpresult_flatphp4ecno=11117

30 Goldenberg L M Wagner J Stumpe J Paulke B-R Goumlrnitz E Langmuir 2002 18

5627-5629

31 httpwwwgoldbiocompdf3761-Protocol201pdf

32 Av Rovisco Pais Lisboa Portugal T amp D Technology West Yorkshire UK Journal

of Molecular Catalysis B Enzymatic 2004 28(2-3) 129-135

33 httpwwwphysicsnyuedugrierlabmethodsnode11html

34 Yoon-Mee Lee1 O-Yul Kwon Yeo-Joon Yoon Keungarp Ryu Biotechnology Letters

2006 28 39ndash43

35 JC Pyun SD Kim JW Chung Analytical Biochemistry 2005 347(2) 227-233

36 George H Pimbley Jr Mathematical Biosciences 1974 20(1-2) 27-51

37 Andy Fenton Sarah E Perkins Parasitology 2010 137 1027-1038

38 Mead James G Brownell Robert L Mammal Species of the World A Taxonomic and

Geographic Reference (3rd ed) Johns Hopkins University Press 2005 2 2142

57

39

Krause KH Schweiz Med Wochenschr 2000 130(4) 97-100

40 Irina Kareva Faina Berezovskaya Carlos Castillo-Chavez Journal of Biological

Dynamics 2010 4(4)315 - 327

41 Delaittre G Reynhout I Cornelissen J Nolte R Chemistry ndash A European Journal

2009 15 12600ndash12603

42 C Buller DH Johnson RC Tschumper Investigative Ophthalmology amp Visual Science

1990 31 2156-2163

43 Robert M Mazo Brownian Motion Fluctuations Dynamics and Applications Oxford

Science Publications 2009 57

44 Vivek K Mutalik KV Venkatesh Theoretical Biology and Medical Modelling 2005 2

19

45 Satoshi Nakamura Kunimasa Koga Biochemical and Biophysical Research

Communications 1977 78(2) 806-810

46 Krishnananda Chattopadhyay Shyamalava Mazumdar Biochemistry 2000 39 (1)

263ndash270

47 Satoshi Nakamura Yasuyuki Ogura J Biochem 1968 63 (3) 308-316

48 van Dongen SF Nallani M Cornelissen JJ Nolte RJ van Hest JC Chemistry 2009

15(5) 1107-1114

49 Ronald Bentley Methods in Enzymology 1955 1 340-345

50 Vartiainen J Raumlttouml M Paulussen S Packaging Technology and Science 2005

18 243ndash251

58

51 GA Sherbeny AA Shindia YMMM Sheriff International Journal of Agriculture

and Biology 2005 7

52 D Rando G-W Kohring F Giffhorn Appl Microbiol Biotechnol 1997 48

53 Worthington enzyme manual Worthington Biochemical Corporation

httpworthington-biochemcomgopdefaulthtml

54 httpwwwbrenda-enzymesorgphpresult_flatphp4ecno=11117

55 httpwwwoxygenphwatercom

56 P Boon Chock Sue Goo Rhee and Earl R Stadtman Annu Rev Biochem 1980 49

813-841

57 Sybil Golden P A Wals Joseph Katz Analytical Biochemistry 1977 77(2) 436-445

58 R L Baehner S K Murrmann J Davis R B Johnston Jr J Clin Invest 1975 56(3)

571ndash576

59 I Bejarano M P Terroacuten S D Paredes C Barriga A B Rodriacuteguez J A Pariente

Molecular and Cellular Biochemistry 2007 296(1-2) 77-84

60 Michael C Moulton Laura K Braydich-Stolle Mallikarjuna N Nadagouda Samantha

Kunzelman Saber M Hussain Rajender S Varma Nanoscale 2010 2 763-770

61 Goya DV Matijevic E Colloids and Surfaces A 1999 146 139

62 Velikov KP Zegers GE van Blaanderen A Langmuir 2003 19 1384

63 Goldenberg L M Wagner J Stumpe J Paulke B-R Goumlrnitz E Langmuir 2002 18

5627-5629

59

64

Banerjee S Gopal J Muraleedharan P Tyagi A K Raj B Curr Sci 2006

901378ndash1383

65 Bogomolov V N Kudinov E K Firsov Y A Sov Phys Solid State 1968 35 555

66 Kudo A Miseki Y Chem Soc Rev 2009 38 253ndash278

67 Gaj T Meyer SC Ghosh I Protein expression and Purification 2007 56 54-61

9

Chapter 2

Immobilized enzymes on Janus particles as catalytic motors

21 Introduction

Enzymes are widely found in biological systems and act on a large variety of substrates

This can make enzymes an ideal choice for powering nanomotors which need to be

biocompatible Several fluids found in the body can act as substrates to the enzymes For

example dissolved glucose which is abundantly found in the body19

is a susbtrate for

enzyme glucose oxidase Urea which is found in the urinary tract20

is a substrate for

urease Hydrogen peroxide produced by phagocytic cells in the body21

is a substrate for

horseradish peroxidase Having motors driven by enzymes can open possibilities of

targeted drug delivery to these parts of the body containing such substrates

Enzymes have already been used to generate motion of carbon nanotubes by mechanism

of bubble propulsion by Feringa etal15

Two other possible mechanisms which may be

able to power motion of nanoparticles using enzymes are asymmetrical decomposition of

substrates into products leading to self-diffusiophoresis (Figure 2 1) and nonreciprocal

changes in enzyme conformation leading to propulsion (Figure 2 2) As seen in Figure 2

1 enzymes which are immobilized on a Janus particle can breakdown the available

substrate in the solution into products causing asymmetric distribution of solutes in the

surrounding fluid This can lead to movement of particle towards the region of lower

10

solute concentration by principle of self-diffusiophoresis Figure 2-2 shows the possible

nonreciprocal changes in the conformation of a theoretical protein which can lead to

motion of the protein22

These nonreciprocal changes in conformation are triggered by the

attachment of substrate molecule and release of product molecules from the enzymersquos

active site

Figure 2 1 Enzyme functionalized Janus particle moves upon catalyzing asymmetrical

decomposition of substrate into product

11

Figure 2 2 Schematic showing idealized nonreciprocal conformation changes in a

theoretical enzyme22

Enhanced diffusion of a single enzyme molecule by catalysis in a substrate solution has

been demonstrated23

The diffusion coefficient of a single urease molecule was measured

using fluorescence correlation spectroscopy and it was observed that the diffusion of the

urease molecule increases by 16-28 by varying urea concentrations from 0001 M to 1

M Addition of enzyme inhibitors to the system curbed the enhanced diffusion This

enhanced diffusion has been attributed to the force generated by self-electrophoresis by

the enzyme in substrate solution

12

Based on the above facts it might be possible to propel a nanoparticle by asymmetric

distribution of enzymes on its surface thereby leading to self-propulsion of the particles

in substrate solution (Figure 2 1) This propulsion may be due to self-

electrophoresisself-diffusiophoresis andor due to nonreciprocal changes in enzyme

conformations Here we aim to investigate a system consisting of polystyrene particles

half coated with enzyme introduced in a suitable substrate solution The enzyme can be

directly linked to the polystyrene particle or it can be linked through a long spacer

molecule (linker) to provide more degrees of freedom to the enzyme

Several factors need to be considered for choosing enzyme for powering motion at the

nanoscale Some of the important factors are the ratio of kcatKm which denotes the

catalytic efficiency of the enzyme possible substrates stability of enzymessubstrates

ease of immobilization relationship with other enzymes in cascade reactions and

commercial availability of the enzymes The enzyme being used in the present study is

horseradish peroxidase It acts on a molecule of hydrogen peroxide and reduces it while

simultaneously oxidizing another substrate molecule In the absence of alternate

oxidizable substrate HRP can oxidize water and oxygen back to hydrogen peroxide24

thus giving an equilibrium reaction

13

211 Horseradish peroxidase

Figure 2 3 Horseradish peroxidase

Horseradish peroxidase belongs to the group of peroxidases which are a group of heme-

containing oxidoreductases that act on peroxide as hydrogen donor25

It is a redox

enzyme which contains a protoheme a complex of trivalent iron with 1358-

tetramethyl-24-divinylporphin-67-dipropionic acid as a prosthetic group It is a 44 kDa

protein which includes 33900 Da polypeptide chain bound with eight oligosaccharide

chains The carbohydrate chains improve the stability of the enzyme by protecting it

against radicals formed in the course of the reaction it catalyzes26

Horseradish

peroxidase is found in horseradish and is used widely in bioanalytical applications such

as ELISA and Western blots It decomposes hydrogen peroxide into water and oxygen

while simultaneously oxidizing another substrate27

The oxidizable substrates can be

chromogenic substrates such as 22rsquo55rsquo-tetramethyl-benzidine (TMB) and 33-

diaminobenzidine (DMB) or chemoluminescent substrates like SuperSignal and ECL

luminal28

14

Figure 2 4 Reaction catalyzed by horseradish peroxidase

The functional parameters for horseradish peroxidase include Km of 015 mM and kcat of

790 s-1

at an optimum temperature of 20 ordmC and an optimum pH of 6429

Some of the

inhibitors of HRP are 110-phenanthroline aminotriazole and bromine

22 Experimental

221 Materials and methods

Uniform carboxyl modified polystyrene microspheres 05 microm in diameter were obtained

from Polysciences Inc The size of the purchased microspheres was verified by Field

Emission Scanning Electron Microscopy (FESEM) Peroxidase Type I from

Horseradish N-(3-dimethyl-aminopropyl)-Nrsquo-ethylcarbodiimide (EDC) and hydrogen

peroxide solution 50 wt in water were purchased from Sigma-Aldrich N-

hydroxysuccinimide (NHS) ge 97 was purchased from Fluka All chemicals were of

reagent grade and used without further purification Nanopure deionized water was used

for all experiments and preparation of solutions All measurements were made at room

15

temperature (25 oC) unless mentioned otherwise The UV-Visible spectroscopy

measurements were performed using Agilent 8453A diode-array UV-Visible

Spectrophotometer with ChemStation UV-Vis Software Diffusion coefficient

measurements were performed using Nanosight LM10

222 Synthesis of Janus particles

A monolayer of particles was prepared according to the procedure (Figure 2 5) by

Goldenberg L et al30

Glass slides were placed on the bottom of a petridish Water was

put into the petridish so that the entire slide was immersed in water A thin layer of

hexane was added on top of water in the petridish A suspension of microspheres in

ethanol (5 ww) was introduced to the water-hexane interface After the monolayer was

formed the glass slide was lifted up through the monolayer with tweezers The

microsphere-covered glass slides were left to dry overnight at room temperature

Following drying a thin film of silica was evaporated on the glass slides in Semicore

evaporator The spheres were partially coated with 30 nm of silica Particles were

removed from the glass slide by gentle sonication The polystyrene-silica (PS-SiO2)

Janus particles thus obtained were washed in deionized water several times before

further immobilization steps were performed FESEM was used to confirm synthesis of

Janus particles

16

Figure 2 5 Preparation of PS-SiO2 Janus particles by evaporation

223 Immobilization of enzymes using EDCNHS coupling method

1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide and N-hydroxysulfosuccinimide

(EDCNHS) coupling procedure was followed for immobilizing horseradish peroxidase

type I on 05 μm PS-SiO2 Janus particles (Figure 2 6) In this process EDC reacts with a

carboxyl groups on the Janus particles to form an amine-reactive O-acylisourea

intermediate If this intermediate does not encounter an amine it can hydrolyze and

regenerate the carboxyl group In the presence of NHS EDC can be used to convert

carboxyl groups to amine-reactive sulfo-NHS esters which are much more stable and do

not readily hydrolyze to regenerate carboxyl groups In the current experiment

immobilization was performed in the presence as well as absence of a long spacer

17

molecule (linker) between Janus particles and enzymes The chemical 6-aminohexanoic

acid was used as the linker (Figure 2 7) HRP immobilized PS-SiO2 Janus particles were

stored in deionized water until required for further experiments

Figure 2 6 EDCNHS coupling method for immobilization of enzyme on carboxylated

polystyrene particles

Figure 2 7 Immobilization of enzyme on Janus particles with and without 6-

aminohexanoic acid linker

18

224 Enzyme assay

Assay for activity of HRP was performed using a spectrophotometric method which

involved using 4-aminoantipyrene as hydrogen donor3132

The reaction rate was

determined by measuring increase in absorbance at 510 nm resulting from the

decomposition of hydrogen peroxide One unit represents the decomposition of 1 μM of

hydrogen peroxide per minute at 25 degC and pH 70 under the specified conditions

The spectrophotometer was set to the wavelength of 510 nm and temperature of 25 degC

Phenol4-aminoantipyrene solution (14 mL) and 00017 M H2O2 solution (15 mL) were

pipetted into each cuvette The cuvette was incubated for 3-4 mins at 25degC and blank was

established A 01 mL solution of diluted enzyme was added to the above mixture and

absorbance was recorded for 4-5 mins Standard graphs of absorbance versus time were

drawn for known concentration of the free enzyme and compared to that of the

immobilized enzyme on the Janus particles The estimate of the amount of enzyme

present on the Janus particles was deduced from the difference between the quantity of

the free enzyme previously added for immobilization and the amount that was measured

to be in the supernatant after some of the enzyme became attached to the Janus particles

The experiment was carried out in triplicate to assess reproducibility

19

The formula used to calculate the activity of enzyme is

where ∆Amin denotes the change in absorbance per minute

225 Diffusion coefficient measurements using Nanosight LM10

Diffusion coefficients for HRP immobilized particles were measured using Nanosight

LM 10 nanoparticle tracking instrument with Nanoparticle Tracking Analysis (NTA) 20

software in the presence of 003 H2O2 solution as the substrate and also in the presence

of only deionized water The videos were recorded for 30 seconds at the rate of 30 frames

per second Each experiment was carried out in triplicate to assess reproducibility

Particles

visualization

zone

Microscope with

LM 10 unit

LM 10 unit

Figure 2 8 Nanosight LM 10 instrumentation

20

Nanosight LM 10 (Figure 2 8) generates videos of population of moving nanoparticles in

a liquid when illuminated by laser light The particles which pass through the laser beam

path are seen by the instrument as small points of light undergoing Brownian motion

(Figure 2 9a)

The videos are further analyzed by NTA (Nanoparticle tracking analysis) software

(Figure 2 9b) which identifies and tracks each particle independently on a frame-by-

frame basis The software also calculates and subtracts any bulk flow in the system The

mean square displacement is calculated for each particle as long as it is visible If the

particle blinks that is if it disappears momentarily and then reappears on the screen the

software will consider the same particle as two separate ones All analysis data is

exported to the excel files in terms of diffusion coefficients of the tracked particles

B A

Figure 2 9 NTA video analysis (a) Particles as they are seen on the screen during

experimentation in Nanosight LM10 (b) NTA screen as the particles are being analyzed for

their diffusion coefficients

21

23 Results

231 Enzyme assay

The activity of free HRP enzyme reported by Sigma Aldrich is 150 unitsmg However

the activity of HRP upon analysis was found to be 52158 unitsmg which is much lower

than the reported value The activity of HRP was found to be further lowered to 2133

unitsmg when it was immobilized on the PS-SiO2 Janus particles in the presence of a

linker The following formula is used to calculate enzyme activity

where ΔAmin is the change in absorbance per minute

22

Figure 2 10 Graph for the assay of solution of free enzyme containing 05mgL of HRP

ΔAmin = 01716 Unitsmg of free enzyme = 52158

During EDCNHS coupling 04 mg enzyme was added to 1 ml of 025 vv suspension

of Janus particles Thus initial concentration of enzyme in the mixture was 04 mgml

Assuming retained activity of the enzyme in supernatant solution the amount of enzyme

in supernatant was calculated to be 03868 mgml from the assay Amount of enzyme

functionalized on the Janus particles was calculated as the difference between enzyme

previously added for immobilization (04 mgml) and the enzyme present in supernatant

(03868 mgml) and was found to be 00132 mgml Enzyme assay was performed on the

enzyme immobilized Janus particles to estimate the activity of immobilized enzyme

(Figure 2 11) and was found to be 2133 unitsmg

y = 0002x + 00516

0

01

02

03

04

05

06

07

08

0 50 100 150 200 250 300 350 400

Ab

sorb

an

ce

51

0 n

m

Time (sec)

Assay of free enzyme HRP

23

Figure 2 11 Graph for the assay of of immobilized enzyme on 05 μm Janus particles

(~025 vv) ΔAmin = 01853 Unitsmg of free enzyme = 2133

232 Diffusion coefficient measurements

No significant increase was observed in diffusion coefficient of HRP-immobilized Janus

particles in the presence of H2O2 as the substrate Similar results were obtained for

diffusion coefficient measurement with and without linker moieties bridging the enzymes

and particles(Table 2 1 Table 2 2) Thus the linker did not seem to assist in increasing

the particle diffusion by improving or retaining the activity of the enzyme

y = 00015x + 00953

0

01

02

03

04

05

06

07

0 50 100 150 200 250 300 350 400

Ab

sorb

an

ce

510 n

m

Time (sec)

Assay of HRP immobilized 05 μm Janus particles

24

Table 2 1 Diffusion coefficient measurements of HRP immobilized Janus particles

without linker

HRP immobilized

Janus particles in

003 H2O2

Control - Janus

particles in

003 H2O2

Control - HRP

immobilized Janus

particles in DI water

Diffusion coefficient

(10-10

cm2s)

1359416 1051777 1308675

Standard deviation 4534801 4122075 4918597

Table 2 2 Diffusion coefficient measurements of HRP immobilized Janus particles in

the presence of linker

HRP immobilized

Janus particles in

003 H2O2

Control - Janus

particles in

003 H2O2

Control - HRP

immobilized Janus

particles in DI water

Diffusion coefficient

(10-10

cm2s)

13838 1339829 1407891

Standard deviation 5644692 5649364 5797218

25

Figure 2 12 Diffusion coefficient measurements of PS-SiO2 Janus particles without and

with 6-aminohexanoic acid linker

24 Discussion

To compare the diffusion coefficients observed during the experiments theoretical

diffusion coefficient was calculated using following equation33

r

kTD altraslation

6 Equation 21

where r is the hydrodynamic radius of particle Dtranslational is translational diffusion

coefficient of the particle T is temperature of the system η is the viscosity of fluid

through which the particles are moving and k is the Boltzmann constant From FE-SEM

images the size of the particles used was determined to be 450 nm For 450 nm diameter

0

20

40

60

80

100

120

140

160

Without linker With linker

Dif

fusio

n c

oeff

icie

nt

(10

-10 c

m2s

)

HRP immobilized 05 μm Janus particles - Diffusion coefficient measurements

HRP immobilized Janus particles in 003 H2O2

Control - Janus particles in 003 H2O2

Control - HRP immobilized Janus particles in DI water

26

particle Dtranslational is equal to 109 μm2s which is comparable to the diffusion

coefficient measured for the enzyme immobilized particles

HRP was found to be active on the Janus particles however the activity of the enzyme

was reduced upon immobilization Lower enzyme activity can be due to the coupling

method used which in this case was EDCNHS covalent binding However EDCNHS

coupling method has been shown to significantly retain the enzyme activity when the

enzyme is immobilized on carboxylated multi-walled carbon nanotubes under appropriate

conditions34

Hence EDCNHS binding method by itself is not believed to be

responsible for reduction of enzyme activity upon immobilization

Direct binding of enzyme to the Janus particle may lead to reduction in the enzyme

activity because of space constraints for the enzyme and difficulty for the passage of

substrate to the active site of the enzyme due to stearic hindrance Hence a linker was

inserted between the enzyme and Janus particle during enzyme immobilization However

inserting a linker between the enzyme and the particle did not lead to enhanced diffusion

of Janus particles or increase in enzyme activity of immobilized enzyme

Lack of enhanced diffusion can be firstly attributed to the reduction in enzyme activity

upon immobilization Enzyme activity may be retained by modifying the conditions

during EDCNHS coupling or changing the binding method altogether The degree of

immobilization of HRP on carboxylated Janus particles can possibly be affected by

several factors such as temperature the concentration of the coupling reagent EDC

27

concentration HRP concentration in solution and incubation time34

Different

combinations of these factors can be tested to find optimum conditions for

immobilization of HRP Alternate binding method can involve thiolation of HRP by the

covalent conjugation of mercaptopropionic acid and then the thiolated HRP can then be

attached on the gold surface of a polystyrene-gold Janus particle35

Thus further

modifications can be made to the system in order to retain enzyme activity on the

immobilized particles and thereby power nanomotors using enzymes

28

Chapter 3

Predator-prey system of enzyme immobilized particles

31 Introduction

Predator-prey systems are widely found in nature and play a major role in biological

processes3637

Predator-prey systems include all the interactions in which one organism

pursues andor consumes another organism One familiar predator-prey system at the

macroscale is that of a tiger chasing and consuming a deer Another not so familiar one

includes Baleen whales which eat millions of microscopic planktons at once38

At the

microscale such kind of interactions can be observed in the case of phagocytes in the

body which are capable of engulfing foreign microorganisms and killing them with a

combination of microbicidal systems39

In the case of cancer the tumor is the prey and

the mature myeloid cells in the body are the predator However the tumor here has a

defense mechanism against the predator in which the tumor produces cytokines which

inhibits the maturation of the myeloid cells and thereby prevents predation40

One similarity that can be observed in all these systems is that the prey possesses some

attribute which is desired by the predator which makes the predator chase the prey A

similar relationship is seen in case of enzymes which are involved in cascade reactions41

In cascade reactions product synthesized by one enzyme is the reactant required by

another enzyme to carry out its own catalytic reaction As such there is a predator-prey

29

kind of dependence between the two enzymes which may cause one enzyme to chase the

other enzyme when placed in suitable substrate solutions However the mechanism

hypothesized for such a system is totally different than the mechanism involved in

biological systems In biological systems such as bacterial chemotaxis or phagocytes

chasing foreign particles in human body complex biochemical reactions are responsible

for the predator-prey behavior42

In the proposed enzyme cascade system there are no

complex biochemical reactions involved The principle hypothesized for predator-prey

behavior of enzyme cascade reactions can be explained as follows Motion of synthetic

motors at nanoscale involves two components translational diffusion and rotational

Brownian diffusion At very short time scales of less than 1Dr where Dr (s-1

) is

rotational Brownian diffusion coefficient the motion of particles is unidirectional For

longer time scales of greater than 1Dr the direction of motion is random giving

poweredactive diffusion43

If the chemical homogeneity is broken that is when there is

higher concentration of a substrate present in one direction of the enzyme the enzyme

might acquire a subtle bias which may lead to a spatial aggregation of the enzymes based

on the substrate concentration gradient The bias can be a result of the relationship

between the speed of enzyme diffusion and substrate concentration where higher

substrate concentration lead to higher enzyme diffusion As a result in an enzyme

cascade system one enzyme will migrate towards higher concentration of substrate

which is present near the other enzyme thus giving rise to a predator-prey behavior

Such enzyme cascades are widely found in signaling networks and metabolic pathways in

living cells and can be used to produce predator-prey like nanomotors systems44

The

30

enzymes being used in the present study are horseradish peroxidase and glucose oxidase

Both enzymes are extremely stable4546

Glucose is the substrate for glucose oxidase

which is widely available and highly stable47

Horseradish peroxidase acts on hydrogen

peroxide It is involved in a cascade reaction with glucose oxidase which produces

hydrogen peroxide by converting glucose into gluconic acid (Figure 3 1)48

Figure 3 1 Concept of particle aggregation due to cascade reactions of immobilized

enzymes

In this experiment glucose oxidase (GOx) and horseradish peroxidase (HRP) were

immobilized on polystyrene carboxylate beads of different sizes using EDCNHS

coupling method Glucose and 3355-Tetramethylbenzidine (TMB) were dissolved

together in water and this solution was used as the substrate solution All the components

were present in a single system in DI water Aggregation of particles and precipitation of

oxidized TMB from the solution was expected to be observed when all these components

were present in such a system

Properties of horseradish peroxidase have been discussed in the previous chapter (Section

211 Horseradish peroxidase)

GOxGOx HRPHRP

Glucose + O2Gluconic acid + H2O2

TMB TMB oxidizedBlue precipitate

H2O

Aggregation

31

311 Glucose oxidase

Figure 3 2 Glucose oxidase

Glucose oxidase (GOx) belongs to the group of oxidoreductases and is also called

glucose aerodehydrogenase49

It is obtained from fungi and has been identified as an

antimicrobial enzyme50

GOx binds to β-D-glucopyranose and catalyzes the oxidation

reaction to from gluconolactone while simultaneously reducing oxygen to hydrogen

peroxide51

GOx has high substrate affinity and specificity high stability and high

turnover number which makes it a suitable biocatalyst for industrial applications52

Figure 3 3 Redox reaction of glucose catalyzed by glucose oxidase53

32

GOx is a glycoprotein with molecular weight of 155 kDa and consists of two identical

polypeptide subunits linked together by a disulfide bond The optimum pH for GOx is

55 though it is stable in a broad pH range of 4 to 7 and can be stored for a long period of

time51

The functional parameters for glucose oxidase include Km of 151 mM and kcat of

337 s-1

at temperature of 20 ordmC and pH of 754

Some of the inhibitors of GOx are 2-

Deoxy-D-glucose adenine nucleotides and NaNO3

32 Experimental

321 Materials and methods

Uniform carboxyl modified polystyrene microspheres 05 microm in diameter were obtained

from Polysciences Inc The size of the purchased microspheres was verified by FESEM

Peroxidase Type I from horseradish glucose oxidase Type II-S from Aspergillus niger

N-(3-dimethyl-aminopropyl)-Nrsquo-ethylcarbodiimide (EDC) and Hydrogen peroxide

solution 50 wt in water were purchased from Sigma-Aldrich N-hydroxysuccinimide

(NHS) was purchased from Fluka D-Glucose (99) was purchased from VWR

International Secure-Seal imaging spacers (9mm diameter) for glass slides were obtained

from Sigma Aldrich All chemicals were of reagent grade and used without further

purification Nanopure deionized water was used for all experiments and preparation of

solutions All measurements were made at room temperature (25 oC) unless mentioned

otherwise The UV-Visible measurements were performed using Agilent 8453A diode-

array UV-Visible Spectrophotometer with ChemStation UV-Vis Software Optical

33

microscopy experiments were performed using Axiovert 200 MAT microscope by Carl

Zeiss Diffusion coefficient measurements were obtained using Nanosight LM10

322 Optical microscopy experiments

The enzyme substrate system was observed under optical microscope to check for

aggregation and precipitation due to cascade reactions Several such systems were

studied Each system in general consisted of GOx and HRP immobilized on different

polystyrene particles The substrate solutions consisted of 01 M glucose and different

concentrations of TMB solution The systems were studied in the presence of all the

constituents and in the absence of glucose GOxHRP and TMB Systems consisting of

different sized HRP immobilized particles in substrate solution of 003 hydrogen

peroxide and TMB were also studied Videos were recorded over a period of 24 hours to

observe changes in the system Each experiment was performed atleast thrice to assess

reproducibility of results

323 Diffusion coefficient measurements using Nanosight LM10

Diffusion coefficient of GOx and HRP immobilized particles were measured using

Nanosight LM10 nanoparticle tracking instrument with Nanoparticle Tracking Analysis

(NTA) 20 software Diffusion coefficients were measured for different systems namely

GOx particles in 01 M glucose and oxygen both GOx and HRP functionalized particles

in 01 M glucose and oxygen along with controls for the individual systems Videos were

34

recorded for 30 seconds at the rate of 30 frames per second Each experiment was carried

out in triplicate to assess reproducibility

33 Results

331 Optical microscopy

According to the enzyme cascade model precipitation of TMB was possible only upon

the interaction of GOx and HRP immobilized particles in presence of appropriate

substrate solution Hence in the first set of experiments precipitation of TMB was used

as an indication of the type of interaction between GOx and HRP immobilized particles

Fan-shaped structures were observed when both HRP and GOx immobilized particles

were present in a substrate solution consisting of glucose and TMB These structures

started forming as a single strand and then branched out and intertwined to form the fan-

shaped structures (Figure 3 4)

Table 3 1 Observations for formation of fan-shaped structures in enzyme cascade

system in presence of different components

GOx HRP Glucose TMB Observation

Yes Yes Yes Yes

Fan-shaped structures observed particles associated with

the fan-shaped structures

No Yes Yes Yes No fan-shaped structures

Yes No Yes Yes No fan-shaped structures

Yes Yes No Yes No fan-shaped structures

Yes Yes Yes No No fan-shaped structures

35

Fan-shaped structures can also be observed in the system consisting of only HRP

immobilized particles in substrate solution of hydrogen peroxide and TMB The size of

the fan-shaped structures depends on the size of the enzyme immobilized particles Free

enzymes show extremely tiny structures which are difficult to view under the optical

microscope

Figure 3 4 Formation of fan-shaped structures when both HRP and GOx

immobilized particles were present in a substrate solution consisting of glucose and

TMB A In the initial stages single strands form on the surface of liquid (5Ox) B

Single strands branch out and intertwine to form fan-shaped structures (50x) C Many

such structures form and settle down after 20 minutes (20x)

A

C

B

36

Experiments were performed in the absence of TMB that is the system consisted of GOx

and HRP immobilized particles in glucose substrate solution In the absence of TMB the

particles were expected to move closer to each other without any precipitation due to the

cascade reaction However there was no conclusive aggregation of particles observed in

the system

Free Enzyme 005 μm HRP - immobilized particle

10 μm HRP - immobilized particle 05 μm HRP - immobilized particle

Figure 3 5 Size dependence of fan-shaped structures on the size of HRP -

immobilized particles (20x)

37

Table 3 2 Observations for particle aggregation in enzyme cascade system in presence

and absence of TMB

Glucose GOx HRP TMB Observation

Present Present Present Present Aggregation and precipitate

Present Present Present Absent No conclusive aggregation

Present Absent Present Present No aggregation or ppt

Absent Present Present Present No aggregation or ppt

Present Present Absent Present No aggregation or ppt

332 Diffusion coefficient measurements

Diffusion coefficients were measured for GOx immobilized particles of 05 μm diameter

in substrate solution of 01 M glucose and oxygen Diffusion coefficients of particles

were also measured for system consisting of GOx and HRP functionalized particles in

glucose and oxygen Each experiment was done in triplicate to assess reproducibility

Nanosight visualization chamber being a closed chamber oxygen was purged into all the

solutions before starting the experiments to ensure sufficient supply of oxygen for

catalytic reaction of GOx

38

Table 3 3 Diffusion coefficients for 05 μm GOx immobilized particles in 01 M

glucose purged with O2

Mean

GOx

particles in

01 M

glucose

Control - GOx

particles in DI

water

Control - GOx

particles in 01 M

glucose without

oxygen purging

Control -

particles in 01

M Glucose

Diffusion

coefficient

(x10-10

cm2s)

13316 12888 13827 13702

Standard

deviation 6307 4926 4424 7468

Table 3 4 Diffusion coefficients for GOx and HRP immobilized particles in 01 M

glucose and purged with O2

Mean

GOx- HRP-

functionalized

particles in 01 M

glucose

Control ndash Gox-

HRP- functionalized

particles in DI water

Control - particles

in 01 M glucose

Diffusion coefficient

(x10-10

cm2s)

13005 14434 13702

Standard deviation 5263 6622 7468

No significant increase was observed in diffusion coefficient of enzyme immobilized

particles in any of the systems

39

34 Discussion

Formation of fan-shaped structures due to patterned deposition of oxidized TMB

indicates some kind of interaction between GOx and HRP immobilized particles Particle

diameter dependence of the size of structures can be explained with a simple model in

Figure 3 6 A larger particle will have a higher surface area as compared to a smaller

particle Hence more enzyme is immobilized on the larger particle As a result higher

quantity of TMB is oxidized and precipitated by the larger particle as compared to the

smaller particle This leads to thicker strands of fan-shaped structures eventually scaling

up the whole system to give bigger fan-shaped structures

The exact reason for the formation of such fan-shaped structures which are dependent on

particle size could not be determined It is hypothesized that hydrogen peroxide andor

Figure 3 6 Model for size dependence of fan-shaped structures on the particle

diameter of enzyme immobilized particles Larger the particle more surface area is

available for precipitation of TMB which eventually leads to bigger fan-shaped

structures

Larger particle Smaller particle

40

TMB gradient in the substrate solution might be responsible for such structures If there

is higher concentration of H2O2 on one side of the particle and assuming an even

concentration distribution of TMB there will be selective precipitation in the direction of

H2O2 When several particles come in the vicinity of higher concentration of H2O2 the

precipitation lines will converge to give fan-shaped structures (Figure 3 7)

Figure 3 7 Hypothesis for formation of fan-shaped structures

In the system consisting of GOx immobilized particles HRP immobilized particles

glucose and TMB it was observed that the fan-shaped structures formed at the liquid-air

interface and eventually settled down to the bottom of the glass slide This makes sense

as oxygen is necessary for the catalytic reaction of conversion of glucose to gluconic acid

and hydrogen peroxide which takes place on the surface of GOx immobilized particle As

a result H2O2 is primarily formed at the liquid-air interface and is then used by HRP for

precipitation of TMB

Following the observations that precipitation of TMB occurs at the surface GOx and

HRP immobilized particles are also expected to interact at the surface in the absence of

TMB Due the cascade reaction it was expected that HRP immobilized particles will be

41

attracted to GOx immobilized particles in appropriate substrate solution Experiments

were carried out to visualize inter-particle interactions at the liquid-air interface

However liquid-air interface not being a stable system to image the precise interaction

of GOx and HRP immobilized particles could not be determined Significant external

disturbances were involved at the interface To enable the cascade reaction in the bulk of

the liquid oxygen was purged into the reaction system before starting the experiment

However this did not lead to significant reaction in the bulk of the liquid and the reaction

prominently took place at the liquid-air interface where the oxygen concentration was

higher

In order to observe the interaction of GOx and HRP immobilized particles better imaging

techniques need to be used to observe diffusion of particles in absence of external

disturbances at the liquid-air interface Large quantity of oxygen needs to be purged into

the system and retained to make the reaction more pronounced in the bulk of the substrate

solution Higher concentration of oxygen in water may also be achieved by using oxygen

enriched water which is commercially available55

Alternatively a different enzyme cascade system may be used which does not require a

gaseous substrate56

For example glycogen synthase synthesizes glycogen from glucose-

1-phosphate and glycogen phosphorylase breaks down glycogen to glucose-1-

phosphate57

This system does not require a gaseous substrate and might be able to

overcome the deficiencies of GOx and HRP enzyme cascade system

42

Chapter 4

Silver-titania nanomotors

41 Introduction

Bimetallic nanowires made of platinum and gold have been developed by WPaxton

etal1 The rods move autonomously in a solution of hydrogen peroxide In the present

study silver-titania (Ag-TiO2) particles are being synthesized and investigated for motion

in presence of hydrogen peroxide and UV light With proper control of conditions and

UV light trigger it can be possible to control the speed and direction of motion of these

nanoparticles

Hydrogen peroxide is produced in the human body by phagocytes as an immune response

to foreign particles and pathogens5859

Also silver nanoparticles have been shown to be

biocompatible and capable of interacting with cells in human body without disrupting

cellular function60

Similarly it has been demonstrated that titania is compatible with

different cells DNA and proteins in human body and does not cause toxic effects Hence

silver-titania nanomotors which swim in hydrogen peroxide may open interesting

avenues in applications like targeted drug delivery in those parts of the body where

hydrogen peroxide is present

43

42 Experimental

421 Materials and methods

Silver nitrate (999995) was purchased from Alfa Aesar Gum arabic was purchased

from MP Biomedicals Hydrogen peroxide solution 50 wt in water and ascorbic acid

were purchased from Sigma-Aldrich Nanopure deionized water was used for all

experiments and preparation of all solutions Secure-Seal imaging spacers (9 mm

diameter) were obtained from Sigma Aldrich All experiments were carried out at room

temperature of 25ordmC unless otherwise mentioned Ag-TiO2 particles were synthesized by

evaporation of titania in Semicore electron beam evaporator Particles were characterized

using field-emission scanning electron microscopy Optical microscopy experiments

were performed using Axiovert 200 MAT microscope by Carl Zeiss Particles were

tracked using Physvis particle tracking software (Figure 4 1) in which the position

information of a particle on screen is stored on a frame by frame basis The data is stored

in a dat file as X and Y co-ordinates of particles tracked on the screen This information

can be converted to diffusion coefficients of the particles by further manipulation using

MathWorks-MATLABreg software

44

Figure 4 1 Physvis particle tracking software

422 Synthesis of silver colloidal particles

Silver particles were synthesized by a nucleationgrowth type reaction developed by

Goya and Matijevic61

and modified by Velikov62

Silver nitrate was used as precursor

metal salt for synthesis of silver particles Solution A was 75 ml of 50 mM AgNO3 and

035 by weight gum arabic Solution B was 75 mL of 100 mM 1-ascorbic acid and

035 by weight gum arabic Solution B was rapidly added to solution A in a 250 mL

reaction flask The reaction flask was covered and particles were stirred for 48 hrs After

48 hours particles were allowed to settle for 2-3 hours in refrigerator and supernatant

was separated from the particles Particles were then rinsed with DI water and centrifuged

thrice to obtain pure silver particles Zeta potential of these particles was reported to be

45

around -50 to -60 mV and the size was around 1μm The size of the particles can be

varied by varying the amounts of ascorbic acid and silver nitrate Using molar ratio of 31

of ascorbic acid silver nitrate can give particles of 200 nm diameter whereas ratio of

151 can give particles of upto 5 μm diameter

423 Preparation of silver-titania (Ag-TiO2) particles

A monolayer of silver particles was prepared according to the procedure by Goldenberg

et al63

Glass slides were placed on the bottom of a petridish Water was put into the

petridish so that the entire slide was immersed in water A thin layer of hexane was then

added on top of water in the petridish A suspension of silver particles in ethanol (5

ww) was slowly introduced to the water-hexane interface in the dish After the

monolayer was formed the glass slide was lifted up through the monolayer with tweezers

The silver particles-covered glass slides were left at room temperature to dry overnight

Following drying a thin film of titania was evaporated on the glass slides in Semicore

evaporator (Figure 2 7) The spheres were partially coated with a 10 nm film of titania

Ag-TiO2 particles were removed from the glass slide by gentle sonication FE-SEM was

used to characterize the particles (Figure 4 2) The thin titania layer was not easily

distinguishable due to roughness of the particles

46

Figure 4 2 FESEM image of Ag-TiO2 particles

424 Optical microscopy experiments

Imaging spacers were placed on a glass slide Suspension of Ag-TiO2 particles was

placed on the glass slide and the particles were allowed to settle Movement of particles

was observed in DI water and videos were recorded at 50x magnification of optical

microscope for 30 sec 05 H2O2 was added to the particle suspension on the slide

Videos were recorded at 50x ranging from 30 sec to 10 min to study the movement of

silver titania particles in hydrogen peroxide solution Particles in both DI water and

hydrogen peroxide were subjected to UV light and videos were recorded to study particle

dynamics in presence of UV radiation Particle tracking was performed using Physvis

particle tracking software Atleast 30 particles in every system were tracked for 10

seconds each Particle tracking data was obtained in terms of X and Y coordinates of

particles on screen This data was manipulated using MathWorks-Matlab software and

graphs of mean square displacement of particles versus time were plotted Diffusion

coefficients of particles were calculated from these graphs

47

43 Results and Discussion

Ag-TiO2 particles demonstrated autonomous motion in the presence of H2O2 No UV

light was required for this motion Motion of the particles was accelerated by addition of

fresh H2O2 to the system When subjected to UV radiation the particles displayed

momentary acceleration for 4-5 seconds

Figure 4 3 Mechanism of motion of Ag-TiO2 nanomotors

In the presence of H2O2 silver part of the Ag-TiO2 particle converts H2O2 to H2O and O2

This may lead to propulsion of the Ag-TiO2 particle due to bubble propulsion andor self-

diffusiophoresis In the presence of UV light additional reaction takes place on the

titania part of Ag-TiO2 particle which increases the velocity of the Ag-TiO2 particles

(Figure 4 4) This UV triggered photoactivity of titania is due to hole-electron separation

caused by photons having energy equal to or greater than the band gap of titania646566

Most of the holes-electrons recombine but a small fraction move to the surface and react

with the available redox species like water and oxygen to form protons (H+) superoxide

(O2-) and hydroxyl (OH) radicals

6566 This may lead to propulsion by diffusiophoresis

as well as electrophoresis

48

Figure 4 4 Silver-titania motors move faster when subjected to UV radiation

In addition to linear motion rotational motion is also observed in several particles and

particle aggregates Aggregation of particles can lead to a net circular propulsive force

which can rotate the aggregate as shown in Figure 4 5

Figure 4 5 Rotation due to particle aggregation of Ag-TiO2 particles

Particle tracking by Physvis software revealed a significant increase in diffusion

coefficient of Ag-TiO2 particles in hydrogen peroxide solution as compared to DI water

49

The Ag-TiO2 particles have a diffusion coefficient of 1491 μm2s in hydrogen peroxide

versus only 180 μm2s in DI water Thus the Ag-TiO2 particles move around more than

10 times their body length per second in hydrogen peroxide (Figure 4 6) When

subjected to UV light the particles experience a significant acceleration in hydrogen

peroxide which lasts only for a short duration of time (4-5 sec) after which the particles

return to their original velocity The diffusion coefficient of the particles during this UV

light trigger is found to be around 5093 μm2s UV light trigger does not have any visible

effect on the Ag-TiO2 particles in DI water

Table 4 1 Translational diffusion coefficients of Ag-TiO2 particles in hydrogen

peroxide and water

Mean 05 H2O2 solution DI water

Diffusion coefficient of Ag-TiO2 (μm2s)

without UV light

1491 180

Diffusion coefficient of Ag-TiO2 (μm2s)

in the presence of UV light

5093 113

50

Figure 4 6 Mean square displacement curves of Ag-TiO2 Janus particles in hydrogen

peroxide and DI water

When higher concentration (~30 vv) of Ag-TiO2 particles in hydrogen peroxide is

subjected to UV radiation the particles exhibit pattern formation (Figure 4 7)

Aggregation of Ag-TiO2 Janus particles is observed when UV light is turned on and the

particles disaggregated when UV light is switched off This can be due to predator-prey

kind of interaction between silver and titania half of the Janus particles in the presence of

UV light Oxygen and water that are produced at the silver end by decomposition of

hydrogen peroxide are used up by titania when subjected to UV radiation This can lead

to attraction between titania and silver parts of neighboring Janus particles causing

aggregation

y = 20373x

y = 59994x

y = 71848x

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10 12

Mean

sq

uare

dis

pla

cem

en

t (μ

m2s

)

Time (sec)

Mean square displacement vs Time for Ag-TiO2

particles

05 Hydrogen peroxide and UV05 Hydrogen peroxide

DI Water

51

A UV OFF B UV ON

C UV OFF

Figure 4 7 Higher concentrations of Ag-TiO2 particles showing aggregation when UV

is turned on

52

Chapter 5

Summary and Conclusion

In summary no significant enhancement in diffusion was observed in case of enzyme

immobilized Janus particles in substrate solution Presence of a linker between enzyme

and particle did not increase the diffusion coefficient of particles Alternative enzyme

immobilization methods need to be tested like thiolation of enzyme and subsequent

binding to gold surface of a Janus particle35

or expressing the enzyme with polyhistidine

or avidin tag67

This can result in higher enzyme activity upon immobilization and may

lead to enhanced diffusion of Janus particles

Predator-prey system of GOx and HRP immobilized particles showed some promising

results Aggregation of particles and formation of fan-shaped structures by TMB

oxidation were observed under optical microscope when GOx and HRP immobilized

particles glucose and TMB were present in the system The size of the fan-shaped

structures was dependent on the size of enzyme immobilized particles This can be

indicative of some kind of interaction between the GOx and HRP immobilized particles

No conclusive aggregation of GOx and HRP immobilized particles was observed in

absence of TMB Better imaging techniques are required to study the interaction of GOx

and HRP immobilized particles at the unstable liquid-air interface where majority of the

reaction takes place Other enzyme cascade systems not requiring gaseous reactants like

glycogen synthase and glycogen phosphorylase should be tested for similar activity

53

Ag-TiO2 particles were found to exhibit autonomous motion in the presence of hydrogen

peroxide solution The motion was momentarily accelerated upon exposure to UV

radiation The particles showed a diffusion coefficient of 1491 μm2s in hydrogen

peroxide versus only 180 μm2s in DI water Upon subjecting particles to UV radiation in

presence of H2O2 diffusion coefficient increased to 5093 μm2s for a period of 4-5

seconds In addition to linear motion rotating motion could also be observed in some

particle aggregates Further development of these aggregate structures can lead to

formation of rotors at nanoscale Higher concentration of silver-titania particles in

hydrogen peroxide displayed aggregation in presence of UV light and disaggregated

when UV light was switched off This may be attributed to the predator-prey kind of

interaction between silver and titania where oxygen and water that are produced by

silver by decomposition of hydrogen peroxide are used up by titania when subjected to

UV radiation This promises to be an interesting system and needs to be further

investigated to explore its properties

54

References

1 Paxton WF Sundararajan S Mallouk TE Sen A Angew Chem Int Ed Engl

2006 45 5420-5429

2 Golestanian R Liverpool TB Adjari A Phys Rev Lett 2005 94 220801

3 httpwwwsilvermedicineorgh2o2html

4 Rami Pedahzur Ovadia Lev Badri Fattal Hillel Shuval Water Science and Technology

1995 31(5-6) 123-129

5 W Went F American Scientist 1968 56

6 Adriano Cavalcanti Tad Hogg Bijan Shirinzadeh IEEE MHS 2006 International

Symposium on Micro-NanoMechatronics and Human Science 2006 226-232

7 EM Purcell American Journal of Physics 1977 45 3-11

8 Golestanian R Phys Rev Lett 2010 105 018103(1)-018103(4)

9 S S Dukhin Z R Ulberg G L Dvornichenko B V Deryagin Russian Chemical

Bulletin 1983 31(8) 1535-1544

10 Dukhin AS Goetz PJ Ultrasound for characterizing colloids Particle sizing zeta

potential rheology Elsevier 2002 59

11 HJKeh HJTu Trends in Colloid and Interface Science XIV Progress in Colloid and

Polymer Science 2000 115 174-180

12 D Zinemanas A Nir International Journal of Multiphase Flow 1995 21(5) 787-800

55

13 Stephen Ebbens Jonathan Howse Soft matter 2010 6 726-739

14 J Vicario R Eelkema W R Browne A Meetsma R M La Crois B L Feringa

Chem Commun 2005 31 3936

15 Davide Pantarotto Wesley R Browne Ben L Feringa Chem Commun 2008 1533 -

1535

16 Lisa A Cameron Matthew J Footer Alexander van Oudenaarden Julie A Theriot

PNAS 1999 96(9) 4908-4913

17 Ali Najafi Ramin Golestanian Phys Rev E 2004 69 062901

18 Jinseok Kim Jungyul Park Sungwook Yang Jeongeun Baek Byungkyu Kim Sang

Ho Lee Eui-Sung Yoon Kukjin Chun Sukho Park Lab Chip 2007 7 1504 - 1508

19 Kurt M Dubowski Clinical Chemistry 2008 5411 1919ndash1920

20 Spector DA Yang Q Wade JB Am J Physiol Renal Physiol 2007 292(1) 467-474

21 Rhee SG Chang TS Bae YS Lee SR Kang SW J Am Soc Nephrol 2003 14(8-3)

211-215

22 Sakaue T Kapral R Mikhailov A S Eur Phys J B 2010 75 381ndash387

23 Muddana H S Sengupta S Mallouk T E Sen A Butler P J J Am Chem Soc

2010 132 (7) 2110ndash2111

24 Baker CJ Deahl K Domek J Orlandi EW Arch Biochem Biophys 2000 382(2) 232-

237

25 V Vojinovic RH Carvalho F Lemos JMS Cabral LP Fonseca BS Ferreira

Biochemical Engineering Journal 2007 35 126-135

56

26

OV Lebedeva NN Ugarova Russian Chemical Bulletin 1996 45(1) 18-25

27 Nigel C Veitch Phytochemistry 2004 65(3) 249-259

28 Kenneth H Petersen Journal of Immunological Methods 2009 340(1) 86-89

29 httpwwwbrenda-enzymesorgphpresult_flatphp4ecno=11117

30 Goldenberg L M Wagner J Stumpe J Paulke B-R Goumlrnitz E Langmuir 2002 18

5627-5629

31 httpwwwgoldbiocompdf3761-Protocol201pdf

32 Av Rovisco Pais Lisboa Portugal T amp D Technology West Yorkshire UK Journal

of Molecular Catalysis B Enzymatic 2004 28(2-3) 129-135

33 httpwwwphysicsnyuedugrierlabmethodsnode11html

34 Yoon-Mee Lee1 O-Yul Kwon Yeo-Joon Yoon Keungarp Ryu Biotechnology Letters

2006 28 39ndash43

35 JC Pyun SD Kim JW Chung Analytical Biochemistry 2005 347(2) 227-233

36 George H Pimbley Jr Mathematical Biosciences 1974 20(1-2) 27-51

37 Andy Fenton Sarah E Perkins Parasitology 2010 137 1027-1038

38 Mead James G Brownell Robert L Mammal Species of the World A Taxonomic and

Geographic Reference (3rd ed) Johns Hopkins University Press 2005 2 2142

57

39

Krause KH Schweiz Med Wochenschr 2000 130(4) 97-100

40 Irina Kareva Faina Berezovskaya Carlos Castillo-Chavez Journal of Biological

Dynamics 2010 4(4)315 - 327

41 Delaittre G Reynhout I Cornelissen J Nolte R Chemistry ndash A European Journal

2009 15 12600ndash12603

42 C Buller DH Johnson RC Tschumper Investigative Ophthalmology amp Visual Science

1990 31 2156-2163

43 Robert M Mazo Brownian Motion Fluctuations Dynamics and Applications Oxford

Science Publications 2009 57

44 Vivek K Mutalik KV Venkatesh Theoretical Biology and Medical Modelling 2005 2

19

45 Satoshi Nakamura Kunimasa Koga Biochemical and Biophysical Research

Communications 1977 78(2) 806-810

46 Krishnananda Chattopadhyay Shyamalava Mazumdar Biochemistry 2000 39 (1)

263ndash270

47 Satoshi Nakamura Yasuyuki Ogura J Biochem 1968 63 (3) 308-316

48 van Dongen SF Nallani M Cornelissen JJ Nolte RJ van Hest JC Chemistry 2009

15(5) 1107-1114

49 Ronald Bentley Methods in Enzymology 1955 1 340-345

50 Vartiainen J Raumlttouml M Paulussen S Packaging Technology and Science 2005

18 243ndash251

58

51 GA Sherbeny AA Shindia YMMM Sheriff International Journal of Agriculture

and Biology 2005 7

52 D Rando G-W Kohring F Giffhorn Appl Microbiol Biotechnol 1997 48

53 Worthington enzyme manual Worthington Biochemical Corporation

httpworthington-biochemcomgopdefaulthtml

54 httpwwwbrenda-enzymesorgphpresult_flatphp4ecno=11117

55 httpwwwoxygenphwatercom

56 P Boon Chock Sue Goo Rhee and Earl R Stadtman Annu Rev Biochem 1980 49

813-841

57 Sybil Golden P A Wals Joseph Katz Analytical Biochemistry 1977 77(2) 436-445

58 R L Baehner S K Murrmann J Davis R B Johnston Jr J Clin Invest 1975 56(3)

571ndash576

59 I Bejarano M P Terroacuten S D Paredes C Barriga A B Rodriacuteguez J A Pariente

Molecular and Cellular Biochemistry 2007 296(1-2) 77-84

60 Michael C Moulton Laura K Braydich-Stolle Mallikarjuna N Nadagouda Samantha

Kunzelman Saber M Hussain Rajender S Varma Nanoscale 2010 2 763-770

61 Goya DV Matijevic E Colloids and Surfaces A 1999 146 139

62 Velikov KP Zegers GE van Blaanderen A Langmuir 2003 19 1384

63 Goldenberg L M Wagner J Stumpe J Paulke B-R Goumlrnitz E Langmuir 2002 18

5627-5629

59

64

Banerjee S Gopal J Muraleedharan P Tyagi A K Raj B Curr Sci 2006

901378ndash1383

65 Bogomolov V N Kudinov E K Firsov Y A Sov Phys Solid State 1968 35 555

66 Kudo A Miseki Y Chem Soc Rev 2009 38 253ndash278

67 Gaj T Meyer SC Ghosh I Protein expression and Purification 2007 56 54-61

10

solute concentration by principle of self-diffusiophoresis Figure 2-2 shows the possible

nonreciprocal changes in the conformation of a theoretical protein which can lead to

motion of the protein22

These nonreciprocal changes in conformation are triggered by the

attachment of substrate molecule and release of product molecules from the enzymersquos

active site

Figure 2 1 Enzyme functionalized Janus particle moves upon catalyzing asymmetrical

decomposition of substrate into product

11

Figure 2 2 Schematic showing idealized nonreciprocal conformation changes in a

theoretical enzyme22

Enhanced diffusion of a single enzyme molecule by catalysis in a substrate solution has

been demonstrated23

The diffusion coefficient of a single urease molecule was measured

using fluorescence correlation spectroscopy and it was observed that the diffusion of the

urease molecule increases by 16-28 by varying urea concentrations from 0001 M to 1

M Addition of enzyme inhibitors to the system curbed the enhanced diffusion This

enhanced diffusion has been attributed to the force generated by self-electrophoresis by

the enzyme in substrate solution

12

Based on the above facts it might be possible to propel a nanoparticle by asymmetric

distribution of enzymes on its surface thereby leading to self-propulsion of the particles

in substrate solution (Figure 2 1) This propulsion may be due to self-

electrophoresisself-diffusiophoresis andor due to nonreciprocal changes in enzyme

conformations Here we aim to investigate a system consisting of polystyrene particles

half coated with enzyme introduced in a suitable substrate solution The enzyme can be

directly linked to the polystyrene particle or it can be linked through a long spacer

molecule (linker) to provide more degrees of freedom to the enzyme

Several factors need to be considered for choosing enzyme for powering motion at the

nanoscale Some of the important factors are the ratio of kcatKm which denotes the

catalytic efficiency of the enzyme possible substrates stability of enzymessubstrates

ease of immobilization relationship with other enzymes in cascade reactions and

commercial availability of the enzymes The enzyme being used in the present study is

horseradish peroxidase It acts on a molecule of hydrogen peroxide and reduces it while

simultaneously oxidizing another substrate molecule In the absence of alternate

oxidizable substrate HRP can oxidize water and oxygen back to hydrogen peroxide24

thus giving an equilibrium reaction

13

211 Horseradish peroxidase

Figure 2 3 Horseradish peroxidase

Horseradish peroxidase belongs to the group of peroxidases which are a group of heme-

containing oxidoreductases that act on peroxide as hydrogen donor25

It is a redox

enzyme which contains a protoheme a complex of trivalent iron with 1358-

tetramethyl-24-divinylporphin-67-dipropionic acid as a prosthetic group It is a 44 kDa

protein which includes 33900 Da polypeptide chain bound with eight oligosaccharide

chains The carbohydrate chains improve the stability of the enzyme by protecting it

against radicals formed in the course of the reaction it catalyzes26

Horseradish

peroxidase is found in horseradish and is used widely in bioanalytical applications such

as ELISA and Western blots It decomposes hydrogen peroxide into water and oxygen

while simultaneously oxidizing another substrate27

The oxidizable substrates can be

chromogenic substrates such as 22rsquo55rsquo-tetramethyl-benzidine (TMB) and 33-

diaminobenzidine (DMB) or chemoluminescent substrates like SuperSignal and ECL

luminal28

14

Figure 2 4 Reaction catalyzed by horseradish peroxidase

The functional parameters for horseradish peroxidase include Km of 015 mM and kcat of

790 s-1

at an optimum temperature of 20 ordmC and an optimum pH of 6429

Some of the

inhibitors of HRP are 110-phenanthroline aminotriazole and bromine

22 Experimental

221 Materials and methods

Uniform carboxyl modified polystyrene microspheres 05 microm in diameter were obtained

from Polysciences Inc The size of the purchased microspheres was verified by Field

Emission Scanning Electron Microscopy (FESEM) Peroxidase Type I from

Horseradish N-(3-dimethyl-aminopropyl)-Nrsquo-ethylcarbodiimide (EDC) and hydrogen

peroxide solution 50 wt in water were purchased from Sigma-Aldrich N-

hydroxysuccinimide (NHS) ge 97 was purchased from Fluka All chemicals were of

reagent grade and used without further purification Nanopure deionized water was used

for all experiments and preparation of solutions All measurements were made at room

15

temperature (25 oC) unless mentioned otherwise The UV-Visible spectroscopy

measurements were performed using Agilent 8453A diode-array UV-Visible

Spectrophotometer with ChemStation UV-Vis Software Diffusion coefficient

measurements were performed using Nanosight LM10

222 Synthesis of Janus particles

A monolayer of particles was prepared according to the procedure (Figure 2 5) by

Goldenberg L et al30

Glass slides were placed on the bottom of a petridish Water was

put into the petridish so that the entire slide was immersed in water A thin layer of

hexane was added on top of water in the petridish A suspension of microspheres in

ethanol (5 ww) was introduced to the water-hexane interface After the monolayer was

formed the glass slide was lifted up through the monolayer with tweezers The

microsphere-covered glass slides were left to dry overnight at room temperature

Following drying a thin film of silica was evaporated on the glass slides in Semicore

evaporator The spheres were partially coated with 30 nm of silica Particles were

removed from the glass slide by gentle sonication The polystyrene-silica (PS-SiO2)

Janus particles thus obtained were washed in deionized water several times before

further immobilization steps were performed FESEM was used to confirm synthesis of

Janus particles

16

Figure 2 5 Preparation of PS-SiO2 Janus particles by evaporation

223 Immobilization of enzymes using EDCNHS coupling method

1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide and N-hydroxysulfosuccinimide

(EDCNHS) coupling procedure was followed for immobilizing horseradish peroxidase

type I on 05 μm PS-SiO2 Janus particles (Figure 2 6) In this process EDC reacts with a

carboxyl groups on the Janus particles to form an amine-reactive O-acylisourea

intermediate If this intermediate does not encounter an amine it can hydrolyze and

regenerate the carboxyl group In the presence of NHS EDC can be used to convert

carboxyl groups to amine-reactive sulfo-NHS esters which are much more stable and do

not readily hydrolyze to regenerate carboxyl groups In the current experiment

immobilization was performed in the presence as well as absence of a long spacer

17

molecule (linker) between Janus particles and enzymes The chemical 6-aminohexanoic

acid was used as the linker (Figure 2 7) HRP immobilized PS-SiO2 Janus particles were

stored in deionized water until required for further experiments

Figure 2 6 EDCNHS coupling method for immobilization of enzyme on carboxylated

polystyrene particles

Figure 2 7 Immobilization of enzyme on Janus particles with and without 6-

aminohexanoic acid linker

18

224 Enzyme assay

Assay for activity of HRP was performed using a spectrophotometric method which

involved using 4-aminoantipyrene as hydrogen donor3132

The reaction rate was

determined by measuring increase in absorbance at 510 nm resulting from the

decomposition of hydrogen peroxide One unit represents the decomposition of 1 μM of

hydrogen peroxide per minute at 25 degC and pH 70 under the specified conditions

The spectrophotometer was set to the wavelength of 510 nm and temperature of 25 degC

Phenol4-aminoantipyrene solution (14 mL) and 00017 M H2O2 solution (15 mL) were

pipetted into each cuvette The cuvette was incubated for 3-4 mins at 25degC and blank was

established A 01 mL solution of diluted enzyme was added to the above mixture and

absorbance was recorded for 4-5 mins Standard graphs of absorbance versus time were

drawn for known concentration of the free enzyme and compared to that of the

immobilized enzyme on the Janus particles The estimate of the amount of enzyme

present on the Janus particles was deduced from the difference between the quantity of

the free enzyme previously added for immobilization and the amount that was measured

to be in the supernatant after some of the enzyme became attached to the Janus particles

The experiment was carried out in triplicate to assess reproducibility

19

The formula used to calculate the activity of enzyme is

where ∆Amin denotes the change in absorbance per minute

225 Diffusion coefficient measurements using Nanosight LM10

Diffusion coefficients for HRP immobilized particles were measured using Nanosight

LM 10 nanoparticle tracking instrument with Nanoparticle Tracking Analysis (NTA) 20

software in the presence of 003 H2O2 solution as the substrate and also in the presence

of only deionized water The videos were recorded for 30 seconds at the rate of 30 frames

per second Each experiment was carried out in triplicate to assess reproducibility

Particles

visualization

zone

Microscope with

LM 10 unit

LM 10 unit

Figure 2 8 Nanosight LM 10 instrumentation

20

Nanosight LM 10 (Figure 2 8) generates videos of population of moving nanoparticles in

a liquid when illuminated by laser light The particles which pass through the laser beam

path are seen by the instrument as small points of light undergoing Brownian motion

(Figure 2 9a)

The videos are further analyzed by NTA (Nanoparticle tracking analysis) software

(Figure 2 9b) which identifies and tracks each particle independently on a frame-by-

frame basis The software also calculates and subtracts any bulk flow in the system The

mean square displacement is calculated for each particle as long as it is visible If the

particle blinks that is if it disappears momentarily and then reappears on the screen the

software will consider the same particle as two separate ones All analysis data is

exported to the excel files in terms of diffusion coefficients of the tracked particles

B A

Figure 2 9 NTA video analysis (a) Particles as they are seen on the screen during

experimentation in Nanosight LM10 (b) NTA screen as the particles are being analyzed for

their diffusion coefficients

21

23 Results

231 Enzyme assay

The activity of free HRP enzyme reported by Sigma Aldrich is 150 unitsmg However

the activity of HRP upon analysis was found to be 52158 unitsmg which is much lower

than the reported value The activity of HRP was found to be further lowered to 2133

unitsmg when it was immobilized on the PS-SiO2 Janus particles in the presence of a

linker The following formula is used to calculate enzyme activity

where ΔAmin is the change in absorbance per minute

22

Figure 2 10 Graph for the assay of solution of free enzyme containing 05mgL of HRP

ΔAmin = 01716 Unitsmg of free enzyme = 52158

During EDCNHS coupling 04 mg enzyme was added to 1 ml of 025 vv suspension

of Janus particles Thus initial concentration of enzyme in the mixture was 04 mgml

Assuming retained activity of the enzyme in supernatant solution the amount of enzyme

in supernatant was calculated to be 03868 mgml from the assay Amount of enzyme

functionalized on the Janus particles was calculated as the difference between enzyme

previously added for immobilization (04 mgml) and the enzyme present in supernatant

(03868 mgml) and was found to be 00132 mgml Enzyme assay was performed on the

enzyme immobilized Janus particles to estimate the activity of immobilized enzyme

(Figure 2 11) and was found to be 2133 unitsmg

y = 0002x + 00516

0

01

02

03

04

05

06

07

08

0 50 100 150 200 250 300 350 400

Ab

sorb

an

ce

51

0 n

m

Time (sec)

Assay of free enzyme HRP

23

Figure 2 11 Graph for the assay of of immobilized enzyme on 05 μm Janus particles

(~025 vv) ΔAmin = 01853 Unitsmg of free enzyme = 2133

232 Diffusion coefficient measurements

No significant increase was observed in diffusion coefficient of HRP-immobilized Janus

particles in the presence of H2O2 as the substrate Similar results were obtained for

diffusion coefficient measurement with and without linker moieties bridging the enzymes

and particles(Table 2 1 Table 2 2) Thus the linker did not seem to assist in increasing

the particle diffusion by improving or retaining the activity of the enzyme

y = 00015x + 00953

0

01

02

03

04

05

06

07

0 50 100 150 200 250 300 350 400

Ab

sorb

an

ce

510 n

m

Time (sec)

Assay of HRP immobilized 05 μm Janus particles

24

Table 2 1 Diffusion coefficient measurements of HRP immobilized Janus particles

without linker

HRP immobilized

Janus particles in

003 H2O2

Control - Janus

particles in

003 H2O2

Control - HRP

immobilized Janus

particles in DI water

Diffusion coefficient

(10-10

cm2s)

1359416 1051777 1308675

Standard deviation 4534801 4122075 4918597

Table 2 2 Diffusion coefficient measurements of HRP immobilized Janus particles in

the presence of linker

HRP immobilized

Janus particles in

003 H2O2

Control - Janus

particles in

003 H2O2

Control - HRP

immobilized Janus

particles in DI water

Diffusion coefficient

(10-10

cm2s)

13838 1339829 1407891

Standard deviation 5644692 5649364 5797218

25

Figure 2 12 Diffusion coefficient measurements of PS-SiO2 Janus particles without and

with 6-aminohexanoic acid linker

24 Discussion

To compare the diffusion coefficients observed during the experiments theoretical

diffusion coefficient was calculated using following equation33

r

kTD altraslation

6 Equation 21

where r is the hydrodynamic radius of particle Dtranslational is translational diffusion

coefficient of the particle T is temperature of the system η is the viscosity of fluid

through which the particles are moving and k is the Boltzmann constant From FE-SEM

images the size of the particles used was determined to be 450 nm For 450 nm diameter

0

20

40

60

80

100

120

140

160

Without linker With linker

Dif

fusio

n c

oeff

icie

nt

(10

-10 c

m2s

)

HRP immobilized 05 μm Janus particles - Diffusion coefficient measurements

HRP immobilized Janus particles in 003 H2O2

Control - Janus particles in 003 H2O2

Control - HRP immobilized Janus particles in DI water

26

particle Dtranslational is equal to 109 μm2s which is comparable to the diffusion

coefficient measured for the enzyme immobilized particles

HRP was found to be active on the Janus particles however the activity of the enzyme

was reduced upon immobilization Lower enzyme activity can be due to the coupling

method used which in this case was EDCNHS covalent binding However EDCNHS

coupling method has been shown to significantly retain the enzyme activity when the

enzyme is immobilized on carboxylated multi-walled carbon nanotubes under appropriate

conditions34

Hence EDCNHS binding method by itself is not believed to be

responsible for reduction of enzyme activity upon immobilization

Direct binding of enzyme to the Janus particle may lead to reduction in the enzyme

activity because of space constraints for the enzyme and difficulty for the passage of

substrate to the active site of the enzyme due to stearic hindrance Hence a linker was

inserted between the enzyme and Janus particle during enzyme immobilization However

inserting a linker between the enzyme and the particle did not lead to enhanced diffusion

of Janus particles or increase in enzyme activity of immobilized enzyme

Lack of enhanced diffusion can be firstly attributed to the reduction in enzyme activity

upon immobilization Enzyme activity may be retained by modifying the conditions

during EDCNHS coupling or changing the binding method altogether The degree of

immobilization of HRP on carboxylated Janus particles can possibly be affected by

several factors such as temperature the concentration of the coupling reagent EDC

27

concentration HRP concentration in solution and incubation time34

Different

combinations of these factors can be tested to find optimum conditions for

immobilization of HRP Alternate binding method can involve thiolation of HRP by the

covalent conjugation of mercaptopropionic acid and then the thiolated HRP can then be

attached on the gold surface of a polystyrene-gold Janus particle35

Thus further

modifications can be made to the system in order to retain enzyme activity on the

immobilized particles and thereby power nanomotors using enzymes

28

Chapter 3

Predator-prey system of enzyme immobilized particles

31 Introduction

Predator-prey systems are widely found in nature and play a major role in biological

processes3637

Predator-prey systems include all the interactions in which one organism

pursues andor consumes another organism One familiar predator-prey system at the

macroscale is that of a tiger chasing and consuming a deer Another not so familiar one

includes Baleen whales which eat millions of microscopic planktons at once38

At the

microscale such kind of interactions can be observed in the case of phagocytes in the

body which are capable of engulfing foreign microorganisms and killing them with a

combination of microbicidal systems39

In the case of cancer the tumor is the prey and

the mature myeloid cells in the body are the predator However the tumor here has a

defense mechanism against the predator in which the tumor produces cytokines which

inhibits the maturation of the myeloid cells and thereby prevents predation40

One similarity that can be observed in all these systems is that the prey possesses some

attribute which is desired by the predator which makes the predator chase the prey A

similar relationship is seen in case of enzymes which are involved in cascade reactions41

In cascade reactions product synthesized by one enzyme is the reactant required by

another enzyme to carry out its own catalytic reaction As such there is a predator-prey

29

kind of dependence between the two enzymes which may cause one enzyme to chase the

other enzyme when placed in suitable substrate solutions However the mechanism

hypothesized for such a system is totally different than the mechanism involved in

biological systems In biological systems such as bacterial chemotaxis or phagocytes

chasing foreign particles in human body complex biochemical reactions are responsible

for the predator-prey behavior42

In the proposed enzyme cascade system there are no

complex biochemical reactions involved The principle hypothesized for predator-prey

behavior of enzyme cascade reactions can be explained as follows Motion of synthetic

motors at nanoscale involves two components translational diffusion and rotational

Brownian diffusion At very short time scales of less than 1Dr where Dr (s-1

) is

rotational Brownian diffusion coefficient the motion of particles is unidirectional For

longer time scales of greater than 1Dr the direction of motion is random giving

poweredactive diffusion43

If the chemical homogeneity is broken that is when there is

higher concentration of a substrate present in one direction of the enzyme the enzyme

might acquire a subtle bias which may lead to a spatial aggregation of the enzymes based

on the substrate concentration gradient The bias can be a result of the relationship

between the speed of enzyme diffusion and substrate concentration where higher

substrate concentration lead to higher enzyme diffusion As a result in an enzyme

cascade system one enzyme will migrate towards higher concentration of substrate

which is present near the other enzyme thus giving rise to a predator-prey behavior

Such enzyme cascades are widely found in signaling networks and metabolic pathways in

living cells and can be used to produce predator-prey like nanomotors systems44

The

30

enzymes being used in the present study are horseradish peroxidase and glucose oxidase

Both enzymes are extremely stable4546

Glucose is the substrate for glucose oxidase

which is widely available and highly stable47

Horseradish peroxidase acts on hydrogen

peroxide It is involved in a cascade reaction with glucose oxidase which produces

hydrogen peroxide by converting glucose into gluconic acid (Figure 3 1)48

Figure 3 1 Concept of particle aggregation due to cascade reactions of immobilized

enzymes

In this experiment glucose oxidase (GOx) and horseradish peroxidase (HRP) were

immobilized on polystyrene carboxylate beads of different sizes using EDCNHS

coupling method Glucose and 3355-Tetramethylbenzidine (TMB) were dissolved

together in water and this solution was used as the substrate solution All the components

were present in a single system in DI water Aggregation of particles and precipitation of

oxidized TMB from the solution was expected to be observed when all these components

were present in such a system

Properties of horseradish peroxidase have been discussed in the previous chapter (Section

211 Horseradish peroxidase)

GOxGOx HRPHRP

Glucose + O2Gluconic acid + H2O2

TMB TMB oxidizedBlue precipitate

H2O

Aggregation

31

311 Glucose oxidase

Figure 3 2 Glucose oxidase

Glucose oxidase (GOx) belongs to the group of oxidoreductases and is also called

glucose aerodehydrogenase49

It is obtained from fungi and has been identified as an

antimicrobial enzyme50

GOx binds to β-D-glucopyranose and catalyzes the oxidation

reaction to from gluconolactone while simultaneously reducing oxygen to hydrogen

peroxide51

GOx has high substrate affinity and specificity high stability and high

turnover number which makes it a suitable biocatalyst for industrial applications52

Figure 3 3 Redox reaction of glucose catalyzed by glucose oxidase53

32

GOx is a glycoprotein with molecular weight of 155 kDa and consists of two identical

polypeptide subunits linked together by a disulfide bond The optimum pH for GOx is

55 though it is stable in a broad pH range of 4 to 7 and can be stored for a long period of

time51

The functional parameters for glucose oxidase include Km of 151 mM and kcat of

337 s-1

at temperature of 20 ordmC and pH of 754

Some of the inhibitors of GOx are 2-

Deoxy-D-glucose adenine nucleotides and NaNO3

32 Experimental

321 Materials and methods

Uniform carboxyl modified polystyrene microspheres 05 microm in diameter were obtained

from Polysciences Inc The size of the purchased microspheres was verified by FESEM

Peroxidase Type I from horseradish glucose oxidase Type II-S from Aspergillus niger

N-(3-dimethyl-aminopropyl)-Nrsquo-ethylcarbodiimide (EDC) and Hydrogen peroxide

solution 50 wt in water were purchased from Sigma-Aldrich N-hydroxysuccinimide

(NHS) was purchased from Fluka D-Glucose (99) was purchased from VWR

International Secure-Seal imaging spacers (9mm diameter) for glass slides were obtained

from Sigma Aldrich All chemicals were of reagent grade and used without further

purification Nanopure deionized water was used for all experiments and preparation of

solutions All measurements were made at room temperature (25 oC) unless mentioned

otherwise The UV-Visible measurements were performed using Agilent 8453A diode-

array UV-Visible Spectrophotometer with ChemStation UV-Vis Software Optical

33

microscopy experiments were performed using Axiovert 200 MAT microscope by Carl

Zeiss Diffusion coefficient measurements were obtained using Nanosight LM10

322 Optical microscopy experiments

The enzyme substrate system was observed under optical microscope to check for

aggregation and precipitation due to cascade reactions Several such systems were

studied Each system in general consisted of GOx and HRP immobilized on different

polystyrene particles The substrate solutions consisted of 01 M glucose and different

concentrations of TMB solution The systems were studied in the presence of all the

constituents and in the absence of glucose GOxHRP and TMB Systems consisting of

different sized HRP immobilized particles in substrate solution of 003 hydrogen

peroxide and TMB were also studied Videos were recorded over a period of 24 hours to

observe changes in the system Each experiment was performed atleast thrice to assess

reproducibility of results

323 Diffusion coefficient measurements using Nanosight LM10

Diffusion coefficient of GOx and HRP immobilized particles were measured using

Nanosight LM10 nanoparticle tracking instrument with Nanoparticle Tracking Analysis

(NTA) 20 software Diffusion coefficients were measured for different systems namely

GOx particles in 01 M glucose and oxygen both GOx and HRP functionalized particles

in 01 M glucose and oxygen along with controls for the individual systems Videos were

34

recorded for 30 seconds at the rate of 30 frames per second Each experiment was carried

out in triplicate to assess reproducibility

33 Results

331 Optical microscopy

According to the enzyme cascade model precipitation of TMB was possible only upon

the interaction of GOx and HRP immobilized particles in presence of appropriate

substrate solution Hence in the first set of experiments precipitation of TMB was used

as an indication of the type of interaction between GOx and HRP immobilized particles

Fan-shaped structures were observed when both HRP and GOx immobilized particles

were present in a substrate solution consisting of glucose and TMB These structures

started forming as a single strand and then branched out and intertwined to form the fan-

shaped structures (Figure 3 4)

Table 3 1 Observations for formation of fan-shaped structures in enzyme cascade

system in presence of different components

GOx HRP Glucose TMB Observation

Yes Yes Yes Yes

Fan-shaped structures observed particles associated with

the fan-shaped structures

No Yes Yes Yes No fan-shaped structures

Yes No Yes Yes No fan-shaped structures

Yes Yes No Yes No fan-shaped structures

Yes Yes Yes No No fan-shaped structures

35

Fan-shaped structures can also be observed in the system consisting of only HRP

immobilized particles in substrate solution of hydrogen peroxide and TMB The size of

the fan-shaped structures depends on the size of the enzyme immobilized particles Free

enzymes show extremely tiny structures which are difficult to view under the optical

microscope

Figure 3 4 Formation of fan-shaped structures when both HRP and GOx

immobilized particles were present in a substrate solution consisting of glucose and

TMB A In the initial stages single strands form on the surface of liquid (5Ox) B

Single strands branch out and intertwine to form fan-shaped structures (50x) C Many

such structures form and settle down after 20 minutes (20x)

A

C

B

36

Experiments were performed in the absence of TMB that is the system consisted of GOx

and HRP immobilized particles in glucose substrate solution In the absence of TMB the

particles were expected to move closer to each other without any precipitation due to the

cascade reaction However there was no conclusive aggregation of particles observed in

the system

Free Enzyme 005 μm HRP - immobilized particle

10 μm HRP - immobilized particle 05 μm HRP - immobilized particle

Figure 3 5 Size dependence of fan-shaped structures on the size of HRP -

immobilized particles (20x)

37

Table 3 2 Observations for particle aggregation in enzyme cascade system in presence

and absence of TMB

Glucose GOx HRP TMB Observation

Present Present Present Present Aggregation and precipitate

Present Present Present Absent No conclusive aggregation

Present Absent Present Present No aggregation or ppt

Absent Present Present Present No aggregation or ppt

Present Present Absent Present No aggregation or ppt

332 Diffusion coefficient measurements

Diffusion coefficients were measured for GOx immobilized particles of 05 μm diameter

in substrate solution of 01 M glucose and oxygen Diffusion coefficients of particles

were also measured for system consisting of GOx and HRP functionalized particles in

glucose and oxygen Each experiment was done in triplicate to assess reproducibility

Nanosight visualization chamber being a closed chamber oxygen was purged into all the

solutions before starting the experiments to ensure sufficient supply of oxygen for

catalytic reaction of GOx

38

Table 3 3 Diffusion coefficients for 05 μm GOx immobilized particles in 01 M

glucose purged with O2

Mean

GOx

particles in

01 M

glucose

Control - GOx

particles in DI

water

Control - GOx

particles in 01 M

glucose without

oxygen purging

Control -

particles in 01

M Glucose

Diffusion

coefficient

(x10-10

cm2s)

13316 12888 13827 13702

Standard

deviation 6307 4926 4424 7468

Table 3 4 Diffusion coefficients for GOx and HRP immobilized particles in 01 M

glucose and purged with O2

Mean

GOx- HRP-

functionalized

particles in 01 M

glucose

Control ndash Gox-

HRP- functionalized

particles in DI water

Control - particles

in 01 M glucose

Diffusion coefficient

(x10-10

cm2s)

13005 14434 13702

Standard deviation 5263 6622 7468

No significant increase was observed in diffusion coefficient of enzyme immobilized

particles in any of the systems

39

34 Discussion

Formation of fan-shaped structures due to patterned deposition of oxidized TMB

indicates some kind of interaction between GOx and HRP immobilized particles Particle

diameter dependence of the size of structures can be explained with a simple model in

Figure 3 6 A larger particle will have a higher surface area as compared to a smaller

particle Hence more enzyme is immobilized on the larger particle As a result higher

quantity of TMB is oxidized and precipitated by the larger particle as compared to the

smaller particle This leads to thicker strands of fan-shaped structures eventually scaling

up the whole system to give bigger fan-shaped structures

The exact reason for the formation of such fan-shaped structures which are dependent on

particle size could not be determined It is hypothesized that hydrogen peroxide andor

Figure 3 6 Model for size dependence of fan-shaped structures on the particle

diameter of enzyme immobilized particles Larger the particle more surface area is

available for precipitation of TMB which eventually leads to bigger fan-shaped

structures

Larger particle Smaller particle

40

TMB gradient in the substrate solution might be responsible for such structures If there

is higher concentration of H2O2 on one side of the particle and assuming an even

concentration distribution of TMB there will be selective precipitation in the direction of

H2O2 When several particles come in the vicinity of higher concentration of H2O2 the

precipitation lines will converge to give fan-shaped structures (Figure 3 7)

Figure 3 7 Hypothesis for formation of fan-shaped structures

In the system consisting of GOx immobilized particles HRP immobilized particles

glucose and TMB it was observed that the fan-shaped structures formed at the liquid-air

interface and eventually settled down to the bottom of the glass slide This makes sense

as oxygen is necessary for the catalytic reaction of conversion of glucose to gluconic acid

and hydrogen peroxide which takes place on the surface of GOx immobilized particle As

a result H2O2 is primarily formed at the liquid-air interface and is then used by HRP for

precipitation of TMB

Following the observations that precipitation of TMB occurs at the surface GOx and

HRP immobilized particles are also expected to interact at the surface in the absence of

TMB Due the cascade reaction it was expected that HRP immobilized particles will be

41

attracted to GOx immobilized particles in appropriate substrate solution Experiments

were carried out to visualize inter-particle interactions at the liquid-air interface

However liquid-air interface not being a stable system to image the precise interaction

of GOx and HRP immobilized particles could not be determined Significant external

disturbances were involved at the interface To enable the cascade reaction in the bulk of

the liquid oxygen was purged into the reaction system before starting the experiment

However this did not lead to significant reaction in the bulk of the liquid and the reaction

prominently took place at the liquid-air interface where the oxygen concentration was

higher

In order to observe the interaction of GOx and HRP immobilized particles better imaging

techniques need to be used to observe diffusion of particles in absence of external

disturbances at the liquid-air interface Large quantity of oxygen needs to be purged into

the system and retained to make the reaction more pronounced in the bulk of the substrate

solution Higher concentration of oxygen in water may also be achieved by using oxygen

enriched water which is commercially available55

Alternatively a different enzyme cascade system may be used which does not require a

gaseous substrate56

For example glycogen synthase synthesizes glycogen from glucose-

1-phosphate and glycogen phosphorylase breaks down glycogen to glucose-1-

phosphate57

This system does not require a gaseous substrate and might be able to

overcome the deficiencies of GOx and HRP enzyme cascade system

42

Chapter 4

Silver-titania nanomotors

41 Introduction

Bimetallic nanowires made of platinum and gold have been developed by WPaxton

etal1 The rods move autonomously in a solution of hydrogen peroxide In the present

study silver-titania (Ag-TiO2) particles are being synthesized and investigated for motion

in presence of hydrogen peroxide and UV light With proper control of conditions and

UV light trigger it can be possible to control the speed and direction of motion of these

nanoparticles

Hydrogen peroxide is produced in the human body by phagocytes as an immune response

to foreign particles and pathogens5859

Also silver nanoparticles have been shown to be

biocompatible and capable of interacting with cells in human body without disrupting

cellular function60

Similarly it has been demonstrated that titania is compatible with

different cells DNA and proteins in human body and does not cause toxic effects Hence

silver-titania nanomotors which swim in hydrogen peroxide may open interesting

avenues in applications like targeted drug delivery in those parts of the body where

hydrogen peroxide is present

43

42 Experimental

421 Materials and methods

Silver nitrate (999995) was purchased from Alfa Aesar Gum arabic was purchased

from MP Biomedicals Hydrogen peroxide solution 50 wt in water and ascorbic acid

were purchased from Sigma-Aldrich Nanopure deionized water was used for all

experiments and preparation of all solutions Secure-Seal imaging spacers (9 mm

diameter) were obtained from Sigma Aldrich All experiments were carried out at room

temperature of 25ordmC unless otherwise mentioned Ag-TiO2 particles were synthesized by

evaporation of titania in Semicore electron beam evaporator Particles were characterized

using field-emission scanning electron microscopy Optical microscopy experiments

were performed using Axiovert 200 MAT microscope by Carl Zeiss Particles were

tracked using Physvis particle tracking software (Figure 4 1) in which the position

information of a particle on screen is stored on a frame by frame basis The data is stored

in a dat file as X and Y co-ordinates of particles tracked on the screen This information

can be converted to diffusion coefficients of the particles by further manipulation using

MathWorks-MATLABreg software

44

Figure 4 1 Physvis particle tracking software

422 Synthesis of silver colloidal particles

Silver particles were synthesized by a nucleationgrowth type reaction developed by

Goya and Matijevic61

and modified by Velikov62

Silver nitrate was used as precursor

metal salt for synthesis of silver particles Solution A was 75 ml of 50 mM AgNO3 and

035 by weight gum arabic Solution B was 75 mL of 100 mM 1-ascorbic acid and

035 by weight gum arabic Solution B was rapidly added to solution A in a 250 mL

reaction flask The reaction flask was covered and particles were stirred for 48 hrs After

48 hours particles were allowed to settle for 2-3 hours in refrigerator and supernatant

was separated from the particles Particles were then rinsed with DI water and centrifuged

thrice to obtain pure silver particles Zeta potential of these particles was reported to be

45

around -50 to -60 mV and the size was around 1μm The size of the particles can be

varied by varying the amounts of ascorbic acid and silver nitrate Using molar ratio of 31

of ascorbic acid silver nitrate can give particles of 200 nm diameter whereas ratio of

151 can give particles of upto 5 μm diameter

423 Preparation of silver-titania (Ag-TiO2) particles

A monolayer of silver particles was prepared according to the procedure by Goldenberg

et al63

Glass slides were placed on the bottom of a petridish Water was put into the

petridish so that the entire slide was immersed in water A thin layer of hexane was then

added on top of water in the petridish A suspension of silver particles in ethanol (5

ww) was slowly introduced to the water-hexane interface in the dish After the

monolayer was formed the glass slide was lifted up through the monolayer with tweezers

The silver particles-covered glass slides were left at room temperature to dry overnight

Following drying a thin film of titania was evaporated on the glass slides in Semicore

evaporator (Figure 2 7) The spheres were partially coated with a 10 nm film of titania

Ag-TiO2 particles were removed from the glass slide by gentle sonication FE-SEM was

used to characterize the particles (Figure 4 2) The thin titania layer was not easily

distinguishable due to roughness of the particles

46

Figure 4 2 FESEM image of Ag-TiO2 particles

424 Optical microscopy experiments

Imaging spacers were placed on a glass slide Suspension of Ag-TiO2 particles was

placed on the glass slide and the particles were allowed to settle Movement of particles

was observed in DI water and videos were recorded at 50x magnification of optical

microscope for 30 sec 05 H2O2 was added to the particle suspension on the slide

Videos were recorded at 50x ranging from 30 sec to 10 min to study the movement of

silver titania particles in hydrogen peroxide solution Particles in both DI water and

hydrogen peroxide were subjected to UV light and videos were recorded to study particle

dynamics in presence of UV radiation Particle tracking was performed using Physvis

particle tracking software Atleast 30 particles in every system were tracked for 10

seconds each Particle tracking data was obtained in terms of X and Y coordinates of

particles on screen This data was manipulated using MathWorks-Matlab software and

graphs of mean square displacement of particles versus time were plotted Diffusion

coefficients of particles were calculated from these graphs

47

43 Results and Discussion

Ag-TiO2 particles demonstrated autonomous motion in the presence of H2O2 No UV

light was required for this motion Motion of the particles was accelerated by addition of

fresh H2O2 to the system When subjected to UV radiation the particles displayed

momentary acceleration for 4-5 seconds

Figure 4 3 Mechanism of motion of Ag-TiO2 nanomotors

In the presence of H2O2 silver part of the Ag-TiO2 particle converts H2O2 to H2O and O2

This may lead to propulsion of the Ag-TiO2 particle due to bubble propulsion andor self-

diffusiophoresis In the presence of UV light additional reaction takes place on the

titania part of Ag-TiO2 particle which increases the velocity of the Ag-TiO2 particles

(Figure 4 4) This UV triggered photoactivity of titania is due to hole-electron separation

caused by photons having energy equal to or greater than the band gap of titania646566

Most of the holes-electrons recombine but a small fraction move to the surface and react

with the available redox species like water and oxygen to form protons (H+) superoxide

(O2-) and hydroxyl (OH) radicals

6566 This may lead to propulsion by diffusiophoresis

as well as electrophoresis

48

Figure 4 4 Silver-titania motors move faster when subjected to UV radiation

In addition to linear motion rotational motion is also observed in several particles and

particle aggregates Aggregation of particles can lead to a net circular propulsive force

which can rotate the aggregate as shown in Figure 4 5

Figure 4 5 Rotation due to particle aggregation of Ag-TiO2 particles

Particle tracking by Physvis software revealed a significant increase in diffusion

coefficient of Ag-TiO2 particles in hydrogen peroxide solution as compared to DI water

49

The Ag-TiO2 particles have a diffusion coefficient of 1491 μm2s in hydrogen peroxide

versus only 180 μm2s in DI water Thus the Ag-TiO2 particles move around more than

10 times their body length per second in hydrogen peroxide (Figure 4 6) When

subjected to UV light the particles experience a significant acceleration in hydrogen

peroxide which lasts only for a short duration of time (4-5 sec) after which the particles

return to their original velocity The diffusion coefficient of the particles during this UV

light trigger is found to be around 5093 μm2s UV light trigger does not have any visible

effect on the Ag-TiO2 particles in DI water

Table 4 1 Translational diffusion coefficients of Ag-TiO2 particles in hydrogen

peroxide and water

Mean 05 H2O2 solution DI water

Diffusion coefficient of Ag-TiO2 (μm2s)

without UV light

1491 180

Diffusion coefficient of Ag-TiO2 (μm2s)

in the presence of UV light

5093 113

50

Figure 4 6 Mean square displacement curves of Ag-TiO2 Janus particles in hydrogen

peroxide and DI water

When higher concentration (~30 vv) of Ag-TiO2 particles in hydrogen peroxide is

subjected to UV radiation the particles exhibit pattern formation (Figure 4 7)

Aggregation of Ag-TiO2 Janus particles is observed when UV light is turned on and the

particles disaggregated when UV light is switched off This can be due to predator-prey

kind of interaction between silver and titania half of the Janus particles in the presence of

UV light Oxygen and water that are produced at the silver end by decomposition of

hydrogen peroxide are used up by titania when subjected to UV radiation This can lead

to attraction between titania and silver parts of neighboring Janus particles causing

aggregation

y = 20373x

y = 59994x

y = 71848x

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10 12

Mean

sq

uare

dis

pla

cem

en

t (μ

m2s

)

Time (sec)

Mean square displacement vs Time for Ag-TiO2

particles

05 Hydrogen peroxide and UV05 Hydrogen peroxide

DI Water

51

A UV OFF B UV ON

C UV OFF

Figure 4 7 Higher concentrations of Ag-TiO2 particles showing aggregation when UV

is turned on

52

Chapter 5

Summary and Conclusion

In summary no significant enhancement in diffusion was observed in case of enzyme

immobilized Janus particles in substrate solution Presence of a linker between enzyme

and particle did not increase the diffusion coefficient of particles Alternative enzyme

immobilization methods need to be tested like thiolation of enzyme and subsequent

binding to gold surface of a Janus particle35

or expressing the enzyme with polyhistidine

or avidin tag67

This can result in higher enzyme activity upon immobilization and may

lead to enhanced diffusion of Janus particles

Predator-prey system of GOx and HRP immobilized particles showed some promising

results Aggregation of particles and formation of fan-shaped structures by TMB

oxidation were observed under optical microscope when GOx and HRP immobilized

particles glucose and TMB were present in the system The size of the fan-shaped

structures was dependent on the size of enzyme immobilized particles This can be

indicative of some kind of interaction between the GOx and HRP immobilized particles

No conclusive aggregation of GOx and HRP immobilized particles was observed in

absence of TMB Better imaging techniques are required to study the interaction of GOx

and HRP immobilized particles at the unstable liquid-air interface where majority of the

reaction takes place Other enzyme cascade systems not requiring gaseous reactants like

glycogen synthase and glycogen phosphorylase should be tested for similar activity

53

Ag-TiO2 particles were found to exhibit autonomous motion in the presence of hydrogen

peroxide solution The motion was momentarily accelerated upon exposure to UV

radiation The particles showed a diffusion coefficient of 1491 μm2s in hydrogen

peroxide versus only 180 μm2s in DI water Upon subjecting particles to UV radiation in

presence of H2O2 diffusion coefficient increased to 5093 μm2s for a period of 4-5

seconds In addition to linear motion rotating motion could also be observed in some

particle aggregates Further development of these aggregate structures can lead to

formation of rotors at nanoscale Higher concentration of silver-titania particles in

hydrogen peroxide displayed aggregation in presence of UV light and disaggregated

when UV light was switched off This may be attributed to the predator-prey kind of

interaction between silver and titania where oxygen and water that are produced by

silver by decomposition of hydrogen peroxide are used up by titania when subjected to

UV radiation This promises to be an interesting system and needs to be further

investigated to explore its properties

54

References

1 Paxton WF Sundararajan S Mallouk TE Sen A Angew Chem Int Ed Engl

2006 45 5420-5429

2 Golestanian R Liverpool TB Adjari A Phys Rev Lett 2005 94 220801

3 httpwwwsilvermedicineorgh2o2html

4 Rami Pedahzur Ovadia Lev Badri Fattal Hillel Shuval Water Science and Technology

1995 31(5-6) 123-129

5 W Went F American Scientist 1968 56

6 Adriano Cavalcanti Tad Hogg Bijan Shirinzadeh IEEE MHS 2006 International

Symposium on Micro-NanoMechatronics and Human Science 2006 226-232

7 EM Purcell American Journal of Physics 1977 45 3-11

8 Golestanian R Phys Rev Lett 2010 105 018103(1)-018103(4)

9 S S Dukhin Z R Ulberg G L Dvornichenko B V Deryagin Russian Chemical

Bulletin 1983 31(8) 1535-1544

10 Dukhin AS Goetz PJ Ultrasound for characterizing colloids Particle sizing zeta

potential rheology Elsevier 2002 59

11 HJKeh HJTu Trends in Colloid and Interface Science XIV Progress in Colloid and

Polymer Science 2000 115 174-180

12 D Zinemanas A Nir International Journal of Multiphase Flow 1995 21(5) 787-800

55

13 Stephen Ebbens Jonathan Howse Soft matter 2010 6 726-739

14 J Vicario R Eelkema W R Browne A Meetsma R M La Crois B L Feringa

Chem Commun 2005 31 3936

15 Davide Pantarotto Wesley R Browne Ben L Feringa Chem Commun 2008 1533 -

1535

16 Lisa A Cameron Matthew J Footer Alexander van Oudenaarden Julie A Theriot

PNAS 1999 96(9) 4908-4913

17 Ali Najafi Ramin Golestanian Phys Rev E 2004 69 062901

18 Jinseok Kim Jungyul Park Sungwook Yang Jeongeun Baek Byungkyu Kim Sang

Ho Lee Eui-Sung Yoon Kukjin Chun Sukho Park Lab Chip 2007 7 1504 - 1508

19 Kurt M Dubowski Clinical Chemistry 2008 5411 1919ndash1920

20 Spector DA Yang Q Wade JB Am J Physiol Renal Physiol 2007 292(1) 467-474

21 Rhee SG Chang TS Bae YS Lee SR Kang SW J Am Soc Nephrol 2003 14(8-3)

211-215

22 Sakaue T Kapral R Mikhailov A S Eur Phys J B 2010 75 381ndash387

23 Muddana H S Sengupta S Mallouk T E Sen A Butler P J J Am Chem Soc

2010 132 (7) 2110ndash2111

24 Baker CJ Deahl K Domek J Orlandi EW Arch Biochem Biophys 2000 382(2) 232-

237

25 V Vojinovic RH Carvalho F Lemos JMS Cabral LP Fonseca BS Ferreira

Biochemical Engineering Journal 2007 35 126-135

56

26

OV Lebedeva NN Ugarova Russian Chemical Bulletin 1996 45(1) 18-25

27 Nigel C Veitch Phytochemistry 2004 65(3) 249-259

28 Kenneth H Petersen Journal of Immunological Methods 2009 340(1) 86-89

29 httpwwwbrenda-enzymesorgphpresult_flatphp4ecno=11117

30 Goldenberg L M Wagner J Stumpe J Paulke B-R Goumlrnitz E Langmuir 2002 18

5627-5629

31 httpwwwgoldbiocompdf3761-Protocol201pdf

32 Av Rovisco Pais Lisboa Portugal T amp D Technology West Yorkshire UK Journal

of Molecular Catalysis B Enzymatic 2004 28(2-3) 129-135

33 httpwwwphysicsnyuedugrierlabmethodsnode11html

34 Yoon-Mee Lee1 O-Yul Kwon Yeo-Joon Yoon Keungarp Ryu Biotechnology Letters

2006 28 39ndash43

35 JC Pyun SD Kim JW Chung Analytical Biochemistry 2005 347(2) 227-233

36 George H Pimbley Jr Mathematical Biosciences 1974 20(1-2) 27-51

37 Andy Fenton Sarah E Perkins Parasitology 2010 137 1027-1038

38 Mead James G Brownell Robert L Mammal Species of the World A Taxonomic and

Geographic Reference (3rd ed) Johns Hopkins University Press 2005 2 2142

57

39

Krause KH Schweiz Med Wochenschr 2000 130(4) 97-100

40 Irina Kareva Faina Berezovskaya Carlos Castillo-Chavez Journal of Biological

Dynamics 2010 4(4)315 - 327

41 Delaittre G Reynhout I Cornelissen J Nolte R Chemistry ndash A European Journal

2009 15 12600ndash12603

42 C Buller DH Johnson RC Tschumper Investigative Ophthalmology amp Visual Science

1990 31 2156-2163

43 Robert M Mazo Brownian Motion Fluctuations Dynamics and Applications Oxford

Science Publications 2009 57

44 Vivek K Mutalik KV Venkatesh Theoretical Biology and Medical Modelling 2005 2

19

45 Satoshi Nakamura Kunimasa Koga Biochemical and Biophysical Research

Communications 1977 78(2) 806-810

46 Krishnananda Chattopadhyay Shyamalava Mazumdar Biochemistry 2000 39 (1)

263ndash270

47 Satoshi Nakamura Yasuyuki Ogura J Biochem 1968 63 (3) 308-316

48 van Dongen SF Nallani M Cornelissen JJ Nolte RJ van Hest JC Chemistry 2009

15(5) 1107-1114

49 Ronald Bentley Methods in Enzymology 1955 1 340-345

50 Vartiainen J Raumlttouml M Paulussen S Packaging Technology and Science 2005

18 243ndash251

58

51 GA Sherbeny AA Shindia YMMM Sheriff International Journal of Agriculture

and Biology 2005 7

52 D Rando G-W Kohring F Giffhorn Appl Microbiol Biotechnol 1997 48

53 Worthington enzyme manual Worthington Biochemical Corporation

httpworthington-biochemcomgopdefaulthtml

54 httpwwwbrenda-enzymesorgphpresult_flatphp4ecno=11117

55 httpwwwoxygenphwatercom

56 P Boon Chock Sue Goo Rhee and Earl R Stadtman Annu Rev Biochem 1980 49

813-841

57 Sybil Golden P A Wals Joseph Katz Analytical Biochemistry 1977 77(2) 436-445

58 R L Baehner S K Murrmann J Davis R B Johnston Jr J Clin Invest 1975 56(3)

571ndash576

59 I Bejarano M P Terroacuten S D Paredes C Barriga A B Rodriacuteguez J A Pariente

Molecular and Cellular Biochemistry 2007 296(1-2) 77-84

60 Michael C Moulton Laura K Braydich-Stolle Mallikarjuna N Nadagouda Samantha

Kunzelman Saber M Hussain Rajender S Varma Nanoscale 2010 2 763-770

61 Goya DV Matijevic E Colloids and Surfaces A 1999 146 139

62 Velikov KP Zegers GE van Blaanderen A Langmuir 2003 19 1384

63 Goldenberg L M Wagner J Stumpe J Paulke B-R Goumlrnitz E Langmuir 2002 18

5627-5629

59

64

Banerjee S Gopal J Muraleedharan P Tyagi A K Raj B Curr Sci 2006

901378ndash1383

65 Bogomolov V N Kudinov E K Firsov Y A Sov Phys Solid State 1968 35 555

66 Kudo A Miseki Y Chem Soc Rev 2009 38 253ndash278

67 Gaj T Meyer SC Ghosh I Protein expression and Purification 2007 56 54-61

11

Figure 2 2 Schematic showing idealized nonreciprocal conformation changes in a

theoretical enzyme22

Enhanced diffusion of a single enzyme molecule by catalysis in a substrate solution has

been demonstrated23

The diffusion coefficient of a single urease molecule was measured

using fluorescence correlation spectroscopy and it was observed that the diffusion of the

urease molecule increases by 16-28 by varying urea concentrations from 0001 M to 1

M Addition of enzyme inhibitors to the system curbed the enhanced diffusion This

enhanced diffusion has been attributed to the force generated by self-electrophoresis by

the enzyme in substrate solution

12

Based on the above facts it might be possible to propel a nanoparticle by asymmetric

distribution of enzymes on its surface thereby leading to self-propulsion of the particles

in substrate solution (Figure 2 1) This propulsion may be due to self-

electrophoresisself-diffusiophoresis andor due to nonreciprocal changes in enzyme

conformations Here we aim to investigate a system consisting of polystyrene particles

half coated with enzyme introduced in a suitable substrate solution The enzyme can be

directly linked to the polystyrene particle or it can be linked through a long spacer

molecule (linker) to provide more degrees of freedom to the enzyme

Several factors need to be considered for choosing enzyme for powering motion at the

nanoscale Some of the important factors are the ratio of kcatKm which denotes the

catalytic efficiency of the enzyme possible substrates stability of enzymessubstrates

ease of immobilization relationship with other enzymes in cascade reactions and

commercial availability of the enzymes The enzyme being used in the present study is

horseradish peroxidase It acts on a molecule of hydrogen peroxide and reduces it while

simultaneously oxidizing another substrate molecule In the absence of alternate

oxidizable substrate HRP can oxidize water and oxygen back to hydrogen peroxide24

thus giving an equilibrium reaction

13

211 Horseradish peroxidase

Figure 2 3 Horseradish peroxidase

Horseradish peroxidase belongs to the group of peroxidases which are a group of heme-

containing oxidoreductases that act on peroxide as hydrogen donor25

It is a redox

enzyme which contains a protoheme a complex of trivalent iron with 1358-

tetramethyl-24-divinylporphin-67-dipropionic acid as a prosthetic group It is a 44 kDa

protein which includes 33900 Da polypeptide chain bound with eight oligosaccharide

chains The carbohydrate chains improve the stability of the enzyme by protecting it

against radicals formed in the course of the reaction it catalyzes26

Horseradish

peroxidase is found in horseradish and is used widely in bioanalytical applications such

as ELISA and Western blots It decomposes hydrogen peroxide into water and oxygen

while simultaneously oxidizing another substrate27

The oxidizable substrates can be

chromogenic substrates such as 22rsquo55rsquo-tetramethyl-benzidine (TMB) and 33-

diaminobenzidine (DMB) or chemoluminescent substrates like SuperSignal and ECL

luminal28

14

Figure 2 4 Reaction catalyzed by horseradish peroxidase

The functional parameters for horseradish peroxidase include Km of 015 mM and kcat of

790 s-1

at an optimum temperature of 20 ordmC and an optimum pH of 6429

Some of the

inhibitors of HRP are 110-phenanthroline aminotriazole and bromine

22 Experimental

221 Materials and methods

Uniform carboxyl modified polystyrene microspheres 05 microm in diameter were obtained

from Polysciences Inc The size of the purchased microspheres was verified by Field

Emission Scanning Electron Microscopy (FESEM) Peroxidase Type I from

Horseradish N-(3-dimethyl-aminopropyl)-Nrsquo-ethylcarbodiimide (EDC) and hydrogen

peroxide solution 50 wt in water were purchased from Sigma-Aldrich N-

hydroxysuccinimide (NHS) ge 97 was purchased from Fluka All chemicals were of

reagent grade and used without further purification Nanopure deionized water was used

for all experiments and preparation of solutions All measurements were made at room

15

temperature (25 oC) unless mentioned otherwise The UV-Visible spectroscopy

measurements were performed using Agilent 8453A diode-array UV-Visible

Spectrophotometer with ChemStation UV-Vis Software Diffusion coefficient

measurements were performed using Nanosight LM10

222 Synthesis of Janus particles

A monolayer of particles was prepared according to the procedure (Figure 2 5) by

Goldenberg L et al30

Glass slides were placed on the bottom of a petridish Water was

put into the petridish so that the entire slide was immersed in water A thin layer of

hexane was added on top of water in the petridish A suspension of microspheres in

ethanol (5 ww) was introduced to the water-hexane interface After the monolayer was

formed the glass slide was lifted up through the monolayer with tweezers The

microsphere-covered glass slides were left to dry overnight at room temperature

Following drying a thin film of silica was evaporated on the glass slides in Semicore

evaporator The spheres were partially coated with 30 nm of silica Particles were

removed from the glass slide by gentle sonication The polystyrene-silica (PS-SiO2)

Janus particles thus obtained were washed in deionized water several times before

further immobilization steps were performed FESEM was used to confirm synthesis of

Janus particles

16

Figure 2 5 Preparation of PS-SiO2 Janus particles by evaporation

223 Immobilization of enzymes using EDCNHS coupling method

1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide and N-hydroxysulfosuccinimide

(EDCNHS) coupling procedure was followed for immobilizing horseradish peroxidase

type I on 05 μm PS-SiO2 Janus particles (Figure 2 6) In this process EDC reacts with a

carboxyl groups on the Janus particles to form an amine-reactive O-acylisourea

intermediate If this intermediate does not encounter an amine it can hydrolyze and

regenerate the carboxyl group In the presence of NHS EDC can be used to convert

carboxyl groups to amine-reactive sulfo-NHS esters which are much more stable and do

not readily hydrolyze to regenerate carboxyl groups In the current experiment

immobilization was performed in the presence as well as absence of a long spacer

17

molecule (linker) between Janus particles and enzymes The chemical 6-aminohexanoic

acid was used as the linker (Figure 2 7) HRP immobilized PS-SiO2 Janus particles were

stored in deionized water until required for further experiments

Figure 2 6 EDCNHS coupling method for immobilization of enzyme on carboxylated

polystyrene particles

Figure 2 7 Immobilization of enzyme on Janus particles with and without 6-

aminohexanoic acid linker

18

224 Enzyme assay

Assay for activity of HRP was performed using a spectrophotometric method which

involved using 4-aminoantipyrene as hydrogen donor3132

The reaction rate was

determined by measuring increase in absorbance at 510 nm resulting from the

decomposition of hydrogen peroxide One unit represents the decomposition of 1 μM of

hydrogen peroxide per minute at 25 degC and pH 70 under the specified conditions

The spectrophotometer was set to the wavelength of 510 nm and temperature of 25 degC

Phenol4-aminoantipyrene solution (14 mL) and 00017 M H2O2 solution (15 mL) were

pipetted into each cuvette The cuvette was incubated for 3-4 mins at 25degC and blank was

established A 01 mL solution of diluted enzyme was added to the above mixture and

absorbance was recorded for 4-5 mins Standard graphs of absorbance versus time were

drawn for known concentration of the free enzyme and compared to that of the

immobilized enzyme on the Janus particles The estimate of the amount of enzyme

present on the Janus particles was deduced from the difference between the quantity of

the free enzyme previously added for immobilization and the amount that was measured

to be in the supernatant after some of the enzyme became attached to the Janus particles

The experiment was carried out in triplicate to assess reproducibility

19

The formula used to calculate the activity of enzyme is

where ∆Amin denotes the change in absorbance per minute

225 Diffusion coefficient measurements using Nanosight LM10

Diffusion coefficients for HRP immobilized particles were measured using Nanosight

LM 10 nanoparticle tracking instrument with Nanoparticle Tracking Analysis (NTA) 20

software in the presence of 003 H2O2 solution as the substrate and also in the presence

of only deionized water The videos were recorded for 30 seconds at the rate of 30 frames

per second Each experiment was carried out in triplicate to assess reproducibility

Particles

visualization

zone

Microscope with

LM 10 unit

LM 10 unit

Figure 2 8 Nanosight LM 10 instrumentation

20

Nanosight LM 10 (Figure 2 8) generates videos of population of moving nanoparticles in

a liquid when illuminated by laser light The particles which pass through the laser beam

path are seen by the instrument as small points of light undergoing Brownian motion

(Figure 2 9a)

The videos are further analyzed by NTA (Nanoparticle tracking analysis) software

(Figure 2 9b) which identifies and tracks each particle independently on a frame-by-

frame basis The software also calculates and subtracts any bulk flow in the system The

mean square displacement is calculated for each particle as long as it is visible If the

particle blinks that is if it disappears momentarily and then reappears on the screen the

software will consider the same particle as two separate ones All analysis data is

exported to the excel files in terms of diffusion coefficients of the tracked particles

B A

Figure 2 9 NTA video analysis (a) Particles as they are seen on the screen during

experimentation in Nanosight LM10 (b) NTA screen as the particles are being analyzed for

their diffusion coefficients

21

23 Results

231 Enzyme assay

The activity of free HRP enzyme reported by Sigma Aldrich is 150 unitsmg However

the activity of HRP upon analysis was found to be 52158 unitsmg which is much lower

than the reported value The activity of HRP was found to be further lowered to 2133

unitsmg when it was immobilized on the PS-SiO2 Janus particles in the presence of a

linker The following formula is used to calculate enzyme activity

where ΔAmin is the change in absorbance per minute

22

Figure 2 10 Graph for the assay of solution of free enzyme containing 05mgL of HRP

ΔAmin = 01716 Unitsmg of free enzyme = 52158

During EDCNHS coupling 04 mg enzyme was added to 1 ml of 025 vv suspension

of Janus particles Thus initial concentration of enzyme in the mixture was 04 mgml

Assuming retained activity of the enzyme in supernatant solution the amount of enzyme

in supernatant was calculated to be 03868 mgml from the assay Amount of enzyme

functionalized on the Janus particles was calculated as the difference between enzyme

previously added for immobilization (04 mgml) and the enzyme present in supernatant

(03868 mgml) and was found to be 00132 mgml Enzyme assay was performed on the

enzyme immobilized Janus particles to estimate the activity of immobilized enzyme

(Figure 2 11) and was found to be 2133 unitsmg

y = 0002x + 00516

0

01

02

03

04

05

06

07

08

0 50 100 150 200 250 300 350 400

Ab

sorb

an

ce

51

0 n

m

Time (sec)

Assay of free enzyme HRP

23

Figure 2 11 Graph for the assay of of immobilized enzyme on 05 μm Janus particles

(~025 vv) ΔAmin = 01853 Unitsmg of free enzyme = 2133

232 Diffusion coefficient measurements

No significant increase was observed in diffusion coefficient of HRP-immobilized Janus

particles in the presence of H2O2 as the substrate Similar results were obtained for

diffusion coefficient measurement with and without linker moieties bridging the enzymes

and particles(Table 2 1 Table 2 2) Thus the linker did not seem to assist in increasing

the particle diffusion by improving or retaining the activity of the enzyme

y = 00015x + 00953

0

01

02

03

04

05

06

07

0 50 100 150 200 250 300 350 400

Ab

sorb

an

ce

510 n

m

Time (sec)

Assay of HRP immobilized 05 μm Janus particles

24

Table 2 1 Diffusion coefficient measurements of HRP immobilized Janus particles

without linker

HRP immobilized

Janus particles in

003 H2O2

Control - Janus

particles in

003 H2O2

Control - HRP

immobilized Janus

particles in DI water

Diffusion coefficient

(10-10

cm2s)

1359416 1051777 1308675

Standard deviation 4534801 4122075 4918597

Table 2 2 Diffusion coefficient measurements of HRP immobilized Janus particles in

the presence of linker

HRP immobilized

Janus particles in

003 H2O2

Control - Janus

particles in

003 H2O2

Control - HRP

immobilized Janus

particles in DI water

Diffusion coefficient

(10-10

cm2s)

13838 1339829 1407891

Standard deviation 5644692 5649364 5797218

25

Figure 2 12 Diffusion coefficient measurements of PS-SiO2 Janus particles without and

with 6-aminohexanoic acid linker

24 Discussion

To compare the diffusion coefficients observed during the experiments theoretical

diffusion coefficient was calculated using following equation33

r

kTD altraslation

6 Equation 21

where r is the hydrodynamic radius of particle Dtranslational is translational diffusion

coefficient of the particle T is temperature of the system η is the viscosity of fluid

through which the particles are moving and k is the Boltzmann constant From FE-SEM

images the size of the particles used was determined to be 450 nm For 450 nm diameter

0

20

40

60

80

100

120

140

160

Without linker With linker

Dif

fusio

n c

oeff

icie

nt

(10

-10 c

m2s

)

HRP immobilized 05 μm Janus particles - Diffusion coefficient measurements

HRP immobilized Janus particles in 003 H2O2

Control - Janus particles in 003 H2O2

Control - HRP immobilized Janus particles in DI water

26

particle Dtranslational is equal to 109 μm2s which is comparable to the diffusion

coefficient measured for the enzyme immobilized particles

HRP was found to be active on the Janus particles however the activity of the enzyme

was reduced upon immobilization Lower enzyme activity can be due to the coupling

method used which in this case was EDCNHS covalent binding However EDCNHS

coupling method has been shown to significantly retain the enzyme activity when the

enzyme is immobilized on carboxylated multi-walled carbon nanotubes under appropriate

conditions34

Hence EDCNHS binding method by itself is not believed to be

responsible for reduction of enzyme activity upon immobilization

Direct binding of enzyme to the Janus particle may lead to reduction in the enzyme

activity because of space constraints for the enzyme and difficulty for the passage of

substrate to the active site of the enzyme due to stearic hindrance Hence a linker was

inserted between the enzyme and Janus particle during enzyme immobilization However

inserting a linker between the enzyme and the particle did not lead to enhanced diffusion

of Janus particles or increase in enzyme activity of immobilized enzyme

Lack of enhanced diffusion can be firstly attributed to the reduction in enzyme activity

upon immobilization Enzyme activity may be retained by modifying the conditions

during EDCNHS coupling or changing the binding method altogether The degree of

immobilization of HRP on carboxylated Janus particles can possibly be affected by

several factors such as temperature the concentration of the coupling reagent EDC

27

concentration HRP concentration in solution and incubation time34

Different

combinations of these factors can be tested to find optimum conditions for

immobilization of HRP Alternate binding method can involve thiolation of HRP by the

covalent conjugation of mercaptopropionic acid and then the thiolated HRP can then be

attached on the gold surface of a polystyrene-gold Janus particle35

Thus further

modifications can be made to the system in order to retain enzyme activity on the

immobilized particles and thereby power nanomotors using enzymes

28

Chapter 3

Predator-prey system of enzyme immobilized particles

31 Introduction

Predator-prey systems are widely found in nature and play a major role in biological

processes3637

Predator-prey systems include all the interactions in which one organism

pursues andor consumes another organism One familiar predator-prey system at the

macroscale is that of a tiger chasing and consuming a deer Another not so familiar one

includes Baleen whales which eat millions of microscopic planktons at once38

At the

microscale such kind of interactions can be observed in the case of phagocytes in the

body which are capable of engulfing foreign microorganisms and killing them with a

combination of microbicidal systems39

In the case of cancer the tumor is the prey and

the mature myeloid cells in the body are the predator However the tumor here has a

defense mechanism against the predator in which the tumor produces cytokines which

inhibits the maturation of the myeloid cells and thereby prevents predation40

One similarity that can be observed in all these systems is that the prey possesses some

attribute which is desired by the predator which makes the predator chase the prey A

similar relationship is seen in case of enzymes which are involved in cascade reactions41

In cascade reactions product synthesized by one enzyme is the reactant required by

another enzyme to carry out its own catalytic reaction As such there is a predator-prey

29

kind of dependence between the two enzymes which may cause one enzyme to chase the

other enzyme when placed in suitable substrate solutions However the mechanism

hypothesized for such a system is totally different than the mechanism involved in

biological systems In biological systems such as bacterial chemotaxis or phagocytes

chasing foreign particles in human body complex biochemical reactions are responsible

for the predator-prey behavior42

In the proposed enzyme cascade system there are no

complex biochemical reactions involved The principle hypothesized for predator-prey

behavior of enzyme cascade reactions can be explained as follows Motion of synthetic

motors at nanoscale involves two components translational diffusion and rotational

Brownian diffusion At very short time scales of less than 1Dr where Dr (s-1

) is

rotational Brownian diffusion coefficient the motion of particles is unidirectional For

longer time scales of greater than 1Dr the direction of motion is random giving

poweredactive diffusion43

If the chemical homogeneity is broken that is when there is

higher concentration of a substrate present in one direction of the enzyme the enzyme

might acquire a subtle bias which may lead to a spatial aggregation of the enzymes based

on the substrate concentration gradient The bias can be a result of the relationship

between the speed of enzyme diffusion and substrate concentration where higher

substrate concentration lead to higher enzyme diffusion As a result in an enzyme

cascade system one enzyme will migrate towards higher concentration of substrate

which is present near the other enzyme thus giving rise to a predator-prey behavior

Such enzyme cascades are widely found in signaling networks and metabolic pathways in

living cells and can be used to produce predator-prey like nanomotors systems44

The

30

enzymes being used in the present study are horseradish peroxidase and glucose oxidase

Both enzymes are extremely stable4546

Glucose is the substrate for glucose oxidase

which is widely available and highly stable47

Horseradish peroxidase acts on hydrogen

peroxide It is involved in a cascade reaction with glucose oxidase which produces

hydrogen peroxide by converting glucose into gluconic acid (Figure 3 1)48

Figure 3 1 Concept of particle aggregation due to cascade reactions of immobilized

enzymes

In this experiment glucose oxidase (GOx) and horseradish peroxidase (HRP) were

immobilized on polystyrene carboxylate beads of different sizes using EDCNHS

coupling method Glucose and 3355-Tetramethylbenzidine (TMB) were dissolved

together in water and this solution was used as the substrate solution All the components

were present in a single system in DI water Aggregation of particles and precipitation of

oxidized TMB from the solution was expected to be observed when all these components

were present in such a system

Properties of horseradish peroxidase have been discussed in the previous chapter (Section

211 Horseradish peroxidase)

GOxGOx HRPHRP

Glucose + O2Gluconic acid + H2O2

TMB TMB oxidizedBlue precipitate

H2O

Aggregation

31

311 Glucose oxidase

Figure 3 2 Glucose oxidase

Glucose oxidase (GOx) belongs to the group of oxidoreductases and is also called

glucose aerodehydrogenase49

It is obtained from fungi and has been identified as an

antimicrobial enzyme50

GOx binds to β-D-glucopyranose and catalyzes the oxidation

reaction to from gluconolactone while simultaneously reducing oxygen to hydrogen

peroxide51

GOx has high substrate affinity and specificity high stability and high

turnover number which makes it a suitable biocatalyst for industrial applications52

Figure 3 3 Redox reaction of glucose catalyzed by glucose oxidase53

32

GOx is a glycoprotein with molecular weight of 155 kDa and consists of two identical

polypeptide subunits linked together by a disulfide bond The optimum pH for GOx is

55 though it is stable in a broad pH range of 4 to 7 and can be stored for a long period of

time51

The functional parameters for glucose oxidase include Km of 151 mM and kcat of

337 s-1

at temperature of 20 ordmC and pH of 754

Some of the inhibitors of GOx are 2-

Deoxy-D-glucose adenine nucleotides and NaNO3

32 Experimental

321 Materials and methods

Uniform carboxyl modified polystyrene microspheres 05 microm in diameter were obtained

from Polysciences Inc The size of the purchased microspheres was verified by FESEM

Peroxidase Type I from horseradish glucose oxidase Type II-S from Aspergillus niger

N-(3-dimethyl-aminopropyl)-Nrsquo-ethylcarbodiimide (EDC) and Hydrogen peroxide

solution 50 wt in water were purchased from Sigma-Aldrich N-hydroxysuccinimide

(NHS) was purchased from Fluka D-Glucose (99) was purchased from VWR

International Secure-Seal imaging spacers (9mm diameter) for glass slides were obtained

from Sigma Aldrich All chemicals were of reagent grade and used without further

purification Nanopure deionized water was used for all experiments and preparation of

solutions All measurements were made at room temperature (25 oC) unless mentioned

otherwise The UV-Visible measurements were performed using Agilent 8453A diode-

array UV-Visible Spectrophotometer with ChemStation UV-Vis Software Optical

33

microscopy experiments were performed using Axiovert 200 MAT microscope by Carl

Zeiss Diffusion coefficient measurements were obtained using Nanosight LM10

322 Optical microscopy experiments

The enzyme substrate system was observed under optical microscope to check for

aggregation and precipitation due to cascade reactions Several such systems were

studied Each system in general consisted of GOx and HRP immobilized on different

polystyrene particles The substrate solutions consisted of 01 M glucose and different

concentrations of TMB solution The systems were studied in the presence of all the

constituents and in the absence of glucose GOxHRP and TMB Systems consisting of

different sized HRP immobilized particles in substrate solution of 003 hydrogen

peroxide and TMB were also studied Videos were recorded over a period of 24 hours to

observe changes in the system Each experiment was performed atleast thrice to assess

reproducibility of results

323 Diffusion coefficient measurements using Nanosight LM10

Diffusion coefficient of GOx and HRP immobilized particles were measured using

Nanosight LM10 nanoparticle tracking instrument with Nanoparticle Tracking Analysis

(NTA) 20 software Diffusion coefficients were measured for different systems namely

GOx particles in 01 M glucose and oxygen both GOx and HRP functionalized particles

in 01 M glucose and oxygen along with controls for the individual systems Videos were

34

recorded for 30 seconds at the rate of 30 frames per second Each experiment was carried

out in triplicate to assess reproducibility

33 Results

331 Optical microscopy

According to the enzyme cascade model precipitation of TMB was possible only upon

the interaction of GOx and HRP immobilized particles in presence of appropriate

substrate solution Hence in the first set of experiments precipitation of TMB was used

as an indication of the type of interaction between GOx and HRP immobilized particles

Fan-shaped structures were observed when both HRP and GOx immobilized particles

were present in a substrate solution consisting of glucose and TMB These structures

started forming as a single strand and then branched out and intertwined to form the fan-

shaped structures (Figure 3 4)

Table 3 1 Observations for formation of fan-shaped structures in enzyme cascade

system in presence of different components

GOx HRP Glucose TMB Observation

Yes Yes Yes Yes

Fan-shaped structures observed particles associated with

the fan-shaped structures

No Yes Yes Yes No fan-shaped structures

Yes No Yes Yes No fan-shaped structures

Yes Yes No Yes No fan-shaped structures

Yes Yes Yes No No fan-shaped structures

35

Fan-shaped structures can also be observed in the system consisting of only HRP

immobilized particles in substrate solution of hydrogen peroxide and TMB The size of

the fan-shaped structures depends on the size of the enzyme immobilized particles Free

enzymes show extremely tiny structures which are difficult to view under the optical

microscope

Figure 3 4 Formation of fan-shaped structures when both HRP and GOx

immobilized particles were present in a substrate solution consisting of glucose and

TMB A In the initial stages single strands form on the surface of liquid (5Ox) B

Single strands branch out and intertwine to form fan-shaped structures (50x) C Many

such structures form and settle down after 20 minutes (20x)

A

C

B

36

Experiments were performed in the absence of TMB that is the system consisted of GOx

and HRP immobilized particles in glucose substrate solution In the absence of TMB the

particles were expected to move closer to each other without any precipitation due to the

cascade reaction However there was no conclusive aggregation of particles observed in

the system

Free Enzyme 005 μm HRP - immobilized particle

10 μm HRP - immobilized particle 05 μm HRP - immobilized particle

Figure 3 5 Size dependence of fan-shaped structures on the size of HRP -

immobilized particles (20x)

37

Table 3 2 Observations for particle aggregation in enzyme cascade system in presence

and absence of TMB

Glucose GOx HRP TMB Observation

Present Present Present Present Aggregation and precipitate

Present Present Present Absent No conclusive aggregation

Present Absent Present Present No aggregation or ppt

Absent Present Present Present No aggregation or ppt

Present Present Absent Present No aggregation or ppt

332 Diffusion coefficient measurements

Diffusion coefficients were measured for GOx immobilized particles of 05 μm diameter

in substrate solution of 01 M glucose and oxygen Diffusion coefficients of particles

were also measured for system consisting of GOx and HRP functionalized particles in

glucose and oxygen Each experiment was done in triplicate to assess reproducibility

Nanosight visualization chamber being a closed chamber oxygen was purged into all the

solutions before starting the experiments to ensure sufficient supply of oxygen for

catalytic reaction of GOx

38

Table 3 3 Diffusion coefficients for 05 μm GOx immobilized particles in 01 M

glucose purged with O2

Mean

GOx

particles in

01 M

glucose

Control - GOx

particles in DI

water

Control - GOx

particles in 01 M

glucose without

oxygen purging

Control -

particles in 01

M Glucose

Diffusion

coefficient

(x10-10

cm2s)

13316 12888 13827 13702

Standard

deviation 6307 4926 4424 7468

Table 3 4 Diffusion coefficients for GOx and HRP immobilized particles in 01 M

glucose and purged with O2

Mean

GOx- HRP-

functionalized

particles in 01 M

glucose

Control ndash Gox-

HRP- functionalized

particles in DI water

Control - particles

in 01 M glucose

Diffusion coefficient

(x10-10

cm2s)

13005 14434 13702

Standard deviation 5263 6622 7468

No significant increase was observed in diffusion coefficient of enzyme immobilized

particles in any of the systems

39

34 Discussion

Formation of fan-shaped structures due to patterned deposition of oxidized TMB

indicates some kind of interaction between GOx and HRP immobilized particles Particle

diameter dependence of the size of structures can be explained with a simple model in

Figure 3 6 A larger particle will have a higher surface area as compared to a smaller

particle Hence more enzyme is immobilized on the larger particle As a result higher

quantity of TMB is oxidized and precipitated by the larger particle as compared to the

smaller particle This leads to thicker strands of fan-shaped structures eventually scaling

up the whole system to give bigger fan-shaped structures

The exact reason for the formation of such fan-shaped structures which are dependent on

particle size could not be determined It is hypothesized that hydrogen peroxide andor

Figure 3 6 Model for size dependence of fan-shaped structures on the particle

diameter of enzyme immobilized particles Larger the particle more surface area is

available for precipitation of TMB which eventually leads to bigger fan-shaped

structures

Larger particle Smaller particle

40

TMB gradient in the substrate solution might be responsible for such structures If there

is higher concentration of H2O2 on one side of the particle and assuming an even

concentration distribution of TMB there will be selective precipitation in the direction of

H2O2 When several particles come in the vicinity of higher concentration of H2O2 the

precipitation lines will converge to give fan-shaped structures (Figure 3 7)

Figure 3 7 Hypothesis for formation of fan-shaped structures

In the system consisting of GOx immobilized particles HRP immobilized particles

glucose and TMB it was observed that the fan-shaped structures formed at the liquid-air

interface and eventually settled down to the bottom of the glass slide This makes sense

as oxygen is necessary for the catalytic reaction of conversion of glucose to gluconic acid

and hydrogen peroxide which takes place on the surface of GOx immobilized particle As

a result H2O2 is primarily formed at the liquid-air interface and is then used by HRP for

precipitation of TMB

Following the observations that precipitation of TMB occurs at the surface GOx and

HRP immobilized particles are also expected to interact at the surface in the absence of

TMB Due the cascade reaction it was expected that HRP immobilized particles will be

41

attracted to GOx immobilized particles in appropriate substrate solution Experiments

were carried out to visualize inter-particle interactions at the liquid-air interface

However liquid-air interface not being a stable system to image the precise interaction

of GOx and HRP immobilized particles could not be determined Significant external

disturbances were involved at the interface To enable the cascade reaction in the bulk of

the liquid oxygen was purged into the reaction system before starting the experiment

However this did not lead to significant reaction in the bulk of the liquid and the reaction

prominently took place at the liquid-air interface where the oxygen concentration was

higher

In order to observe the interaction of GOx and HRP immobilized particles better imaging

techniques need to be used to observe diffusion of particles in absence of external

disturbances at the liquid-air interface Large quantity of oxygen needs to be purged into

the system and retained to make the reaction more pronounced in the bulk of the substrate

solution Higher concentration of oxygen in water may also be achieved by using oxygen

enriched water which is commercially available55

Alternatively a different enzyme cascade system may be used which does not require a

gaseous substrate56

For example glycogen synthase synthesizes glycogen from glucose-

1-phosphate and glycogen phosphorylase breaks down glycogen to glucose-1-

phosphate57

This system does not require a gaseous substrate and might be able to

overcome the deficiencies of GOx and HRP enzyme cascade system

42

Chapter 4

Silver-titania nanomotors

41 Introduction

Bimetallic nanowires made of platinum and gold have been developed by WPaxton

etal1 The rods move autonomously in a solution of hydrogen peroxide In the present

study silver-titania (Ag-TiO2) particles are being synthesized and investigated for motion

in presence of hydrogen peroxide and UV light With proper control of conditions and

UV light trigger it can be possible to control the speed and direction of motion of these

nanoparticles

Hydrogen peroxide is produced in the human body by phagocytes as an immune response

to foreign particles and pathogens5859

Also silver nanoparticles have been shown to be

biocompatible and capable of interacting with cells in human body without disrupting

cellular function60

Similarly it has been demonstrated that titania is compatible with

different cells DNA and proteins in human body and does not cause toxic effects Hence

silver-titania nanomotors which swim in hydrogen peroxide may open interesting

avenues in applications like targeted drug delivery in those parts of the body where

hydrogen peroxide is present

43

42 Experimental

421 Materials and methods

Silver nitrate (999995) was purchased from Alfa Aesar Gum arabic was purchased

from MP Biomedicals Hydrogen peroxide solution 50 wt in water and ascorbic acid

were purchased from Sigma-Aldrich Nanopure deionized water was used for all

experiments and preparation of all solutions Secure-Seal imaging spacers (9 mm

diameter) were obtained from Sigma Aldrich All experiments were carried out at room

temperature of 25ordmC unless otherwise mentioned Ag-TiO2 particles were synthesized by

evaporation of titania in Semicore electron beam evaporator Particles were characterized

using field-emission scanning electron microscopy Optical microscopy experiments

were performed using Axiovert 200 MAT microscope by Carl Zeiss Particles were

tracked using Physvis particle tracking software (Figure 4 1) in which the position

information of a particle on screen is stored on a frame by frame basis The data is stored

in a dat file as X and Y co-ordinates of particles tracked on the screen This information

can be converted to diffusion coefficients of the particles by further manipulation using

MathWorks-MATLABreg software

44

Figure 4 1 Physvis particle tracking software

422 Synthesis of silver colloidal particles

Silver particles were synthesized by a nucleationgrowth type reaction developed by

Goya and Matijevic61

and modified by Velikov62

Silver nitrate was used as precursor

metal salt for synthesis of silver particles Solution A was 75 ml of 50 mM AgNO3 and

035 by weight gum arabic Solution B was 75 mL of 100 mM 1-ascorbic acid and

035 by weight gum arabic Solution B was rapidly added to solution A in a 250 mL

reaction flask The reaction flask was covered and particles were stirred for 48 hrs After

48 hours particles were allowed to settle for 2-3 hours in refrigerator and supernatant

was separated from the particles Particles were then rinsed with DI water and centrifuged

thrice to obtain pure silver particles Zeta potential of these particles was reported to be

45

around -50 to -60 mV and the size was around 1μm The size of the particles can be

varied by varying the amounts of ascorbic acid and silver nitrate Using molar ratio of 31

of ascorbic acid silver nitrate can give particles of 200 nm diameter whereas ratio of

151 can give particles of upto 5 μm diameter

423 Preparation of silver-titania (Ag-TiO2) particles

A monolayer of silver particles was prepared according to the procedure by Goldenberg

et al63

Glass slides were placed on the bottom of a petridish Water was put into the

petridish so that the entire slide was immersed in water A thin layer of hexane was then

added on top of water in the petridish A suspension of silver particles in ethanol (5

ww) was slowly introduced to the water-hexane interface in the dish After the

monolayer was formed the glass slide was lifted up through the monolayer with tweezers

The silver particles-covered glass slides were left at room temperature to dry overnight

Following drying a thin film of titania was evaporated on the glass slides in Semicore

evaporator (Figure 2 7) The spheres were partially coated with a 10 nm film of titania

Ag-TiO2 particles were removed from the glass slide by gentle sonication FE-SEM was

used to characterize the particles (Figure 4 2) The thin titania layer was not easily

distinguishable due to roughness of the particles

46

Figure 4 2 FESEM image of Ag-TiO2 particles

424 Optical microscopy experiments

Imaging spacers were placed on a glass slide Suspension of Ag-TiO2 particles was

placed on the glass slide and the particles were allowed to settle Movement of particles

was observed in DI water and videos were recorded at 50x magnification of optical

microscope for 30 sec 05 H2O2 was added to the particle suspension on the slide

Videos were recorded at 50x ranging from 30 sec to 10 min to study the movement of

silver titania particles in hydrogen peroxide solution Particles in both DI water and

hydrogen peroxide were subjected to UV light and videos were recorded to study particle

dynamics in presence of UV radiation Particle tracking was performed using Physvis

particle tracking software Atleast 30 particles in every system were tracked for 10

seconds each Particle tracking data was obtained in terms of X and Y coordinates of

particles on screen This data was manipulated using MathWorks-Matlab software and

graphs of mean square displacement of particles versus time were plotted Diffusion

coefficients of particles were calculated from these graphs

47

43 Results and Discussion

Ag-TiO2 particles demonstrated autonomous motion in the presence of H2O2 No UV

light was required for this motion Motion of the particles was accelerated by addition of

fresh H2O2 to the system When subjected to UV radiation the particles displayed

momentary acceleration for 4-5 seconds

Figure 4 3 Mechanism of motion of Ag-TiO2 nanomotors

In the presence of H2O2 silver part of the Ag-TiO2 particle converts H2O2 to H2O and O2

This may lead to propulsion of the Ag-TiO2 particle due to bubble propulsion andor self-

diffusiophoresis In the presence of UV light additional reaction takes place on the

titania part of Ag-TiO2 particle which increases the velocity of the Ag-TiO2 particles

(Figure 4 4) This UV triggered photoactivity of titania is due to hole-electron separation

caused by photons having energy equal to or greater than the band gap of titania646566

Most of the holes-electrons recombine but a small fraction move to the surface and react

with the available redox species like water and oxygen to form protons (H+) superoxide

(O2-) and hydroxyl (OH) radicals

6566 This may lead to propulsion by diffusiophoresis

as well as electrophoresis

48

Figure 4 4 Silver-titania motors move faster when subjected to UV radiation

In addition to linear motion rotational motion is also observed in several particles and

particle aggregates Aggregation of particles can lead to a net circular propulsive force

which can rotate the aggregate as shown in Figure 4 5

Figure 4 5 Rotation due to particle aggregation of Ag-TiO2 particles

Particle tracking by Physvis software revealed a significant increase in diffusion

coefficient of Ag-TiO2 particles in hydrogen peroxide solution as compared to DI water

49

The Ag-TiO2 particles have a diffusion coefficient of 1491 μm2s in hydrogen peroxide

versus only 180 μm2s in DI water Thus the Ag-TiO2 particles move around more than

10 times their body length per second in hydrogen peroxide (Figure 4 6) When

subjected to UV light the particles experience a significant acceleration in hydrogen

peroxide which lasts only for a short duration of time (4-5 sec) after which the particles

return to their original velocity The diffusion coefficient of the particles during this UV

light trigger is found to be around 5093 μm2s UV light trigger does not have any visible

effect on the Ag-TiO2 particles in DI water

Table 4 1 Translational diffusion coefficients of Ag-TiO2 particles in hydrogen

peroxide and water

Mean 05 H2O2 solution DI water

Diffusion coefficient of Ag-TiO2 (μm2s)

without UV light

1491 180

Diffusion coefficient of Ag-TiO2 (μm2s)

in the presence of UV light

5093 113

50

Figure 4 6 Mean square displacement curves of Ag-TiO2 Janus particles in hydrogen

peroxide and DI water

When higher concentration (~30 vv) of Ag-TiO2 particles in hydrogen peroxide is

subjected to UV radiation the particles exhibit pattern formation (Figure 4 7)

Aggregation of Ag-TiO2 Janus particles is observed when UV light is turned on and the

particles disaggregated when UV light is switched off This can be due to predator-prey

kind of interaction between silver and titania half of the Janus particles in the presence of

UV light Oxygen and water that are produced at the silver end by decomposition of

hydrogen peroxide are used up by titania when subjected to UV radiation This can lead

to attraction between titania and silver parts of neighboring Janus particles causing

aggregation

y = 20373x

y = 59994x

y = 71848x

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10 12

Mean

sq

uare

dis

pla

cem

en

t (μ

m2s

)

Time (sec)

Mean square displacement vs Time for Ag-TiO2

particles

05 Hydrogen peroxide and UV05 Hydrogen peroxide

DI Water

51

A UV OFF B UV ON

C UV OFF

Figure 4 7 Higher concentrations of Ag-TiO2 particles showing aggregation when UV

is turned on

52

Chapter 5

Summary and Conclusion

In summary no significant enhancement in diffusion was observed in case of enzyme

immobilized Janus particles in substrate solution Presence of a linker between enzyme

and particle did not increase the diffusion coefficient of particles Alternative enzyme

immobilization methods need to be tested like thiolation of enzyme and subsequent

binding to gold surface of a Janus particle35

or expressing the enzyme with polyhistidine

or avidin tag67

This can result in higher enzyme activity upon immobilization and may

lead to enhanced diffusion of Janus particles

Predator-prey system of GOx and HRP immobilized particles showed some promising

results Aggregation of particles and formation of fan-shaped structures by TMB

oxidation were observed under optical microscope when GOx and HRP immobilized

particles glucose and TMB were present in the system The size of the fan-shaped

structures was dependent on the size of enzyme immobilized particles This can be

indicative of some kind of interaction between the GOx and HRP immobilized particles

No conclusive aggregation of GOx and HRP immobilized particles was observed in

absence of TMB Better imaging techniques are required to study the interaction of GOx

and HRP immobilized particles at the unstable liquid-air interface where majority of the

reaction takes place Other enzyme cascade systems not requiring gaseous reactants like

glycogen synthase and glycogen phosphorylase should be tested for similar activity

53

Ag-TiO2 particles were found to exhibit autonomous motion in the presence of hydrogen

peroxide solution The motion was momentarily accelerated upon exposure to UV

radiation The particles showed a diffusion coefficient of 1491 μm2s in hydrogen

peroxide versus only 180 μm2s in DI water Upon subjecting particles to UV radiation in

presence of H2O2 diffusion coefficient increased to 5093 μm2s for a period of 4-5

seconds In addition to linear motion rotating motion could also be observed in some

particle aggregates Further development of these aggregate structures can lead to

formation of rotors at nanoscale Higher concentration of silver-titania particles in

hydrogen peroxide displayed aggregation in presence of UV light and disaggregated

when UV light was switched off This may be attributed to the predator-prey kind of

interaction between silver and titania where oxygen and water that are produced by

silver by decomposition of hydrogen peroxide are used up by titania when subjected to

UV radiation This promises to be an interesting system and needs to be further

investigated to explore its properties

54

References

1 Paxton WF Sundararajan S Mallouk TE Sen A Angew Chem Int Ed Engl

2006 45 5420-5429

2 Golestanian R Liverpool TB Adjari A Phys Rev Lett 2005 94 220801

3 httpwwwsilvermedicineorgh2o2html

4 Rami Pedahzur Ovadia Lev Badri Fattal Hillel Shuval Water Science and Technology

1995 31(5-6) 123-129

5 W Went F American Scientist 1968 56

6 Adriano Cavalcanti Tad Hogg Bijan Shirinzadeh IEEE MHS 2006 International

Symposium on Micro-NanoMechatronics and Human Science 2006 226-232

7 EM Purcell American Journal of Physics 1977 45 3-11

8 Golestanian R Phys Rev Lett 2010 105 018103(1)-018103(4)

9 S S Dukhin Z R Ulberg G L Dvornichenko B V Deryagin Russian Chemical

Bulletin 1983 31(8) 1535-1544

10 Dukhin AS Goetz PJ Ultrasound for characterizing colloids Particle sizing zeta

potential rheology Elsevier 2002 59

11 HJKeh HJTu Trends in Colloid and Interface Science XIV Progress in Colloid and

Polymer Science 2000 115 174-180

12 D Zinemanas A Nir International Journal of Multiphase Flow 1995 21(5) 787-800

55

13 Stephen Ebbens Jonathan Howse Soft matter 2010 6 726-739

14 J Vicario R Eelkema W R Browne A Meetsma R M La Crois B L Feringa

Chem Commun 2005 31 3936

15 Davide Pantarotto Wesley R Browne Ben L Feringa Chem Commun 2008 1533 -

1535

16 Lisa A Cameron Matthew J Footer Alexander van Oudenaarden Julie A Theriot

PNAS 1999 96(9) 4908-4913

17 Ali Najafi Ramin Golestanian Phys Rev E 2004 69 062901

18 Jinseok Kim Jungyul Park Sungwook Yang Jeongeun Baek Byungkyu Kim Sang

Ho Lee Eui-Sung Yoon Kukjin Chun Sukho Park Lab Chip 2007 7 1504 - 1508

19 Kurt M Dubowski Clinical Chemistry 2008 5411 1919ndash1920

20 Spector DA Yang Q Wade JB Am J Physiol Renal Physiol 2007 292(1) 467-474

21 Rhee SG Chang TS Bae YS Lee SR Kang SW J Am Soc Nephrol 2003 14(8-3)

211-215

22 Sakaue T Kapral R Mikhailov A S Eur Phys J B 2010 75 381ndash387

23 Muddana H S Sengupta S Mallouk T E Sen A Butler P J J Am Chem Soc

2010 132 (7) 2110ndash2111

24 Baker CJ Deahl K Domek J Orlandi EW Arch Biochem Biophys 2000 382(2) 232-

237

25 V Vojinovic RH Carvalho F Lemos JMS Cabral LP Fonseca BS Ferreira

Biochemical Engineering Journal 2007 35 126-135

56

26

OV Lebedeva NN Ugarova Russian Chemical Bulletin 1996 45(1) 18-25

27 Nigel C Veitch Phytochemistry 2004 65(3) 249-259

28 Kenneth H Petersen Journal of Immunological Methods 2009 340(1) 86-89

29 httpwwwbrenda-enzymesorgphpresult_flatphp4ecno=11117

30 Goldenberg L M Wagner J Stumpe J Paulke B-R Goumlrnitz E Langmuir 2002 18

5627-5629

31 httpwwwgoldbiocompdf3761-Protocol201pdf

32 Av Rovisco Pais Lisboa Portugal T amp D Technology West Yorkshire UK Journal

of Molecular Catalysis B Enzymatic 2004 28(2-3) 129-135

33 httpwwwphysicsnyuedugrierlabmethodsnode11html

34 Yoon-Mee Lee1 O-Yul Kwon Yeo-Joon Yoon Keungarp Ryu Biotechnology Letters

2006 28 39ndash43

35 JC Pyun SD Kim JW Chung Analytical Biochemistry 2005 347(2) 227-233

36 George H Pimbley Jr Mathematical Biosciences 1974 20(1-2) 27-51

37 Andy Fenton Sarah E Perkins Parasitology 2010 137 1027-1038

38 Mead James G Brownell Robert L Mammal Species of the World A Taxonomic and

Geographic Reference (3rd ed) Johns Hopkins University Press 2005 2 2142

57

39

Krause KH Schweiz Med Wochenschr 2000 130(4) 97-100

40 Irina Kareva Faina Berezovskaya Carlos Castillo-Chavez Journal of Biological

Dynamics 2010 4(4)315 - 327

41 Delaittre G Reynhout I Cornelissen J Nolte R Chemistry ndash A European Journal

2009 15 12600ndash12603

42 C Buller DH Johnson RC Tschumper Investigative Ophthalmology amp Visual Science

1990 31 2156-2163

43 Robert M Mazo Brownian Motion Fluctuations Dynamics and Applications Oxford

Science Publications 2009 57

44 Vivek K Mutalik KV Venkatesh Theoretical Biology and Medical Modelling 2005 2

19

45 Satoshi Nakamura Kunimasa Koga Biochemical and Biophysical Research

Communications 1977 78(2) 806-810

46 Krishnananda Chattopadhyay Shyamalava Mazumdar Biochemistry 2000 39 (1)

263ndash270

47 Satoshi Nakamura Yasuyuki Ogura J Biochem 1968 63 (3) 308-316

48 van Dongen SF Nallani M Cornelissen JJ Nolte RJ van Hest JC Chemistry 2009

15(5) 1107-1114

49 Ronald Bentley Methods in Enzymology 1955 1 340-345

50 Vartiainen J Raumlttouml M Paulussen S Packaging Technology and Science 2005

18 243ndash251

58

51 GA Sherbeny AA Shindia YMMM Sheriff International Journal of Agriculture

and Biology 2005 7

52 D Rando G-W Kohring F Giffhorn Appl Microbiol Biotechnol 1997 48

53 Worthington enzyme manual Worthington Biochemical Corporation

httpworthington-biochemcomgopdefaulthtml

54 httpwwwbrenda-enzymesorgphpresult_flatphp4ecno=11117

55 httpwwwoxygenphwatercom

56 P Boon Chock Sue Goo Rhee and Earl R Stadtman Annu Rev Biochem 1980 49

813-841

57 Sybil Golden P A Wals Joseph Katz Analytical Biochemistry 1977 77(2) 436-445

58 R L Baehner S K Murrmann J Davis R B Johnston Jr J Clin Invest 1975 56(3)

571ndash576

59 I Bejarano M P Terroacuten S D Paredes C Barriga A B Rodriacuteguez J A Pariente

Molecular and Cellular Biochemistry 2007 296(1-2) 77-84

60 Michael C Moulton Laura K Braydich-Stolle Mallikarjuna N Nadagouda Samantha

Kunzelman Saber M Hussain Rajender S Varma Nanoscale 2010 2 763-770

61 Goya DV Matijevic E Colloids and Surfaces A 1999 146 139

62 Velikov KP Zegers GE van Blaanderen A Langmuir 2003 19 1384

63 Goldenberg L M Wagner J Stumpe J Paulke B-R Goumlrnitz E Langmuir 2002 18

5627-5629

59

64

Banerjee S Gopal J Muraleedharan P Tyagi A K Raj B Curr Sci 2006

901378ndash1383

65 Bogomolov V N Kudinov E K Firsov Y A Sov Phys Solid State 1968 35 555

66 Kudo A Miseki Y Chem Soc Rev 2009 38 253ndash278

67 Gaj T Meyer SC Ghosh I Protein expression and Purification 2007 56 54-61

12

Based on the above facts it might be possible to propel a nanoparticle by asymmetric

distribution of enzymes on its surface thereby leading to self-propulsion of the particles

in substrate solution (Figure 2 1) This propulsion may be due to self-

electrophoresisself-diffusiophoresis andor due to nonreciprocal changes in enzyme

conformations Here we aim to investigate a system consisting of polystyrene particles

half coated with enzyme introduced in a suitable substrate solution The enzyme can be

directly linked to the polystyrene particle or it can be linked through a long spacer

molecule (linker) to provide more degrees of freedom to the enzyme

Several factors need to be considered for choosing enzyme for powering motion at the

nanoscale Some of the important factors are the ratio of kcatKm which denotes the

catalytic efficiency of the enzyme possible substrates stability of enzymessubstrates

ease of immobilization relationship with other enzymes in cascade reactions and

commercial availability of the enzymes The enzyme being used in the present study is

horseradish peroxidase It acts on a molecule of hydrogen peroxide and reduces it while

simultaneously oxidizing another substrate molecule In the absence of alternate

oxidizable substrate HRP can oxidize water and oxygen back to hydrogen peroxide24

thus giving an equilibrium reaction

13

211 Horseradish peroxidase

Figure 2 3 Horseradish peroxidase

Horseradish peroxidase belongs to the group of peroxidases which are a group of heme-

containing oxidoreductases that act on peroxide as hydrogen donor25

It is a redox

enzyme which contains a protoheme a complex of trivalent iron with 1358-

tetramethyl-24-divinylporphin-67-dipropionic acid as a prosthetic group It is a 44 kDa

protein which includes 33900 Da polypeptide chain bound with eight oligosaccharide

chains The carbohydrate chains improve the stability of the enzyme by protecting it

against radicals formed in the course of the reaction it catalyzes26

Horseradish

peroxidase is found in horseradish and is used widely in bioanalytical applications such

as ELISA and Western blots It decomposes hydrogen peroxide into water and oxygen

while simultaneously oxidizing another substrate27

The oxidizable substrates can be

chromogenic substrates such as 22rsquo55rsquo-tetramethyl-benzidine (TMB) and 33-

diaminobenzidine (DMB) or chemoluminescent substrates like SuperSignal and ECL

luminal28

14

Figure 2 4 Reaction catalyzed by horseradish peroxidase

The functional parameters for horseradish peroxidase include Km of 015 mM and kcat of

790 s-1

at an optimum temperature of 20 ordmC and an optimum pH of 6429

Some of the

inhibitors of HRP are 110-phenanthroline aminotriazole and bromine

22 Experimental

221 Materials and methods

Uniform carboxyl modified polystyrene microspheres 05 microm in diameter were obtained

from Polysciences Inc The size of the purchased microspheres was verified by Field

Emission Scanning Electron Microscopy (FESEM) Peroxidase Type I from

Horseradish N-(3-dimethyl-aminopropyl)-Nrsquo-ethylcarbodiimide (EDC) and hydrogen

peroxide solution 50 wt in water were purchased from Sigma-Aldrich N-

hydroxysuccinimide (NHS) ge 97 was purchased from Fluka All chemicals were of

reagent grade and used without further purification Nanopure deionized water was used

for all experiments and preparation of solutions All measurements were made at room

15

temperature (25 oC) unless mentioned otherwise The UV-Visible spectroscopy

measurements were performed using Agilent 8453A diode-array UV-Visible

Spectrophotometer with ChemStation UV-Vis Software Diffusion coefficient

measurements were performed using Nanosight LM10

222 Synthesis of Janus particles

A monolayer of particles was prepared according to the procedure (Figure 2 5) by

Goldenberg L et al30

Glass slides were placed on the bottom of a petridish Water was

put into the petridish so that the entire slide was immersed in water A thin layer of

hexane was added on top of water in the petridish A suspension of microspheres in

ethanol (5 ww) was introduced to the water-hexane interface After the monolayer was

formed the glass slide was lifted up through the monolayer with tweezers The

microsphere-covered glass slides were left to dry overnight at room temperature

Following drying a thin film of silica was evaporated on the glass slides in Semicore

evaporator The spheres were partially coated with 30 nm of silica Particles were

removed from the glass slide by gentle sonication The polystyrene-silica (PS-SiO2)

Janus particles thus obtained were washed in deionized water several times before

further immobilization steps were performed FESEM was used to confirm synthesis of

Janus particles

16

Figure 2 5 Preparation of PS-SiO2 Janus particles by evaporation

223 Immobilization of enzymes using EDCNHS coupling method

1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide and N-hydroxysulfosuccinimide

(EDCNHS) coupling procedure was followed for immobilizing horseradish peroxidase

type I on 05 μm PS-SiO2 Janus particles (Figure 2 6) In this process EDC reacts with a

carboxyl groups on the Janus particles to form an amine-reactive O-acylisourea

intermediate If this intermediate does not encounter an amine it can hydrolyze and

regenerate the carboxyl group In the presence of NHS EDC can be used to convert

carboxyl groups to amine-reactive sulfo-NHS esters which are much more stable and do

not readily hydrolyze to regenerate carboxyl groups In the current experiment

immobilization was performed in the presence as well as absence of a long spacer

17

molecule (linker) between Janus particles and enzymes The chemical 6-aminohexanoic

acid was used as the linker (Figure 2 7) HRP immobilized PS-SiO2 Janus particles were

stored in deionized water until required for further experiments

Figure 2 6 EDCNHS coupling method for immobilization of enzyme on carboxylated

polystyrene particles

Figure 2 7 Immobilization of enzyme on Janus particles with and without 6-

aminohexanoic acid linker

18

224 Enzyme assay

Assay for activity of HRP was performed using a spectrophotometric method which

involved using 4-aminoantipyrene as hydrogen donor3132

The reaction rate was

determined by measuring increase in absorbance at 510 nm resulting from the

decomposition of hydrogen peroxide One unit represents the decomposition of 1 μM of

hydrogen peroxide per minute at 25 degC and pH 70 under the specified conditions

The spectrophotometer was set to the wavelength of 510 nm and temperature of 25 degC

Phenol4-aminoantipyrene solution (14 mL) and 00017 M H2O2 solution (15 mL) were

pipetted into each cuvette The cuvette was incubated for 3-4 mins at 25degC and blank was

established A 01 mL solution of diluted enzyme was added to the above mixture and

absorbance was recorded for 4-5 mins Standard graphs of absorbance versus time were

drawn for known concentration of the free enzyme and compared to that of the

immobilized enzyme on the Janus particles The estimate of the amount of enzyme

present on the Janus particles was deduced from the difference between the quantity of

the free enzyme previously added for immobilization and the amount that was measured

to be in the supernatant after some of the enzyme became attached to the Janus particles

The experiment was carried out in triplicate to assess reproducibility

19

The formula used to calculate the activity of enzyme is

where ∆Amin denotes the change in absorbance per minute

225 Diffusion coefficient measurements using Nanosight LM10

Diffusion coefficients for HRP immobilized particles were measured using Nanosight

LM 10 nanoparticle tracking instrument with Nanoparticle Tracking Analysis (NTA) 20

software in the presence of 003 H2O2 solution as the substrate and also in the presence

of only deionized water The videos were recorded for 30 seconds at the rate of 30 frames

per second Each experiment was carried out in triplicate to assess reproducibility

Particles

visualization

zone

Microscope with

LM 10 unit

LM 10 unit

Figure 2 8 Nanosight LM 10 instrumentation

20

Nanosight LM 10 (Figure 2 8) generates videos of population of moving nanoparticles in

a liquid when illuminated by laser light The particles which pass through the laser beam

path are seen by the instrument as small points of light undergoing Brownian motion

(Figure 2 9a)

The videos are further analyzed by NTA (Nanoparticle tracking analysis) software

(Figure 2 9b) which identifies and tracks each particle independently on a frame-by-

frame basis The software also calculates and subtracts any bulk flow in the system The

mean square displacement is calculated for each particle as long as it is visible If the

particle blinks that is if it disappears momentarily and then reappears on the screen the

software will consider the same particle as two separate ones All analysis data is

exported to the excel files in terms of diffusion coefficients of the tracked particles

B A

Figure 2 9 NTA video analysis (a) Particles as they are seen on the screen during

experimentation in Nanosight LM10 (b) NTA screen as the particles are being analyzed for

their diffusion coefficients

21

23 Results

231 Enzyme assay

The activity of free HRP enzyme reported by Sigma Aldrich is 150 unitsmg However

the activity of HRP upon analysis was found to be 52158 unitsmg which is much lower

than the reported value The activity of HRP was found to be further lowered to 2133

unitsmg when it was immobilized on the PS-SiO2 Janus particles in the presence of a

linker The following formula is used to calculate enzyme activity

where ΔAmin is the change in absorbance per minute

22

Figure 2 10 Graph for the assay of solution of free enzyme containing 05mgL of HRP

ΔAmin = 01716 Unitsmg of free enzyme = 52158

During EDCNHS coupling 04 mg enzyme was added to 1 ml of 025 vv suspension

of Janus particles Thus initial concentration of enzyme in the mixture was 04 mgml

Assuming retained activity of the enzyme in supernatant solution the amount of enzyme

in supernatant was calculated to be 03868 mgml from the assay Amount of enzyme

functionalized on the Janus particles was calculated as the difference between enzyme

previously added for immobilization (04 mgml) and the enzyme present in supernatant

(03868 mgml) and was found to be 00132 mgml Enzyme assay was performed on the

enzyme immobilized Janus particles to estimate the activity of immobilized enzyme

(Figure 2 11) and was found to be 2133 unitsmg

y = 0002x + 00516

0

01

02

03

04

05

06

07

08

0 50 100 150 200 250 300 350 400

Ab

sorb

an

ce

51

0 n

m

Time (sec)

Assay of free enzyme HRP

23

Figure 2 11 Graph for the assay of of immobilized enzyme on 05 μm Janus particles

(~025 vv) ΔAmin = 01853 Unitsmg of free enzyme = 2133

232 Diffusion coefficient measurements

No significant increase was observed in diffusion coefficient of HRP-immobilized Janus

particles in the presence of H2O2 as the substrate Similar results were obtained for

diffusion coefficient measurement with and without linker moieties bridging the enzymes

and particles(Table 2 1 Table 2 2) Thus the linker did not seem to assist in increasing

the particle diffusion by improving or retaining the activity of the enzyme

y = 00015x + 00953

0

01

02

03

04

05

06

07

0 50 100 150 200 250 300 350 400

Ab

sorb

an

ce

510 n

m

Time (sec)

Assay of HRP immobilized 05 μm Janus particles

24

Table 2 1 Diffusion coefficient measurements of HRP immobilized Janus particles

without linker

HRP immobilized

Janus particles in

003 H2O2

Control - Janus

particles in

003 H2O2

Control - HRP

immobilized Janus

particles in DI water

Diffusion coefficient

(10-10

cm2s)

1359416 1051777 1308675

Standard deviation 4534801 4122075 4918597

Table 2 2 Diffusion coefficient measurements of HRP immobilized Janus particles in

the presence of linker

HRP immobilized

Janus particles in

003 H2O2

Control - Janus

particles in

003 H2O2

Control - HRP

immobilized Janus

particles in DI water

Diffusion coefficient

(10-10

cm2s)

13838 1339829 1407891

Standard deviation 5644692 5649364 5797218

25

Figure 2 12 Diffusion coefficient measurements of PS-SiO2 Janus particles without and

with 6-aminohexanoic acid linker

24 Discussion

To compare the diffusion coefficients observed during the experiments theoretical

diffusion coefficient was calculated using following equation33

r

kTD altraslation

6 Equation 21

where r is the hydrodynamic radius of particle Dtranslational is translational diffusion

coefficient of the particle T is temperature of the system η is the viscosity of fluid

through which the particles are moving and k is the Boltzmann constant From FE-SEM

images the size of the particles used was determined to be 450 nm For 450 nm diameter

0

20

40

60

80

100

120

140

160

Without linker With linker

Dif

fusio

n c

oeff

icie

nt

(10

-10 c

m2s

)

HRP immobilized 05 μm Janus particles - Diffusion coefficient measurements

HRP immobilized Janus particles in 003 H2O2

Control - Janus particles in 003 H2O2

Control - HRP immobilized Janus particles in DI water

26

particle Dtranslational is equal to 109 μm2s which is comparable to the diffusion

coefficient measured for the enzyme immobilized particles

HRP was found to be active on the Janus particles however the activity of the enzyme

was reduced upon immobilization Lower enzyme activity can be due to the coupling

method used which in this case was EDCNHS covalent binding However EDCNHS

coupling method has been shown to significantly retain the enzyme activity when the

enzyme is immobilized on carboxylated multi-walled carbon nanotubes under appropriate

conditions34

Hence EDCNHS binding method by itself is not believed to be

responsible for reduction of enzyme activity upon immobilization

Direct binding of enzyme to the Janus particle may lead to reduction in the enzyme

activity because of space constraints for the enzyme and difficulty for the passage of

substrate to the active site of the enzyme due to stearic hindrance Hence a linker was

inserted between the enzyme and Janus particle during enzyme immobilization However

inserting a linker between the enzyme and the particle did not lead to enhanced diffusion

of Janus particles or increase in enzyme activity of immobilized enzyme

Lack of enhanced diffusion can be firstly attributed to the reduction in enzyme activity

upon immobilization Enzyme activity may be retained by modifying the conditions

during EDCNHS coupling or changing the binding method altogether The degree of

immobilization of HRP on carboxylated Janus particles can possibly be affected by

several factors such as temperature the concentration of the coupling reagent EDC

27

concentration HRP concentration in solution and incubation time34

Different

combinations of these factors can be tested to find optimum conditions for

immobilization of HRP Alternate binding method can involve thiolation of HRP by the

covalent conjugation of mercaptopropionic acid and then the thiolated HRP can then be

attached on the gold surface of a polystyrene-gold Janus particle35

Thus further

modifications can be made to the system in order to retain enzyme activity on the

immobilized particles and thereby power nanomotors using enzymes

28

Chapter 3

Predator-prey system of enzyme immobilized particles

31 Introduction

Predator-prey systems are widely found in nature and play a major role in biological

processes3637

Predator-prey systems include all the interactions in which one organism

pursues andor consumes another organism One familiar predator-prey system at the

macroscale is that of a tiger chasing and consuming a deer Another not so familiar one

includes Baleen whales which eat millions of microscopic planktons at once38

At the

microscale such kind of interactions can be observed in the case of phagocytes in the

body which are capable of engulfing foreign microorganisms and killing them with a

combination of microbicidal systems39

In the case of cancer the tumor is the prey and

the mature myeloid cells in the body are the predator However the tumor here has a

defense mechanism against the predator in which the tumor produces cytokines which

inhibits the maturation of the myeloid cells and thereby prevents predation40

One similarity that can be observed in all these systems is that the prey possesses some

attribute which is desired by the predator which makes the predator chase the prey A

similar relationship is seen in case of enzymes which are involved in cascade reactions41

In cascade reactions product synthesized by one enzyme is the reactant required by

another enzyme to carry out its own catalytic reaction As such there is a predator-prey

29

kind of dependence between the two enzymes which may cause one enzyme to chase the

other enzyme when placed in suitable substrate solutions However the mechanism

hypothesized for such a system is totally different than the mechanism involved in

biological systems In biological systems such as bacterial chemotaxis or phagocytes

chasing foreign particles in human body complex biochemical reactions are responsible

for the predator-prey behavior42

In the proposed enzyme cascade system there are no

complex biochemical reactions involved The principle hypothesized for predator-prey

behavior of enzyme cascade reactions can be explained as follows Motion of synthetic

motors at nanoscale involves two components translational diffusion and rotational

Brownian diffusion At very short time scales of less than 1Dr where Dr (s-1

) is

rotational Brownian diffusion coefficient the motion of particles is unidirectional For

longer time scales of greater than 1Dr the direction of motion is random giving

poweredactive diffusion43

If the chemical homogeneity is broken that is when there is

higher concentration of a substrate present in one direction of the enzyme the enzyme

might acquire a subtle bias which may lead to a spatial aggregation of the enzymes based

on the substrate concentration gradient The bias can be a result of the relationship

between the speed of enzyme diffusion and substrate concentration where higher

substrate concentration lead to higher enzyme diffusion As a result in an enzyme

cascade system one enzyme will migrate towards higher concentration of substrate

which is present near the other enzyme thus giving rise to a predator-prey behavior

Such enzyme cascades are widely found in signaling networks and metabolic pathways in

living cells and can be used to produce predator-prey like nanomotors systems44

The

30

enzymes being used in the present study are horseradish peroxidase and glucose oxidase

Both enzymes are extremely stable4546

Glucose is the substrate for glucose oxidase

which is widely available and highly stable47

Horseradish peroxidase acts on hydrogen

peroxide It is involved in a cascade reaction with glucose oxidase which produces

hydrogen peroxide by converting glucose into gluconic acid (Figure 3 1)48

Figure 3 1 Concept of particle aggregation due to cascade reactions of immobilized

enzymes

In this experiment glucose oxidase (GOx) and horseradish peroxidase (HRP) were

immobilized on polystyrene carboxylate beads of different sizes using EDCNHS

coupling method Glucose and 3355-Tetramethylbenzidine (TMB) were dissolved

together in water and this solution was used as the substrate solution All the components

were present in a single system in DI water Aggregation of particles and precipitation of

oxidized TMB from the solution was expected to be observed when all these components

were present in such a system

Properties of horseradish peroxidase have been discussed in the previous chapter (Section

211 Horseradish peroxidase)

GOxGOx HRPHRP

Glucose + O2Gluconic acid + H2O2

TMB TMB oxidizedBlue precipitate

H2O

Aggregation

31

311 Glucose oxidase

Figure 3 2 Glucose oxidase

Glucose oxidase (GOx) belongs to the group of oxidoreductases and is also called

glucose aerodehydrogenase49

It is obtained from fungi and has been identified as an

antimicrobial enzyme50

GOx binds to β-D-glucopyranose and catalyzes the oxidation

reaction to from gluconolactone while simultaneously reducing oxygen to hydrogen

peroxide51

GOx has high substrate affinity and specificity high stability and high

turnover number which makes it a suitable biocatalyst for industrial applications52

Figure 3 3 Redox reaction of glucose catalyzed by glucose oxidase53

32

GOx is a glycoprotein with molecular weight of 155 kDa and consists of two identical

polypeptide subunits linked together by a disulfide bond The optimum pH for GOx is

55 though it is stable in a broad pH range of 4 to 7 and can be stored for a long period of

time51

The functional parameters for glucose oxidase include Km of 151 mM and kcat of

337 s-1

at temperature of 20 ordmC and pH of 754

Some of the inhibitors of GOx are 2-

Deoxy-D-glucose adenine nucleotides and NaNO3

32 Experimental

321 Materials and methods

Uniform carboxyl modified polystyrene microspheres 05 microm in diameter were obtained

from Polysciences Inc The size of the purchased microspheres was verified by FESEM

Peroxidase Type I from horseradish glucose oxidase Type II-S from Aspergillus niger

N-(3-dimethyl-aminopropyl)-Nrsquo-ethylcarbodiimide (EDC) and Hydrogen peroxide

solution 50 wt in water were purchased from Sigma-Aldrich N-hydroxysuccinimide

(NHS) was purchased from Fluka D-Glucose (99) was purchased from VWR

International Secure-Seal imaging spacers (9mm diameter) for glass slides were obtained

from Sigma Aldrich All chemicals were of reagent grade and used without further

purification Nanopure deionized water was used for all experiments and preparation of

solutions All measurements were made at room temperature (25 oC) unless mentioned

otherwise The UV-Visible measurements were performed using Agilent 8453A diode-

array UV-Visible Spectrophotometer with ChemStation UV-Vis Software Optical

33

microscopy experiments were performed using Axiovert 200 MAT microscope by Carl

Zeiss Diffusion coefficient measurements were obtained using Nanosight LM10

322 Optical microscopy experiments

The enzyme substrate system was observed under optical microscope to check for

aggregation and precipitation due to cascade reactions Several such systems were

studied Each system in general consisted of GOx and HRP immobilized on different

polystyrene particles The substrate solutions consisted of 01 M glucose and different

concentrations of TMB solution The systems were studied in the presence of all the

constituents and in the absence of glucose GOxHRP and TMB Systems consisting of

different sized HRP immobilized particles in substrate solution of 003 hydrogen

peroxide and TMB were also studied Videos were recorded over a period of 24 hours to

observe changes in the system Each experiment was performed atleast thrice to assess

reproducibility of results

323 Diffusion coefficient measurements using Nanosight LM10

Diffusion coefficient of GOx and HRP immobilized particles were measured using

Nanosight LM10 nanoparticle tracking instrument with Nanoparticle Tracking Analysis

(NTA) 20 software Diffusion coefficients were measured for different systems namely

GOx particles in 01 M glucose and oxygen both GOx and HRP functionalized particles

in 01 M glucose and oxygen along with controls for the individual systems Videos were

34

recorded for 30 seconds at the rate of 30 frames per second Each experiment was carried

out in triplicate to assess reproducibility

33 Results

331 Optical microscopy

According to the enzyme cascade model precipitation of TMB was possible only upon

the interaction of GOx and HRP immobilized particles in presence of appropriate

substrate solution Hence in the first set of experiments precipitation of TMB was used

as an indication of the type of interaction between GOx and HRP immobilized particles

Fan-shaped structures were observed when both HRP and GOx immobilized particles

were present in a substrate solution consisting of glucose and TMB These structures

started forming as a single strand and then branched out and intertwined to form the fan-

shaped structures (Figure 3 4)

Table 3 1 Observations for formation of fan-shaped structures in enzyme cascade

system in presence of different components

GOx HRP Glucose TMB Observation

Yes Yes Yes Yes

Fan-shaped structures observed particles associated with

the fan-shaped structures

No Yes Yes Yes No fan-shaped structures

Yes No Yes Yes No fan-shaped structures

Yes Yes No Yes No fan-shaped structures

Yes Yes Yes No No fan-shaped structures

35

Fan-shaped structures can also be observed in the system consisting of only HRP

immobilized particles in substrate solution of hydrogen peroxide and TMB The size of

the fan-shaped structures depends on the size of the enzyme immobilized particles Free

enzymes show extremely tiny structures which are difficult to view under the optical

microscope

Figure 3 4 Formation of fan-shaped structures when both HRP and GOx

immobilized particles were present in a substrate solution consisting of glucose and

TMB A In the initial stages single strands form on the surface of liquid (5Ox) B

Single strands branch out and intertwine to form fan-shaped structures (50x) C Many

such structures form and settle down after 20 minutes (20x)

A

C

B

36

Experiments were performed in the absence of TMB that is the system consisted of GOx

and HRP immobilized particles in glucose substrate solution In the absence of TMB the

particles were expected to move closer to each other without any precipitation due to the

cascade reaction However there was no conclusive aggregation of particles observed in

the system

Free Enzyme 005 μm HRP - immobilized particle

10 μm HRP - immobilized particle 05 μm HRP - immobilized particle

Figure 3 5 Size dependence of fan-shaped structures on the size of HRP -

immobilized particles (20x)

37

Table 3 2 Observations for particle aggregation in enzyme cascade system in presence

and absence of TMB

Glucose GOx HRP TMB Observation

Present Present Present Present Aggregation and precipitate

Present Present Present Absent No conclusive aggregation

Present Absent Present Present No aggregation or ppt

Absent Present Present Present No aggregation or ppt

Present Present Absent Present No aggregation or ppt

332 Diffusion coefficient measurements

Diffusion coefficients were measured for GOx immobilized particles of 05 μm diameter

in substrate solution of 01 M glucose and oxygen Diffusion coefficients of particles

were also measured for system consisting of GOx and HRP functionalized particles in

glucose and oxygen Each experiment was done in triplicate to assess reproducibility

Nanosight visualization chamber being a closed chamber oxygen was purged into all the

solutions before starting the experiments to ensure sufficient supply of oxygen for

catalytic reaction of GOx

38

Table 3 3 Diffusion coefficients for 05 μm GOx immobilized particles in 01 M

glucose purged with O2

Mean

GOx

particles in

01 M

glucose

Control - GOx

particles in DI

water

Control - GOx

particles in 01 M

glucose without

oxygen purging

Control -

particles in 01

M Glucose

Diffusion

coefficient

(x10-10

cm2s)

13316 12888 13827 13702

Standard

deviation 6307 4926 4424 7468

Table 3 4 Diffusion coefficients for GOx and HRP immobilized particles in 01 M

glucose and purged with O2

Mean

GOx- HRP-

functionalized

particles in 01 M

glucose

Control ndash Gox-

HRP- functionalized

particles in DI water

Control - particles

in 01 M glucose

Diffusion coefficient

(x10-10

cm2s)

13005 14434 13702

Standard deviation 5263 6622 7468

No significant increase was observed in diffusion coefficient of enzyme immobilized

particles in any of the systems

39

34 Discussion

Formation of fan-shaped structures due to patterned deposition of oxidized TMB

indicates some kind of interaction between GOx and HRP immobilized particles Particle

diameter dependence of the size of structures can be explained with a simple model in

Figure 3 6 A larger particle will have a higher surface area as compared to a smaller

particle Hence more enzyme is immobilized on the larger particle As a result higher

quantity of TMB is oxidized and precipitated by the larger particle as compared to the

smaller particle This leads to thicker strands of fan-shaped structures eventually scaling

up the whole system to give bigger fan-shaped structures

The exact reason for the formation of such fan-shaped structures which are dependent on

particle size could not be determined It is hypothesized that hydrogen peroxide andor

Figure 3 6 Model for size dependence of fan-shaped structures on the particle

diameter of enzyme immobilized particles Larger the particle more surface area is

available for precipitation of TMB which eventually leads to bigger fan-shaped

structures

Larger particle Smaller particle

40

TMB gradient in the substrate solution might be responsible for such structures If there

is higher concentration of H2O2 on one side of the particle and assuming an even

concentration distribution of TMB there will be selective precipitation in the direction of

H2O2 When several particles come in the vicinity of higher concentration of H2O2 the

precipitation lines will converge to give fan-shaped structures (Figure 3 7)

Figure 3 7 Hypothesis for formation of fan-shaped structures

In the system consisting of GOx immobilized particles HRP immobilized particles

glucose and TMB it was observed that the fan-shaped structures formed at the liquid-air

interface and eventually settled down to the bottom of the glass slide This makes sense

as oxygen is necessary for the catalytic reaction of conversion of glucose to gluconic acid

and hydrogen peroxide which takes place on the surface of GOx immobilized particle As

a result H2O2 is primarily formed at the liquid-air interface and is then used by HRP for

precipitation of TMB

Following the observations that precipitation of TMB occurs at the surface GOx and

HRP immobilized particles are also expected to interact at the surface in the absence of

TMB Due the cascade reaction it was expected that HRP immobilized particles will be

41

attracted to GOx immobilized particles in appropriate substrate solution Experiments

were carried out to visualize inter-particle interactions at the liquid-air interface

However liquid-air interface not being a stable system to image the precise interaction

of GOx and HRP immobilized particles could not be determined Significant external

disturbances were involved at the interface To enable the cascade reaction in the bulk of

the liquid oxygen was purged into the reaction system before starting the experiment

However this did not lead to significant reaction in the bulk of the liquid and the reaction

prominently took place at the liquid-air interface where the oxygen concentration was

higher

In order to observe the interaction of GOx and HRP immobilized particles better imaging

techniques need to be used to observe diffusion of particles in absence of external

disturbances at the liquid-air interface Large quantity of oxygen needs to be purged into

the system and retained to make the reaction more pronounced in the bulk of the substrate

solution Higher concentration of oxygen in water may also be achieved by using oxygen

enriched water which is commercially available55

Alternatively a different enzyme cascade system may be used which does not require a

gaseous substrate56

For example glycogen synthase synthesizes glycogen from glucose-

1-phosphate and glycogen phosphorylase breaks down glycogen to glucose-1-

phosphate57

This system does not require a gaseous substrate and might be able to

overcome the deficiencies of GOx and HRP enzyme cascade system

42

Chapter 4

Silver-titania nanomotors

41 Introduction

Bimetallic nanowires made of platinum and gold have been developed by WPaxton

etal1 The rods move autonomously in a solution of hydrogen peroxide In the present

study silver-titania (Ag-TiO2) particles are being synthesized and investigated for motion

in presence of hydrogen peroxide and UV light With proper control of conditions and

UV light trigger it can be possible to control the speed and direction of motion of these

nanoparticles

Hydrogen peroxide is produced in the human body by phagocytes as an immune response

to foreign particles and pathogens5859

Also silver nanoparticles have been shown to be

biocompatible and capable of interacting with cells in human body without disrupting

cellular function60

Similarly it has been demonstrated that titania is compatible with

different cells DNA and proteins in human body and does not cause toxic effects Hence

silver-titania nanomotors which swim in hydrogen peroxide may open interesting

avenues in applications like targeted drug delivery in those parts of the body where

hydrogen peroxide is present

43

42 Experimental

421 Materials and methods

Silver nitrate (999995) was purchased from Alfa Aesar Gum arabic was purchased

from MP Biomedicals Hydrogen peroxide solution 50 wt in water and ascorbic acid

were purchased from Sigma-Aldrich Nanopure deionized water was used for all

experiments and preparation of all solutions Secure-Seal imaging spacers (9 mm

diameter) were obtained from Sigma Aldrich All experiments were carried out at room

temperature of 25ordmC unless otherwise mentioned Ag-TiO2 particles were synthesized by

evaporation of titania in Semicore electron beam evaporator Particles were characterized

using field-emission scanning electron microscopy Optical microscopy experiments

were performed using Axiovert 200 MAT microscope by Carl Zeiss Particles were

tracked using Physvis particle tracking software (Figure 4 1) in which the position

information of a particle on screen is stored on a frame by frame basis The data is stored

in a dat file as X and Y co-ordinates of particles tracked on the screen This information

can be converted to diffusion coefficients of the particles by further manipulation using

MathWorks-MATLABreg software

44

Figure 4 1 Physvis particle tracking software

422 Synthesis of silver colloidal particles

Silver particles were synthesized by a nucleationgrowth type reaction developed by

Goya and Matijevic61

and modified by Velikov62

Silver nitrate was used as precursor

metal salt for synthesis of silver particles Solution A was 75 ml of 50 mM AgNO3 and

035 by weight gum arabic Solution B was 75 mL of 100 mM 1-ascorbic acid and

035 by weight gum arabic Solution B was rapidly added to solution A in a 250 mL

reaction flask The reaction flask was covered and particles were stirred for 48 hrs After

48 hours particles were allowed to settle for 2-3 hours in refrigerator and supernatant

was separated from the particles Particles were then rinsed with DI water and centrifuged

thrice to obtain pure silver particles Zeta potential of these particles was reported to be

45

around -50 to -60 mV and the size was around 1μm The size of the particles can be

varied by varying the amounts of ascorbic acid and silver nitrate Using molar ratio of 31

of ascorbic acid silver nitrate can give particles of 200 nm diameter whereas ratio of

151 can give particles of upto 5 μm diameter

423 Preparation of silver-titania (Ag-TiO2) particles

A monolayer of silver particles was prepared according to the procedure by Goldenberg

et al63

Glass slides were placed on the bottom of a petridish Water was put into the

petridish so that the entire slide was immersed in water A thin layer of hexane was then

added on top of water in the petridish A suspension of silver particles in ethanol (5

ww) was slowly introduced to the water-hexane interface in the dish After the

monolayer was formed the glass slide was lifted up through the monolayer with tweezers

The silver particles-covered glass slides were left at room temperature to dry overnight

Following drying a thin film of titania was evaporated on the glass slides in Semicore

evaporator (Figure 2 7) The spheres were partially coated with a 10 nm film of titania

Ag-TiO2 particles were removed from the glass slide by gentle sonication FE-SEM was

used to characterize the particles (Figure 4 2) The thin titania layer was not easily

distinguishable due to roughness of the particles

46

Figure 4 2 FESEM image of Ag-TiO2 particles

424 Optical microscopy experiments

Imaging spacers were placed on a glass slide Suspension of Ag-TiO2 particles was

placed on the glass slide and the particles were allowed to settle Movement of particles

was observed in DI water and videos were recorded at 50x magnification of optical

microscope for 30 sec 05 H2O2 was added to the particle suspension on the slide

Videos were recorded at 50x ranging from 30 sec to 10 min to study the movement of

silver titania particles in hydrogen peroxide solution Particles in both DI water and

hydrogen peroxide were subjected to UV light and videos were recorded to study particle

dynamics in presence of UV radiation Particle tracking was performed using Physvis

particle tracking software Atleast 30 particles in every system were tracked for 10

seconds each Particle tracking data was obtained in terms of X and Y coordinates of

particles on screen This data was manipulated using MathWorks-Matlab software and

graphs of mean square displacement of particles versus time were plotted Diffusion

coefficients of particles were calculated from these graphs

47

43 Results and Discussion

Ag-TiO2 particles demonstrated autonomous motion in the presence of H2O2 No UV

light was required for this motion Motion of the particles was accelerated by addition of

fresh H2O2 to the system When subjected to UV radiation the particles displayed

momentary acceleration for 4-5 seconds

Figure 4 3 Mechanism of motion of Ag-TiO2 nanomotors

In the presence of H2O2 silver part of the Ag-TiO2 particle converts H2O2 to H2O and O2

This may lead to propulsion of the Ag-TiO2 particle due to bubble propulsion andor self-

diffusiophoresis In the presence of UV light additional reaction takes place on the

titania part of Ag-TiO2 particle which increases the velocity of the Ag-TiO2 particles

(Figure 4 4) This UV triggered photoactivity of titania is due to hole-electron separation

caused by photons having energy equal to or greater than the band gap of titania646566

Most of the holes-electrons recombine but a small fraction move to the surface and react

with the available redox species like water and oxygen to form protons (H+) superoxide

(O2-) and hydroxyl (OH) radicals

6566 This may lead to propulsion by diffusiophoresis

as well as electrophoresis

48

Figure 4 4 Silver-titania motors move faster when subjected to UV radiation

In addition to linear motion rotational motion is also observed in several particles and

particle aggregates Aggregation of particles can lead to a net circular propulsive force

which can rotate the aggregate as shown in Figure 4 5

Figure 4 5 Rotation due to particle aggregation of Ag-TiO2 particles

Particle tracking by Physvis software revealed a significant increase in diffusion

coefficient of Ag-TiO2 particles in hydrogen peroxide solution as compared to DI water

49

The Ag-TiO2 particles have a diffusion coefficient of 1491 μm2s in hydrogen peroxide

versus only 180 μm2s in DI water Thus the Ag-TiO2 particles move around more than

10 times their body length per second in hydrogen peroxide (Figure 4 6) When

subjected to UV light the particles experience a significant acceleration in hydrogen

peroxide which lasts only for a short duration of time (4-5 sec) after which the particles

return to their original velocity The diffusion coefficient of the particles during this UV

light trigger is found to be around 5093 μm2s UV light trigger does not have any visible

effect on the Ag-TiO2 particles in DI water

Table 4 1 Translational diffusion coefficients of Ag-TiO2 particles in hydrogen

peroxide and water

Mean 05 H2O2 solution DI water

Diffusion coefficient of Ag-TiO2 (μm2s)

without UV light

1491 180

Diffusion coefficient of Ag-TiO2 (μm2s)

in the presence of UV light

5093 113

50

Figure 4 6 Mean square displacement curves of Ag-TiO2 Janus particles in hydrogen

peroxide and DI water

When higher concentration (~30 vv) of Ag-TiO2 particles in hydrogen peroxide is

subjected to UV radiation the particles exhibit pattern formation (Figure 4 7)

Aggregation of Ag-TiO2 Janus particles is observed when UV light is turned on and the

particles disaggregated when UV light is switched off This can be due to predator-prey

kind of interaction between silver and titania half of the Janus particles in the presence of

UV light Oxygen and water that are produced at the silver end by decomposition of

hydrogen peroxide are used up by titania when subjected to UV radiation This can lead

to attraction between titania and silver parts of neighboring Janus particles causing

aggregation

y = 20373x

y = 59994x

y = 71848x

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10 12

Mean

sq

uare

dis

pla

cem

en

t (μ

m2s

)

Time (sec)

Mean square displacement vs Time for Ag-TiO2

particles

05 Hydrogen peroxide and UV05 Hydrogen peroxide

DI Water

51

A UV OFF B UV ON

C UV OFF

Figure 4 7 Higher concentrations of Ag-TiO2 particles showing aggregation when UV

is turned on

52

Chapter 5

Summary and Conclusion

In summary no significant enhancement in diffusion was observed in case of enzyme

immobilized Janus particles in substrate solution Presence of a linker between enzyme

and particle did not increase the diffusion coefficient of particles Alternative enzyme

immobilization methods need to be tested like thiolation of enzyme and subsequent

binding to gold surface of a Janus particle35

or expressing the enzyme with polyhistidine

or avidin tag67

This can result in higher enzyme activity upon immobilization and may

lead to enhanced diffusion of Janus particles

Predator-prey system of GOx and HRP immobilized particles showed some promising

results Aggregation of particles and formation of fan-shaped structures by TMB

oxidation were observed under optical microscope when GOx and HRP immobilized

particles glucose and TMB were present in the system The size of the fan-shaped

structures was dependent on the size of enzyme immobilized particles This can be

indicative of some kind of interaction between the GOx and HRP immobilized particles

No conclusive aggregation of GOx and HRP immobilized particles was observed in

absence of TMB Better imaging techniques are required to study the interaction of GOx

and HRP immobilized particles at the unstable liquid-air interface where majority of the

reaction takes place Other enzyme cascade systems not requiring gaseous reactants like

glycogen synthase and glycogen phosphorylase should be tested for similar activity

53

Ag-TiO2 particles were found to exhibit autonomous motion in the presence of hydrogen

peroxide solution The motion was momentarily accelerated upon exposure to UV

radiation The particles showed a diffusion coefficient of 1491 μm2s in hydrogen

peroxide versus only 180 μm2s in DI water Upon subjecting particles to UV radiation in

presence of H2O2 diffusion coefficient increased to 5093 μm2s for a period of 4-5

seconds In addition to linear motion rotating motion could also be observed in some

particle aggregates Further development of these aggregate structures can lead to

formation of rotors at nanoscale Higher concentration of silver-titania particles in

hydrogen peroxide displayed aggregation in presence of UV light and disaggregated

when UV light was switched off This may be attributed to the predator-prey kind of

interaction between silver and titania where oxygen and water that are produced by

silver by decomposition of hydrogen peroxide are used up by titania when subjected to

UV radiation This promises to be an interesting system and needs to be further

investigated to explore its properties

54

References

1 Paxton WF Sundararajan S Mallouk TE Sen A Angew Chem Int Ed Engl

2006 45 5420-5429

2 Golestanian R Liverpool TB Adjari A Phys Rev Lett 2005 94 220801

3 httpwwwsilvermedicineorgh2o2html

4 Rami Pedahzur Ovadia Lev Badri Fattal Hillel Shuval Water Science and Technology

1995 31(5-6) 123-129

5 W Went F American Scientist 1968 56

6 Adriano Cavalcanti Tad Hogg Bijan Shirinzadeh IEEE MHS 2006 International

Symposium on Micro-NanoMechatronics and Human Science 2006 226-232

7 EM Purcell American Journal of Physics 1977 45 3-11

8 Golestanian R Phys Rev Lett 2010 105 018103(1)-018103(4)

9 S S Dukhin Z R Ulberg G L Dvornichenko B V Deryagin Russian Chemical

Bulletin 1983 31(8) 1535-1544

10 Dukhin AS Goetz PJ Ultrasound for characterizing colloids Particle sizing zeta

potential rheology Elsevier 2002 59

11 HJKeh HJTu Trends in Colloid and Interface Science XIV Progress in Colloid and

Polymer Science 2000 115 174-180

12 D Zinemanas A Nir International Journal of Multiphase Flow 1995 21(5) 787-800

55

13 Stephen Ebbens Jonathan Howse Soft matter 2010 6 726-739

14 J Vicario R Eelkema W R Browne A Meetsma R M La Crois B L Feringa

Chem Commun 2005 31 3936

15 Davide Pantarotto Wesley R Browne Ben L Feringa Chem Commun 2008 1533 -

1535

16 Lisa A Cameron Matthew J Footer Alexander van Oudenaarden Julie A Theriot

PNAS 1999 96(9) 4908-4913

17 Ali Najafi Ramin Golestanian Phys Rev E 2004 69 062901

18 Jinseok Kim Jungyul Park Sungwook Yang Jeongeun Baek Byungkyu Kim Sang

Ho Lee Eui-Sung Yoon Kukjin Chun Sukho Park Lab Chip 2007 7 1504 - 1508

19 Kurt M Dubowski Clinical Chemistry 2008 5411 1919ndash1920

20 Spector DA Yang Q Wade JB Am J Physiol Renal Physiol 2007 292(1) 467-474

21 Rhee SG Chang TS Bae YS Lee SR Kang SW J Am Soc Nephrol 2003 14(8-3)

211-215

22 Sakaue T Kapral R Mikhailov A S Eur Phys J B 2010 75 381ndash387

23 Muddana H S Sengupta S Mallouk T E Sen A Butler P J J Am Chem Soc

2010 132 (7) 2110ndash2111

24 Baker CJ Deahl K Domek J Orlandi EW Arch Biochem Biophys 2000 382(2) 232-

237

25 V Vojinovic RH Carvalho F Lemos JMS Cabral LP Fonseca BS Ferreira

Biochemical Engineering Journal 2007 35 126-135

56

26

OV Lebedeva NN Ugarova Russian Chemical Bulletin 1996 45(1) 18-25

27 Nigel C Veitch Phytochemistry 2004 65(3) 249-259

28 Kenneth H Petersen Journal of Immunological Methods 2009 340(1) 86-89

29 httpwwwbrenda-enzymesorgphpresult_flatphp4ecno=11117

30 Goldenberg L M Wagner J Stumpe J Paulke B-R Goumlrnitz E Langmuir 2002 18

5627-5629

31 httpwwwgoldbiocompdf3761-Protocol201pdf

32 Av Rovisco Pais Lisboa Portugal T amp D Technology West Yorkshire UK Journal

of Molecular Catalysis B Enzymatic 2004 28(2-3) 129-135

33 httpwwwphysicsnyuedugrierlabmethodsnode11html

34 Yoon-Mee Lee1 O-Yul Kwon Yeo-Joon Yoon Keungarp Ryu Biotechnology Letters

2006 28 39ndash43

35 JC Pyun SD Kim JW Chung Analytical Biochemistry 2005 347(2) 227-233

36 George H Pimbley Jr Mathematical Biosciences 1974 20(1-2) 27-51

37 Andy Fenton Sarah E Perkins Parasitology 2010 137 1027-1038

38 Mead James G Brownell Robert L Mammal Species of the World A Taxonomic and

Geographic Reference (3rd ed) Johns Hopkins University Press 2005 2 2142

57

39

Krause KH Schweiz Med Wochenschr 2000 130(4) 97-100

40 Irina Kareva Faina Berezovskaya Carlos Castillo-Chavez Journal of Biological

Dynamics 2010 4(4)315 - 327

41 Delaittre G Reynhout I Cornelissen J Nolte R Chemistry ndash A European Journal

2009 15 12600ndash12603

42 C Buller DH Johnson RC Tschumper Investigative Ophthalmology amp Visual Science

1990 31 2156-2163

43 Robert M Mazo Brownian Motion Fluctuations Dynamics and Applications Oxford

Science Publications 2009 57

44 Vivek K Mutalik KV Venkatesh Theoretical Biology and Medical Modelling 2005 2

19

45 Satoshi Nakamura Kunimasa Koga Biochemical and Biophysical Research

Communications 1977 78(2) 806-810

46 Krishnananda Chattopadhyay Shyamalava Mazumdar Biochemistry 2000 39 (1)

263ndash270

47 Satoshi Nakamura Yasuyuki Ogura J Biochem 1968 63 (3) 308-316

48 van Dongen SF Nallani M Cornelissen JJ Nolte RJ van Hest JC Chemistry 2009

15(5) 1107-1114

49 Ronald Bentley Methods in Enzymology 1955 1 340-345

50 Vartiainen J Raumlttouml M Paulussen S Packaging Technology and Science 2005

18 243ndash251

58

51 GA Sherbeny AA Shindia YMMM Sheriff International Journal of Agriculture

and Biology 2005 7

52 D Rando G-W Kohring F Giffhorn Appl Microbiol Biotechnol 1997 48

53 Worthington enzyme manual Worthington Biochemical Corporation

httpworthington-biochemcomgopdefaulthtml

54 httpwwwbrenda-enzymesorgphpresult_flatphp4ecno=11117

55 httpwwwoxygenphwatercom

56 P Boon Chock Sue Goo Rhee and Earl R Stadtman Annu Rev Biochem 1980 49

813-841

57 Sybil Golden P A Wals Joseph Katz Analytical Biochemistry 1977 77(2) 436-445

58 R L Baehner S K Murrmann J Davis R B Johnston Jr J Clin Invest 1975 56(3)

571ndash576

59 I Bejarano M P Terroacuten S D Paredes C Barriga A B Rodriacuteguez J A Pariente

Molecular and Cellular Biochemistry 2007 296(1-2) 77-84

60 Michael C Moulton Laura K Braydich-Stolle Mallikarjuna N Nadagouda Samantha

Kunzelman Saber M Hussain Rajender S Varma Nanoscale 2010 2 763-770

61 Goya DV Matijevic E Colloids and Surfaces A 1999 146 139

62 Velikov KP Zegers GE van Blaanderen A Langmuir 2003 19 1384

63 Goldenberg L M Wagner J Stumpe J Paulke B-R Goumlrnitz E Langmuir 2002 18

5627-5629

59

64

Banerjee S Gopal J Muraleedharan P Tyagi A K Raj B Curr Sci 2006

901378ndash1383

65 Bogomolov V N Kudinov E K Firsov Y A Sov Phys Solid State 1968 35 555

66 Kudo A Miseki Y Chem Soc Rev 2009 38 253ndash278

67 Gaj T Meyer SC Ghosh I Protein expression and Purification 2007 56 54-61

13

211 Horseradish peroxidase

Figure 2 3 Horseradish peroxidase

Horseradish peroxidase belongs to the group of peroxidases which are a group of heme-

containing oxidoreductases that act on peroxide as hydrogen donor25

It is a redox

enzyme which contains a protoheme a complex of trivalent iron with 1358-

tetramethyl-24-divinylporphin-67-dipropionic acid as a prosthetic group It is a 44 kDa

protein which includes 33900 Da polypeptide chain bound with eight oligosaccharide

chains The carbohydrate chains improve the stability of the enzyme by protecting it

against radicals formed in the course of the reaction it catalyzes26

Horseradish

peroxidase is found in horseradish and is used widely in bioanalytical applications such

as ELISA and Western blots It decomposes hydrogen peroxide into water and oxygen

while simultaneously oxidizing another substrate27

The oxidizable substrates can be

chromogenic substrates such as 22rsquo55rsquo-tetramethyl-benzidine (TMB) and 33-

diaminobenzidine (DMB) or chemoluminescent substrates like SuperSignal and ECL

luminal28

14

Figure 2 4 Reaction catalyzed by horseradish peroxidase

The functional parameters for horseradish peroxidase include Km of 015 mM and kcat of

790 s-1

at an optimum temperature of 20 ordmC and an optimum pH of 6429

Some of the

inhibitors of HRP are 110-phenanthroline aminotriazole and bromine

22 Experimental

221 Materials and methods

Uniform carboxyl modified polystyrene microspheres 05 microm in diameter were obtained

from Polysciences Inc The size of the purchased microspheres was verified by Field

Emission Scanning Electron Microscopy (FESEM) Peroxidase Type I from

Horseradish N-(3-dimethyl-aminopropyl)-Nrsquo-ethylcarbodiimide (EDC) and hydrogen

peroxide solution 50 wt in water were purchased from Sigma-Aldrich N-

hydroxysuccinimide (NHS) ge 97 was purchased from Fluka All chemicals were of

reagent grade and used without further purification Nanopure deionized water was used

for all experiments and preparation of solutions All measurements were made at room

15

temperature (25 oC) unless mentioned otherwise The UV-Visible spectroscopy

measurements were performed using Agilent 8453A diode-array UV-Visible

Spectrophotometer with ChemStation UV-Vis Software Diffusion coefficient

measurements were performed using Nanosight LM10

222 Synthesis of Janus particles

A monolayer of particles was prepared according to the procedure (Figure 2 5) by

Goldenberg L et al30

Glass slides were placed on the bottom of a petridish Water was

put into the petridish so that the entire slide was immersed in water A thin layer of

hexane was added on top of water in the petridish A suspension of microspheres in

ethanol (5 ww) was introduced to the water-hexane interface After the monolayer was

formed the glass slide was lifted up through the monolayer with tweezers The

microsphere-covered glass slides were left to dry overnight at room temperature

Following drying a thin film of silica was evaporated on the glass slides in Semicore

evaporator The spheres were partially coated with 30 nm of silica Particles were

removed from the glass slide by gentle sonication The polystyrene-silica (PS-SiO2)

Janus particles thus obtained were washed in deionized water several times before

further immobilization steps were performed FESEM was used to confirm synthesis of

Janus particles

16

Figure 2 5 Preparation of PS-SiO2 Janus particles by evaporation

223 Immobilization of enzymes using EDCNHS coupling method

1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide and N-hydroxysulfosuccinimide

(EDCNHS) coupling procedure was followed for immobilizing horseradish peroxidase

type I on 05 μm PS-SiO2 Janus particles (Figure 2 6) In this process EDC reacts with a

carboxyl groups on the Janus particles to form an amine-reactive O-acylisourea

intermediate If this intermediate does not encounter an amine it can hydrolyze and

regenerate the carboxyl group In the presence of NHS EDC can be used to convert

carboxyl groups to amine-reactive sulfo-NHS esters which are much more stable and do

not readily hydrolyze to regenerate carboxyl groups In the current experiment

immobilization was performed in the presence as well as absence of a long spacer

17

molecule (linker) between Janus particles and enzymes The chemical 6-aminohexanoic

acid was used as the linker (Figure 2 7) HRP immobilized PS-SiO2 Janus particles were

stored in deionized water until required for further experiments

Figure 2 6 EDCNHS coupling method for immobilization of enzyme on carboxylated

polystyrene particles

Figure 2 7 Immobilization of enzyme on Janus particles with and without 6-

aminohexanoic acid linker

18

224 Enzyme assay

Assay for activity of HRP was performed using a spectrophotometric method which

involved using 4-aminoantipyrene as hydrogen donor3132

The reaction rate was

determined by measuring increase in absorbance at 510 nm resulting from the

decomposition of hydrogen peroxide One unit represents the decomposition of 1 μM of

hydrogen peroxide per minute at 25 degC and pH 70 under the specified conditions

The spectrophotometer was set to the wavelength of 510 nm and temperature of 25 degC

Phenol4-aminoantipyrene solution (14 mL) and 00017 M H2O2 solution (15 mL) were

pipetted into each cuvette The cuvette was incubated for 3-4 mins at 25degC and blank was

established A 01 mL solution of diluted enzyme was added to the above mixture and

absorbance was recorded for 4-5 mins Standard graphs of absorbance versus time were

drawn for known concentration of the free enzyme and compared to that of the

immobilized enzyme on the Janus particles The estimate of the amount of enzyme

present on the Janus particles was deduced from the difference between the quantity of

the free enzyme previously added for immobilization and the amount that was measured

to be in the supernatant after some of the enzyme became attached to the Janus particles

The experiment was carried out in triplicate to assess reproducibility

19

The formula used to calculate the activity of enzyme is

where ∆Amin denotes the change in absorbance per minute

225 Diffusion coefficient measurements using Nanosight LM10

Diffusion coefficients for HRP immobilized particles were measured using Nanosight

LM 10 nanoparticle tracking instrument with Nanoparticle Tracking Analysis (NTA) 20

software in the presence of 003 H2O2 solution as the substrate and also in the presence

of only deionized water The videos were recorded for 30 seconds at the rate of 30 frames

per second Each experiment was carried out in triplicate to assess reproducibility

Particles

visualization

zone

Microscope with

LM 10 unit

LM 10 unit

Figure 2 8 Nanosight LM 10 instrumentation

20

Nanosight LM 10 (Figure 2 8) generates videos of population of moving nanoparticles in

a liquid when illuminated by laser light The particles which pass through the laser beam

path are seen by the instrument as small points of light undergoing Brownian motion

(Figure 2 9a)

The videos are further analyzed by NTA (Nanoparticle tracking analysis) software

(Figure 2 9b) which identifies and tracks each particle independently on a frame-by-

frame basis The software also calculates and subtracts any bulk flow in the system The

mean square displacement is calculated for each particle as long as it is visible If the

particle blinks that is if it disappears momentarily and then reappears on the screen the

software will consider the same particle as two separate ones All analysis data is

exported to the excel files in terms of diffusion coefficients of the tracked particles

B A

Figure 2 9 NTA video analysis (a) Particles as they are seen on the screen during

experimentation in Nanosight LM10 (b) NTA screen as the particles are being analyzed for

their diffusion coefficients

21

23 Results

231 Enzyme assay

The activity of free HRP enzyme reported by Sigma Aldrich is 150 unitsmg However

the activity of HRP upon analysis was found to be 52158 unitsmg which is much lower

than the reported value The activity of HRP was found to be further lowered to 2133

unitsmg when it was immobilized on the PS-SiO2 Janus particles in the presence of a

linker The following formula is used to calculate enzyme activity

where ΔAmin is the change in absorbance per minute

22

Figure 2 10 Graph for the assay of solution of free enzyme containing 05mgL of HRP

ΔAmin = 01716 Unitsmg of free enzyme = 52158

During EDCNHS coupling 04 mg enzyme was added to 1 ml of 025 vv suspension

of Janus particles Thus initial concentration of enzyme in the mixture was 04 mgml

Assuming retained activity of the enzyme in supernatant solution the amount of enzyme

in supernatant was calculated to be 03868 mgml from the assay Amount of enzyme

functionalized on the Janus particles was calculated as the difference between enzyme

previously added for immobilization (04 mgml) and the enzyme present in supernatant

(03868 mgml) and was found to be 00132 mgml Enzyme assay was performed on the

enzyme immobilized Janus particles to estimate the activity of immobilized enzyme

(Figure 2 11) and was found to be 2133 unitsmg

y = 0002x + 00516

0

01

02

03

04

05

06

07

08

0 50 100 150 200 250 300 350 400

Ab

sorb

an

ce

51

0 n

m

Time (sec)

Assay of free enzyme HRP

23

Figure 2 11 Graph for the assay of of immobilized enzyme on 05 μm Janus particles

(~025 vv) ΔAmin = 01853 Unitsmg of free enzyme = 2133

232 Diffusion coefficient measurements

No significant increase was observed in diffusion coefficient of HRP-immobilized Janus

particles in the presence of H2O2 as the substrate Similar results were obtained for

diffusion coefficient measurement with and without linker moieties bridging the enzymes

and particles(Table 2 1 Table 2 2) Thus the linker did not seem to assist in increasing

the particle diffusion by improving or retaining the activity of the enzyme

y = 00015x + 00953

0

01

02

03

04

05

06

07

0 50 100 150 200 250 300 350 400

Ab

sorb

an

ce

510 n

m

Time (sec)

Assay of HRP immobilized 05 μm Janus particles

24

Table 2 1 Diffusion coefficient measurements of HRP immobilized Janus particles

without linker

HRP immobilized

Janus particles in

003 H2O2

Control - Janus

particles in

003 H2O2

Control - HRP

immobilized Janus

particles in DI water

Diffusion coefficient

(10-10

cm2s)

1359416 1051777 1308675

Standard deviation 4534801 4122075 4918597

Table 2 2 Diffusion coefficient measurements of HRP immobilized Janus particles in

the presence of linker

HRP immobilized

Janus particles in

003 H2O2

Control - Janus

particles in

003 H2O2

Control - HRP

immobilized Janus

particles in DI water

Diffusion coefficient

(10-10

cm2s)

13838 1339829 1407891

Standard deviation 5644692 5649364 5797218

25

Figure 2 12 Diffusion coefficient measurements of PS-SiO2 Janus particles without and

with 6-aminohexanoic acid linker

24 Discussion

To compare the diffusion coefficients observed during the experiments theoretical

diffusion coefficient was calculated using following equation33

r

kTD altraslation

6 Equation 21

where r is the hydrodynamic radius of particle Dtranslational is translational diffusion

coefficient of the particle T is temperature of the system η is the viscosity of fluid

through which the particles are moving and k is the Boltzmann constant From FE-SEM

images the size of the particles used was determined to be 450 nm For 450 nm diameter

0

20

40

60

80

100

120

140

160

Without linker With linker

Dif

fusio

n c

oeff

icie

nt

(10

-10 c

m2s

)

HRP immobilized 05 μm Janus particles - Diffusion coefficient measurements

HRP immobilized Janus particles in 003 H2O2

Control - Janus particles in 003 H2O2

Control - HRP immobilized Janus particles in DI water

26

particle Dtranslational is equal to 109 μm2s which is comparable to the diffusion

coefficient measured for the enzyme immobilized particles

HRP was found to be active on the Janus particles however the activity of the enzyme

was reduced upon immobilization Lower enzyme activity can be due to the coupling

method used which in this case was EDCNHS covalent binding However EDCNHS

coupling method has been shown to significantly retain the enzyme activity when the

enzyme is immobilized on carboxylated multi-walled carbon nanotubes under appropriate

conditions34

Hence EDCNHS binding method by itself is not believed to be

responsible for reduction of enzyme activity upon immobilization

Direct binding of enzyme to the Janus particle may lead to reduction in the enzyme

activity because of space constraints for the enzyme and difficulty for the passage of

substrate to the active site of the enzyme due to stearic hindrance Hence a linker was

inserted between the enzyme and Janus particle during enzyme immobilization However

inserting a linker between the enzyme and the particle did not lead to enhanced diffusion

of Janus particles or increase in enzyme activity of immobilized enzyme

Lack of enhanced diffusion can be firstly attributed to the reduction in enzyme activity

upon immobilization Enzyme activity may be retained by modifying the conditions

during EDCNHS coupling or changing the binding method altogether The degree of

immobilization of HRP on carboxylated Janus particles can possibly be affected by

several factors such as temperature the concentration of the coupling reagent EDC

27

concentration HRP concentration in solution and incubation time34

Different

combinations of these factors can be tested to find optimum conditions for

immobilization of HRP Alternate binding method can involve thiolation of HRP by the

covalent conjugation of mercaptopropionic acid and then the thiolated HRP can then be

attached on the gold surface of a polystyrene-gold Janus particle35

Thus further

modifications can be made to the system in order to retain enzyme activity on the

immobilized particles and thereby power nanomotors using enzymes

28

Chapter 3

Predator-prey system of enzyme immobilized particles

31 Introduction

Predator-prey systems are widely found in nature and play a major role in biological

processes3637

Predator-prey systems include all the interactions in which one organism

pursues andor consumes another organism One familiar predator-prey system at the

macroscale is that of a tiger chasing and consuming a deer Another not so familiar one

includes Baleen whales which eat millions of microscopic planktons at once38

At the

microscale such kind of interactions can be observed in the case of phagocytes in the

body which are capable of engulfing foreign microorganisms and killing them with a

combination of microbicidal systems39

In the case of cancer the tumor is the prey and

the mature myeloid cells in the body are the predator However the tumor here has a

defense mechanism against the predator in which the tumor produces cytokines which

inhibits the maturation of the myeloid cells and thereby prevents predation40

One similarity that can be observed in all these systems is that the prey possesses some

attribute which is desired by the predator which makes the predator chase the prey A

similar relationship is seen in case of enzymes which are involved in cascade reactions41

In cascade reactions product synthesized by one enzyme is the reactant required by

another enzyme to carry out its own catalytic reaction As such there is a predator-prey

29

kind of dependence between the two enzymes which may cause one enzyme to chase the

other enzyme when placed in suitable substrate solutions However the mechanism

hypothesized for such a system is totally different than the mechanism involved in

biological systems In biological systems such as bacterial chemotaxis or phagocytes

chasing foreign particles in human body complex biochemical reactions are responsible

for the predator-prey behavior42

In the proposed enzyme cascade system there are no

complex biochemical reactions involved The principle hypothesized for predator-prey

behavior of enzyme cascade reactions can be explained as follows Motion of synthetic

motors at nanoscale involves two components translational diffusion and rotational

Brownian diffusion At very short time scales of less than 1Dr where Dr (s-1

) is

rotational Brownian diffusion coefficient the motion of particles is unidirectional For

longer time scales of greater than 1Dr the direction of motion is random giving

poweredactive diffusion43

If the chemical homogeneity is broken that is when there is

higher concentration of a substrate present in one direction of the enzyme the enzyme

might acquire a subtle bias which may lead to a spatial aggregation of the enzymes based

on the substrate concentration gradient The bias can be a result of the relationship

between the speed of enzyme diffusion and substrate concentration where higher

substrate concentration lead to higher enzyme diffusion As a result in an enzyme

cascade system one enzyme will migrate towards higher concentration of substrate

which is present near the other enzyme thus giving rise to a predator-prey behavior

Such enzyme cascades are widely found in signaling networks and metabolic pathways in

living cells and can be used to produce predator-prey like nanomotors systems44

The

30

enzymes being used in the present study are horseradish peroxidase and glucose oxidase

Both enzymes are extremely stable4546

Glucose is the substrate for glucose oxidase

which is widely available and highly stable47

Horseradish peroxidase acts on hydrogen

peroxide It is involved in a cascade reaction with glucose oxidase which produces

hydrogen peroxide by converting glucose into gluconic acid (Figure 3 1)48

Figure 3 1 Concept of particle aggregation due to cascade reactions of immobilized

enzymes

In this experiment glucose oxidase (GOx) and horseradish peroxidase (HRP) were

immobilized on polystyrene carboxylate beads of different sizes using EDCNHS

coupling method Glucose and 3355-Tetramethylbenzidine (TMB) were dissolved

together in water and this solution was used as the substrate solution All the components

were present in a single system in DI water Aggregation of particles and precipitation of

oxidized TMB from the solution was expected to be observed when all these components

were present in such a system

Properties of horseradish peroxidase have been discussed in the previous chapter (Section

211 Horseradish peroxidase)

GOxGOx HRPHRP

Glucose + O2Gluconic acid + H2O2

TMB TMB oxidizedBlue precipitate

H2O

Aggregation

31

311 Glucose oxidase

Figure 3 2 Glucose oxidase

Glucose oxidase (GOx) belongs to the group of oxidoreductases and is also called

glucose aerodehydrogenase49

It is obtained from fungi and has been identified as an

antimicrobial enzyme50

GOx binds to β-D-glucopyranose and catalyzes the oxidation

reaction to from gluconolactone while simultaneously reducing oxygen to hydrogen

peroxide51

GOx has high substrate affinity and specificity high stability and high

turnover number which makes it a suitable biocatalyst for industrial applications52

Figure 3 3 Redox reaction of glucose catalyzed by glucose oxidase53

32

GOx is a glycoprotein with molecular weight of 155 kDa and consists of two identical

polypeptide subunits linked together by a disulfide bond The optimum pH for GOx is

55 though it is stable in a broad pH range of 4 to 7 and can be stored for a long period of

time51

The functional parameters for glucose oxidase include Km of 151 mM and kcat of

337 s-1

at temperature of 20 ordmC and pH of 754

Some of the inhibitors of GOx are 2-

Deoxy-D-glucose adenine nucleotides and NaNO3

32 Experimental

321 Materials and methods

Uniform carboxyl modified polystyrene microspheres 05 microm in diameter were obtained

from Polysciences Inc The size of the purchased microspheres was verified by FESEM

Peroxidase Type I from horseradish glucose oxidase Type II-S from Aspergillus niger

N-(3-dimethyl-aminopropyl)-Nrsquo-ethylcarbodiimide (EDC) and Hydrogen peroxide

solution 50 wt in water were purchased from Sigma-Aldrich N-hydroxysuccinimide

(NHS) was purchased from Fluka D-Glucose (99) was purchased from VWR

International Secure-Seal imaging spacers (9mm diameter) for glass slides were obtained

from Sigma Aldrich All chemicals were of reagent grade and used without further

purification Nanopure deionized water was used for all experiments and preparation of

solutions All measurements were made at room temperature (25 oC) unless mentioned

otherwise The UV-Visible measurements were performed using Agilent 8453A diode-

array UV-Visible Spectrophotometer with ChemStation UV-Vis Software Optical

33

microscopy experiments were performed using Axiovert 200 MAT microscope by Carl

Zeiss Diffusion coefficient measurements were obtained using Nanosight LM10

322 Optical microscopy experiments

The enzyme substrate system was observed under optical microscope to check for

aggregation and precipitation due to cascade reactions Several such systems were

studied Each system in general consisted of GOx and HRP immobilized on different

polystyrene particles The substrate solutions consisted of 01 M glucose and different

concentrations of TMB solution The systems were studied in the presence of all the

constituents and in the absence of glucose GOxHRP and TMB Systems consisting of

different sized HRP immobilized particles in substrate solution of 003 hydrogen

peroxide and TMB were also studied Videos were recorded over a period of 24 hours to

observe changes in the system Each experiment was performed atleast thrice to assess

reproducibility of results

323 Diffusion coefficient measurements using Nanosight LM10

Diffusion coefficient of GOx and HRP immobilized particles were measured using

Nanosight LM10 nanoparticle tracking instrument with Nanoparticle Tracking Analysis

(NTA) 20 software Diffusion coefficients were measured for different systems namely

GOx particles in 01 M glucose and oxygen both GOx and HRP functionalized particles

in 01 M glucose and oxygen along with controls for the individual systems Videos were

34

recorded for 30 seconds at the rate of 30 frames per second Each experiment was carried

out in triplicate to assess reproducibility

33 Results

331 Optical microscopy

According to the enzyme cascade model precipitation of TMB was possible only upon

the interaction of GOx and HRP immobilized particles in presence of appropriate

substrate solution Hence in the first set of experiments precipitation of TMB was used

as an indication of the type of interaction between GOx and HRP immobilized particles

Fan-shaped structures were observed when both HRP and GOx immobilized particles

were present in a substrate solution consisting of glucose and TMB These structures

started forming as a single strand and then branched out and intertwined to form the fan-

shaped structures (Figure 3 4)

Table 3 1 Observations for formation of fan-shaped structures in enzyme cascade

system in presence of different components

GOx HRP Glucose TMB Observation

Yes Yes Yes Yes

Fan-shaped structures observed particles associated with

the fan-shaped structures

No Yes Yes Yes No fan-shaped structures

Yes No Yes Yes No fan-shaped structures

Yes Yes No Yes No fan-shaped structures

Yes Yes Yes No No fan-shaped structures

35

Fan-shaped structures can also be observed in the system consisting of only HRP

immobilized particles in substrate solution of hydrogen peroxide and TMB The size of

the fan-shaped structures depends on the size of the enzyme immobilized particles Free

enzymes show extremely tiny structures which are difficult to view under the optical

microscope

Figure 3 4 Formation of fan-shaped structures when both HRP and GOx

immobilized particles were present in a substrate solution consisting of glucose and

TMB A In the initial stages single strands form on the surface of liquid (5Ox) B

Single strands branch out and intertwine to form fan-shaped structures (50x) C Many

such structures form and settle down after 20 minutes (20x)

A

C

B

36

Experiments were performed in the absence of TMB that is the system consisted of GOx

and HRP immobilized particles in glucose substrate solution In the absence of TMB the

particles were expected to move closer to each other without any precipitation due to the

cascade reaction However there was no conclusive aggregation of particles observed in

the system

Free Enzyme 005 μm HRP - immobilized particle

10 μm HRP - immobilized particle 05 μm HRP - immobilized particle

Figure 3 5 Size dependence of fan-shaped structures on the size of HRP -

immobilized particles (20x)

37

Table 3 2 Observations for particle aggregation in enzyme cascade system in presence

and absence of TMB

Glucose GOx HRP TMB Observation

Present Present Present Present Aggregation and precipitate

Present Present Present Absent No conclusive aggregation

Present Absent Present Present No aggregation or ppt

Absent Present Present Present No aggregation or ppt

Present Present Absent Present No aggregation or ppt

332 Diffusion coefficient measurements

Diffusion coefficients were measured for GOx immobilized particles of 05 μm diameter

in substrate solution of 01 M glucose and oxygen Diffusion coefficients of particles

were also measured for system consisting of GOx and HRP functionalized particles in

glucose and oxygen Each experiment was done in triplicate to assess reproducibility

Nanosight visualization chamber being a closed chamber oxygen was purged into all the

solutions before starting the experiments to ensure sufficient supply of oxygen for

catalytic reaction of GOx

38

Table 3 3 Diffusion coefficients for 05 μm GOx immobilized particles in 01 M

glucose purged with O2

Mean

GOx

particles in

01 M

glucose

Control - GOx

particles in DI

water

Control - GOx

particles in 01 M

glucose without

oxygen purging

Control -

particles in 01

M Glucose

Diffusion

coefficient

(x10-10

cm2s)

13316 12888 13827 13702

Standard

deviation 6307 4926 4424 7468

Table 3 4 Diffusion coefficients for GOx and HRP immobilized particles in 01 M

glucose and purged with O2

Mean

GOx- HRP-

functionalized

particles in 01 M

glucose

Control ndash Gox-

HRP- functionalized

particles in DI water

Control - particles

in 01 M glucose

Diffusion coefficient

(x10-10

cm2s)

13005 14434 13702

Standard deviation 5263 6622 7468

No significant increase was observed in diffusion coefficient of enzyme immobilized

particles in any of the systems

39

34 Discussion

Formation of fan-shaped structures due to patterned deposition of oxidized TMB

indicates some kind of interaction between GOx and HRP immobilized particles Particle

diameter dependence of the size of structures can be explained with a simple model in

Figure 3 6 A larger particle will have a higher surface area as compared to a smaller

particle Hence more enzyme is immobilized on the larger particle As a result higher

quantity of TMB is oxidized and precipitated by the larger particle as compared to the

smaller particle This leads to thicker strands of fan-shaped structures eventually scaling

up the whole system to give bigger fan-shaped structures

The exact reason for the formation of such fan-shaped structures which are dependent on

particle size could not be determined It is hypothesized that hydrogen peroxide andor

Figure 3 6 Model for size dependence of fan-shaped structures on the particle

diameter of enzyme immobilized particles Larger the particle more surface area is

available for precipitation of TMB which eventually leads to bigger fan-shaped

structures

Larger particle Smaller particle

40

TMB gradient in the substrate solution might be responsible for such structures If there

is higher concentration of H2O2 on one side of the particle and assuming an even

concentration distribution of TMB there will be selective precipitation in the direction of

H2O2 When several particles come in the vicinity of higher concentration of H2O2 the

precipitation lines will converge to give fan-shaped structures (Figure 3 7)

Figure 3 7 Hypothesis for formation of fan-shaped structures

In the system consisting of GOx immobilized particles HRP immobilized particles

glucose and TMB it was observed that the fan-shaped structures formed at the liquid-air

interface and eventually settled down to the bottom of the glass slide This makes sense

as oxygen is necessary for the catalytic reaction of conversion of glucose to gluconic acid

and hydrogen peroxide which takes place on the surface of GOx immobilized particle As

a result H2O2 is primarily formed at the liquid-air interface and is then used by HRP for

precipitation of TMB

Following the observations that precipitation of TMB occurs at the surface GOx and

HRP immobilized particles are also expected to interact at the surface in the absence of

TMB Due the cascade reaction it was expected that HRP immobilized particles will be

41

attracted to GOx immobilized particles in appropriate substrate solution Experiments

were carried out to visualize inter-particle interactions at the liquid-air interface

However liquid-air interface not being a stable system to image the precise interaction

of GOx and HRP immobilized particles could not be determined Significant external

disturbances were involved at the interface To enable the cascade reaction in the bulk of

the liquid oxygen was purged into the reaction system before starting the experiment

However this did not lead to significant reaction in the bulk of the liquid and the reaction

prominently took place at the liquid-air interface where the oxygen concentration was

higher

In order to observe the interaction of GOx and HRP immobilized particles better imaging

techniques need to be used to observe diffusion of particles in absence of external

disturbances at the liquid-air interface Large quantity of oxygen needs to be purged into

the system and retained to make the reaction more pronounced in the bulk of the substrate

solution Higher concentration of oxygen in water may also be achieved by using oxygen

enriched water which is commercially available55

Alternatively a different enzyme cascade system may be used which does not require a

gaseous substrate56

For example glycogen synthase synthesizes glycogen from glucose-

1-phosphate and glycogen phosphorylase breaks down glycogen to glucose-1-

phosphate57

This system does not require a gaseous substrate and might be able to

overcome the deficiencies of GOx and HRP enzyme cascade system

42

Chapter 4

Silver-titania nanomotors

41 Introduction

Bimetallic nanowires made of platinum and gold have been developed by WPaxton

etal1 The rods move autonomously in a solution of hydrogen peroxide In the present

study silver-titania (Ag-TiO2) particles are being synthesized and investigated for motion

in presence of hydrogen peroxide and UV light With proper control of conditions and

UV light trigger it can be possible to control the speed and direction of motion of these

nanoparticles

Hydrogen peroxide is produced in the human body by phagocytes as an immune response

to foreign particles and pathogens5859

Also silver nanoparticles have been shown to be

biocompatible and capable of interacting with cells in human body without disrupting

cellular function60

Similarly it has been demonstrated that titania is compatible with

different cells DNA and proteins in human body and does not cause toxic effects Hence

silver-titania nanomotors which swim in hydrogen peroxide may open interesting

avenues in applications like targeted drug delivery in those parts of the body where

hydrogen peroxide is present

43

42 Experimental

421 Materials and methods

Silver nitrate (999995) was purchased from Alfa Aesar Gum arabic was purchased

from MP Biomedicals Hydrogen peroxide solution 50 wt in water and ascorbic acid

were purchased from Sigma-Aldrich Nanopure deionized water was used for all

experiments and preparation of all solutions Secure-Seal imaging spacers (9 mm

diameter) were obtained from Sigma Aldrich All experiments were carried out at room

temperature of 25ordmC unless otherwise mentioned Ag-TiO2 particles were synthesized by

evaporation of titania in Semicore electron beam evaporator Particles were characterized

using field-emission scanning electron microscopy Optical microscopy experiments

were performed using Axiovert 200 MAT microscope by Carl Zeiss Particles were

tracked using Physvis particle tracking software (Figure 4 1) in which the position

information of a particle on screen is stored on a frame by frame basis The data is stored

in a dat file as X and Y co-ordinates of particles tracked on the screen This information

can be converted to diffusion coefficients of the particles by further manipulation using

MathWorks-MATLABreg software

44

Figure 4 1 Physvis particle tracking software

422 Synthesis of silver colloidal particles

Silver particles were synthesized by a nucleationgrowth type reaction developed by

Goya and Matijevic61

and modified by Velikov62

Silver nitrate was used as precursor

metal salt for synthesis of silver particles Solution A was 75 ml of 50 mM AgNO3 and

035 by weight gum arabic Solution B was 75 mL of 100 mM 1-ascorbic acid and

035 by weight gum arabic Solution B was rapidly added to solution A in a 250 mL

reaction flask The reaction flask was covered and particles were stirred for 48 hrs After

48 hours particles were allowed to settle for 2-3 hours in refrigerator and supernatant

was separated from the particles Particles were then rinsed with DI water and centrifuged

thrice to obtain pure silver particles Zeta potential of these particles was reported to be

45

around -50 to -60 mV and the size was around 1μm The size of the particles can be

varied by varying the amounts of ascorbic acid and silver nitrate Using molar ratio of 31

of ascorbic acid silver nitrate can give particles of 200 nm diameter whereas ratio of

151 can give particles of upto 5 μm diameter

423 Preparation of silver-titania (Ag-TiO2) particles

A monolayer of silver particles was prepared according to the procedure by Goldenberg

et al63

Glass slides were placed on the bottom of a petridish Water was put into the

petridish so that the entire slide was immersed in water A thin layer of hexane was then

added on top of water in the petridish A suspension of silver particles in ethanol (5

ww) was slowly introduced to the water-hexane interface in the dish After the

monolayer was formed the glass slide was lifted up through the monolayer with tweezers

The silver particles-covered glass slides were left at room temperature to dry overnight

Following drying a thin film of titania was evaporated on the glass slides in Semicore

evaporator (Figure 2 7) The spheres were partially coated with a 10 nm film of titania

Ag-TiO2 particles were removed from the glass slide by gentle sonication FE-SEM was

used to characterize the particles (Figure 4 2) The thin titania layer was not easily

distinguishable due to roughness of the particles

46

Figure 4 2 FESEM image of Ag-TiO2 particles

424 Optical microscopy experiments

Imaging spacers were placed on a glass slide Suspension of Ag-TiO2 particles was

placed on the glass slide and the particles were allowed to settle Movement of particles

was observed in DI water and videos were recorded at 50x magnification of optical

microscope for 30 sec 05 H2O2 was added to the particle suspension on the slide

Videos were recorded at 50x ranging from 30 sec to 10 min to study the movement of

silver titania particles in hydrogen peroxide solution Particles in both DI water and

hydrogen peroxide were subjected to UV light and videos were recorded to study particle

dynamics in presence of UV radiation Particle tracking was performed using Physvis

particle tracking software Atleast 30 particles in every system were tracked for 10

seconds each Particle tracking data was obtained in terms of X and Y coordinates of

particles on screen This data was manipulated using MathWorks-Matlab software and

graphs of mean square displacement of particles versus time were plotted Diffusion

coefficients of particles were calculated from these graphs

47

43 Results and Discussion

Ag-TiO2 particles demonstrated autonomous motion in the presence of H2O2 No UV

light was required for this motion Motion of the particles was accelerated by addition of

fresh H2O2 to the system When subjected to UV radiation the particles displayed

momentary acceleration for 4-5 seconds

Figure 4 3 Mechanism of motion of Ag-TiO2 nanomotors

In the presence of H2O2 silver part of the Ag-TiO2 particle converts H2O2 to H2O and O2

This may lead to propulsion of the Ag-TiO2 particle due to bubble propulsion andor self-

diffusiophoresis In the presence of UV light additional reaction takes place on the

titania part of Ag-TiO2 particle which increases the velocity of the Ag-TiO2 particles

(Figure 4 4) This UV triggered photoactivity of titania is due to hole-electron separation

caused by photons having energy equal to or greater than the band gap of titania646566

Most of the holes-electrons recombine but a small fraction move to the surface and react

with the available redox species like water and oxygen to form protons (H+) superoxide

(O2-) and hydroxyl (OH) radicals

6566 This may lead to propulsion by diffusiophoresis

as well as electrophoresis

48

Figure 4 4 Silver-titania motors move faster when subjected to UV radiation

In addition to linear motion rotational motion is also observed in several particles and

particle aggregates Aggregation of particles can lead to a net circular propulsive force

which can rotate the aggregate as shown in Figure 4 5

Figure 4 5 Rotation due to particle aggregation of Ag-TiO2 particles

Particle tracking by Physvis software revealed a significant increase in diffusion

coefficient of Ag-TiO2 particles in hydrogen peroxide solution as compared to DI water

49

The Ag-TiO2 particles have a diffusion coefficient of 1491 μm2s in hydrogen peroxide

versus only 180 μm2s in DI water Thus the Ag-TiO2 particles move around more than

10 times their body length per second in hydrogen peroxide (Figure 4 6) When

subjected to UV light the particles experience a significant acceleration in hydrogen

peroxide which lasts only for a short duration of time (4-5 sec) after which the particles

return to their original velocity The diffusion coefficient of the particles during this UV

light trigger is found to be around 5093 μm2s UV light trigger does not have any visible

effect on the Ag-TiO2 particles in DI water

Table 4 1 Translational diffusion coefficients of Ag-TiO2 particles in hydrogen

peroxide and water

Mean 05 H2O2 solution DI water

Diffusion coefficient of Ag-TiO2 (μm2s)

without UV light

1491 180

Diffusion coefficient of Ag-TiO2 (μm2s)

in the presence of UV light

5093 113

50

Figure 4 6 Mean square displacement curves of Ag-TiO2 Janus particles in hydrogen

peroxide and DI water

When higher concentration (~30 vv) of Ag-TiO2 particles in hydrogen peroxide is

subjected to UV radiation the particles exhibit pattern formation (Figure 4 7)

Aggregation of Ag-TiO2 Janus particles is observed when UV light is turned on and the

particles disaggregated when UV light is switched off This can be due to predator-prey

kind of interaction between silver and titania half of the Janus particles in the presence of

UV light Oxygen and water that are produced at the silver end by decomposition of

hydrogen peroxide are used up by titania when subjected to UV radiation This can lead

to attraction between titania and silver parts of neighboring Janus particles causing

aggregation

y = 20373x

y = 59994x

y = 71848x

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10 12

Mean

sq

uare

dis

pla

cem

en

t (μ

m2s

)

Time (sec)

Mean square displacement vs Time for Ag-TiO2

particles

05 Hydrogen peroxide and UV05 Hydrogen peroxide

DI Water

51

A UV OFF B UV ON

C UV OFF

Figure 4 7 Higher concentrations of Ag-TiO2 particles showing aggregation when UV

is turned on

52

Chapter 5

Summary and Conclusion

In summary no significant enhancement in diffusion was observed in case of enzyme

immobilized Janus particles in substrate solution Presence of a linker between enzyme

and particle did not increase the diffusion coefficient of particles Alternative enzyme

immobilization methods need to be tested like thiolation of enzyme and subsequent

binding to gold surface of a Janus particle35

or expressing the enzyme with polyhistidine

or avidin tag67

This can result in higher enzyme activity upon immobilization and may

lead to enhanced diffusion of Janus particles

Predator-prey system of GOx and HRP immobilized particles showed some promising

results Aggregation of particles and formation of fan-shaped structures by TMB

oxidation were observed under optical microscope when GOx and HRP immobilized

particles glucose and TMB were present in the system The size of the fan-shaped

structures was dependent on the size of enzyme immobilized particles This can be

indicative of some kind of interaction between the GOx and HRP immobilized particles

No conclusive aggregation of GOx and HRP immobilized particles was observed in

absence of TMB Better imaging techniques are required to study the interaction of GOx

and HRP immobilized particles at the unstable liquid-air interface where majority of the

reaction takes place Other enzyme cascade systems not requiring gaseous reactants like

glycogen synthase and glycogen phosphorylase should be tested for similar activity

53

Ag-TiO2 particles were found to exhibit autonomous motion in the presence of hydrogen

peroxide solution The motion was momentarily accelerated upon exposure to UV

radiation The particles showed a diffusion coefficient of 1491 μm2s in hydrogen

peroxide versus only 180 μm2s in DI water Upon subjecting particles to UV radiation in

presence of H2O2 diffusion coefficient increased to 5093 μm2s for a period of 4-5

seconds In addition to linear motion rotating motion could also be observed in some

particle aggregates Further development of these aggregate structures can lead to

formation of rotors at nanoscale Higher concentration of silver-titania particles in

hydrogen peroxide displayed aggregation in presence of UV light and disaggregated

when UV light was switched off This may be attributed to the predator-prey kind of

interaction between silver and titania where oxygen and water that are produced by

silver by decomposition of hydrogen peroxide are used up by titania when subjected to

UV radiation This promises to be an interesting system and needs to be further

investigated to explore its properties

54

References

1 Paxton WF Sundararajan S Mallouk TE Sen A Angew Chem Int Ed Engl

2006 45 5420-5429

2 Golestanian R Liverpool TB Adjari A Phys Rev Lett 2005 94 220801

3 httpwwwsilvermedicineorgh2o2html

4 Rami Pedahzur Ovadia Lev Badri Fattal Hillel Shuval Water Science and Technology

1995 31(5-6) 123-129

5 W Went F American Scientist 1968 56

6 Adriano Cavalcanti Tad Hogg Bijan Shirinzadeh IEEE MHS 2006 International

Symposium on Micro-NanoMechatronics and Human Science 2006 226-232

7 EM Purcell American Journal of Physics 1977 45 3-11

8 Golestanian R Phys Rev Lett 2010 105 018103(1)-018103(4)

9 S S Dukhin Z R Ulberg G L Dvornichenko B V Deryagin Russian Chemical

Bulletin 1983 31(8) 1535-1544

10 Dukhin AS Goetz PJ Ultrasound for characterizing colloids Particle sizing zeta

potential rheology Elsevier 2002 59

11 HJKeh HJTu Trends in Colloid and Interface Science XIV Progress in Colloid and

Polymer Science 2000 115 174-180

12 D Zinemanas A Nir International Journal of Multiphase Flow 1995 21(5) 787-800

55

13 Stephen Ebbens Jonathan Howse Soft matter 2010 6 726-739

14 J Vicario R Eelkema W R Browne A Meetsma R M La Crois B L Feringa

Chem Commun 2005 31 3936

15 Davide Pantarotto Wesley R Browne Ben L Feringa Chem Commun 2008 1533 -

1535

16 Lisa A Cameron Matthew J Footer Alexander van Oudenaarden Julie A Theriot

PNAS 1999 96(9) 4908-4913

17 Ali Najafi Ramin Golestanian Phys Rev E 2004 69 062901

18 Jinseok Kim Jungyul Park Sungwook Yang Jeongeun Baek Byungkyu Kim Sang

Ho Lee Eui-Sung Yoon Kukjin Chun Sukho Park Lab Chip 2007 7 1504 - 1508

19 Kurt M Dubowski Clinical Chemistry 2008 5411 1919ndash1920

20 Spector DA Yang Q Wade JB Am J Physiol Renal Physiol 2007 292(1) 467-474

21 Rhee SG Chang TS Bae YS Lee SR Kang SW J Am Soc Nephrol 2003 14(8-3)

211-215

22 Sakaue T Kapral R Mikhailov A S Eur Phys J B 2010 75 381ndash387

23 Muddana H S Sengupta S Mallouk T E Sen A Butler P J J Am Chem Soc

2010 132 (7) 2110ndash2111

24 Baker CJ Deahl K Domek J Orlandi EW Arch Biochem Biophys 2000 382(2) 232-

237

25 V Vojinovic RH Carvalho F Lemos JMS Cabral LP Fonseca BS Ferreira

Biochemical Engineering Journal 2007 35 126-135

56

26

OV Lebedeva NN Ugarova Russian Chemical Bulletin 1996 45(1) 18-25

27 Nigel C Veitch Phytochemistry 2004 65(3) 249-259

28 Kenneth H Petersen Journal of Immunological Methods 2009 340(1) 86-89

29 httpwwwbrenda-enzymesorgphpresult_flatphp4ecno=11117

30 Goldenberg L M Wagner J Stumpe J Paulke B-R Goumlrnitz E Langmuir 2002 18

5627-5629

31 httpwwwgoldbiocompdf3761-Protocol201pdf

32 Av Rovisco Pais Lisboa Portugal T amp D Technology West Yorkshire UK Journal

of Molecular Catalysis B Enzymatic 2004 28(2-3) 129-135

33 httpwwwphysicsnyuedugrierlabmethodsnode11html

34 Yoon-Mee Lee1 O-Yul Kwon Yeo-Joon Yoon Keungarp Ryu Biotechnology Letters

2006 28 39ndash43

35 JC Pyun SD Kim JW Chung Analytical Biochemistry 2005 347(2) 227-233

36 George H Pimbley Jr Mathematical Biosciences 1974 20(1-2) 27-51

37 Andy Fenton Sarah E Perkins Parasitology 2010 137 1027-1038

38 Mead James G Brownell Robert L Mammal Species of the World A Taxonomic and

Geographic Reference (3rd ed) Johns Hopkins University Press 2005 2 2142

57

39

Krause KH Schweiz Med Wochenschr 2000 130(4) 97-100

40 Irina Kareva Faina Berezovskaya Carlos Castillo-Chavez Journal of Biological

Dynamics 2010 4(4)315 - 327

41 Delaittre G Reynhout I Cornelissen J Nolte R Chemistry ndash A European Journal

2009 15 12600ndash12603

42 C Buller DH Johnson RC Tschumper Investigative Ophthalmology amp Visual Science

1990 31 2156-2163

43 Robert M Mazo Brownian Motion Fluctuations Dynamics and Applications Oxford

Science Publications 2009 57

44 Vivek K Mutalik KV Venkatesh Theoretical Biology and Medical Modelling 2005 2

19

45 Satoshi Nakamura Kunimasa Koga Biochemical and Biophysical Research

Communications 1977 78(2) 806-810

46 Krishnananda Chattopadhyay Shyamalava Mazumdar Biochemistry 2000 39 (1)

263ndash270

47 Satoshi Nakamura Yasuyuki Ogura J Biochem 1968 63 (3) 308-316

48 van Dongen SF Nallani M Cornelissen JJ Nolte RJ van Hest JC Chemistry 2009

15(5) 1107-1114

49 Ronald Bentley Methods in Enzymology 1955 1 340-345

50 Vartiainen J Raumlttouml M Paulussen S Packaging Technology and Science 2005

18 243ndash251

58

51 GA Sherbeny AA Shindia YMMM Sheriff International Journal of Agriculture

and Biology 2005 7

52 D Rando G-W Kohring F Giffhorn Appl Microbiol Biotechnol 1997 48

53 Worthington enzyme manual Worthington Biochemical Corporation

httpworthington-biochemcomgopdefaulthtml

54 httpwwwbrenda-enzymesorgphpresult_flatphp4ecno=11117

55 httpwwwoxygenphwatercom

56 P Boon Chock Sue Goo Rhee and Earl R Stadtman Annu Rev Biochem 1980 49

813-841

57 Sybil Golden P A Wals Joseph Katz Analytical Biochemistry 1977 77(2) 436-445

58 R L Baehner S K Murrmann J Davis R B Johnston Jr J Clin Invest 1975 56(3)

571ndash576

59 I Bejarano M P Terroacuten S D Paredes C Barriga A B Rodriacuteguez J A Pariente

Molecular and Cellular Biochemistry 2007 296(1-2) 77-84

60 Michael C Moulton Laura K Braydich-Stolle Mallikarjuna N Nadagouda Samantha

Kunzelman Saber M Hussain Rajender S Varma Nanoscale 2010 2 763-770

61 Goya DV Matijevic E Colloids and Surfaces A 1999 146 139

62 Velikov KP Zegers GE van Blaanderen A Langmuir 2003 19 1384

63 Goldenberg L M Wagner J Stumpe J Paulke B-R Goumlrnitz E Langmuir 2002 18

5627-5629

59

64

Banerjee S Gopal J Muraleedharan P Tyagi A K Raj B Curr Sci 2006

901378ndash1383

65 Bogomolov V N Kudinov E K Firsov Y A Sov Phys Solid State 1968 35 555

66 Kudo A Miseki Y Chem Soc Rev 2009 38 253ndash278

67 Gaj T Meyer SC Ghosh I Protein expression and Purification 2007 56 54-61

14

Figure 2 4 Reaction catalyzed by horseradish peroxidase

The functional parameters for horseradish peroxidase include Km of 015 mM and kcat of

790 s-1

at an optimum temperature of 20 ordmC and an optimum pH of 6429

Some of the

inhibitors of HRP are 110-phenanthroline aminotriazole and bromine

22 Experimental

221 Materials and methods

Uniform carboxyl modified polystyrene microspheres 05 microm in diameter were obtained

from Polysciences Inc The size of the purchased microspheres was verified by Field

Emission Scanning Electron Microscopy (FESEM) Peroxidase Type I from

Horseradish N-(3-dimethyl-aminopropyl)-Nrsquo-ethylcarbodiimide (EDC) and hydrogen

peroxide solution 50 wt in water were purchased from Sigma-Aldrich N-

hydroxysuccinimide (NHS) ge 97 was purchased from Fluka All chemicals were of

reagent grade and used without further purification Nanopure deionized water was used

for all experiments and preparation of solutions All measurements were made at room

15

temperature (25 oC) unless mentioned otherwise The UV-Visible spectroscopy

measurements were performed using Agilent 8453A diode-array UV-Visible

Spectrophotometer with ChemStation UV-Vis Software Diffusion coefficient

measurements were performed using Nanosight LM10

222 Synthesis of Janus particles

A monolayer of particles was prepared according to the procedure (Figure 2 5) by

Goldenberg L et al30

Glass slides were placed on the bottom of a petridish Water was

put into the petridish so that the entire slide was immersed in water A thin layer of

hexane was added on top of water in the petridish A suspension of microspheres in

ethanol (5 ww) was introduced to the water-hexane interface After the monolayer was

formed the glass slide was lifted up through the monolayer with tweezers The

microsphere-covered glass slides were left to dry overnight at room temperature

Following drying a thin film of silica was evaporated on the glass slides in Semicore

evaporator The spheres were partially coated with 30 nm of silica Particles were

removed from the glass slide by gentle sonication The polystyrene-silica (PS-SiO2)

Janus particles thus obtained were washed in deionized water several times before

further immobilization steps were performed FESEM was used to confirm synthesis of

Janus particles

16

Figure 2 5 Preparation of PS-SiO2 Janus particles by evaporation

223 Immobilization of enzymes using EDCNHS coupling method

1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide and N-hydroxysulfosuccinimide

(EDCNHS) coupling procedure was followed for immobilizing horseradish peroxidase

type I on 05 μm PS-SiO2 Janus particles (Figure 2 6) In this process EDC reacts with a

carboxyl groups on the Janus particles to form an amine-reactive O-acylisourea

intermediate If this intermediate does not encounter an amine it can hydrolyze and

regenerate the carboxyl group In the presence of NHS EDC can be used to convert

carboxyl groups to amine-reactive sulfo-NHS esters which are much more stable and do

not readily hydrolyze to regenerate carboxyl groups In the current experiment

immobilization was performed in the presence as well as absence of a long spacer

17

molecule (linker) between Janus particles and enzymes The chemical 6-aminohexanoic

acid was used as the linker (Figure 2 7) HRP immobilized PS-SiO2 Janus particles were

stored in deionized water until required for further experiments

Figure 2 6 EDCNHS coupling method for immobilization of enzyme on carboxylated

polystyrene particles

Figure 2 7 Immobilization of enzyme on Janus particles with and without 6-

aminohexanoic acid linker

18

224 Enzyme assay

Assay for activity of HRP was performed using a spectrophotometric method which

involved using 4-aminoantipyrene as hydrogen donor3132

The reaction rate was

determined by measuring increase in absorbance at 510 nm resulting from the

decomposition of hydrogen peroxide One unit represents the decomposition of 1 μM of

hydrogen peroxide per minute at 25 degC and pH 70 under the specified conditions

The spectrophotometer was set to the wavelength of 510 nm and temperature of 25 degC

Phenol4-aminoantipyrene solution (14 mL) and 00017 M H2O2 solution (15 mL) were

pipetted into each cuvette The cuvette was incubated for 3-4 mins at 25degC and blank was

established A 01 mL solution of diluted enzyme was added to the above mixture and

absorbance was recorded for 4-5 mins Standard graphs of absorbance versus time were

drawn for known concentration of the free enzyme and compared to that of the

immobilized enzyme on the Janus particles The estimate of the amount of enzyme

present on the Janus particles was deduced from the difference between the quantity of

the free enzyme previously added for immobilization and the amount that was measured

to be in the supernatant after some of the enzyme became attached to the Janus particles

The experiment was carried out in triplicate to assess reproducibility

19

The formula used to calculate the activity of enzyme is

where ∆Amin denotes the change in absorbance per minute

225 Diffusion coefficient measurements using Nanosight LM10

Diffusion coefficients for HRP immobilized particles were measured using Nanosight

LM 10 nanoparticle tracking instrument with Nanoparticle Tracking Analysis (NTA) 20

software in the presence of 003 H2O2 solution as the substrate and also in the presence

of only deionized water The videos were recorded for 30 seconds at the rate of 30 frames

per second Each experiment was carried out in triplicate to assess reproducibility

Particles

visualization

zone

Microscope with

LM 10 unit

LM 10 unit

Figure 2 8 Nanosight LM 10 instrumentation

20

Nanosight LM 10 (Figure 2 8) generates videos of population of moving nanoparticles in

a liquid when illuminated by laser light The particles which pass through the laser beam

path are seen by the instrument as small points of light undergoing Brownian motion

(Figure 2 9a)

The videos are further analyzed by NTA (Nanoparticle tracking analysis) software

(Figure 2 9b) which identifies and tracks each particle independently on a frame-by-

frame basis The software also calculates and subtracts any bulk flow in the system The

mean square displacement is calculated for each particle as long as it is visible If the

particle blinks that is if it disappears momentarily and then reappears on the screen the

software will consider the same particle as two separate ones All analysis data is

exported to the excel files in terms of diffusion coefficients of the tracked particles

B A

Figure 2 9 NTA video analysis (a) Particles as they are seen on the screen during

experimentation in Nanosight LM10 (b) NTA screen as the particles are being analyzed for

their diffusion coefficients

21

23 Results

231 Enzyme assay

The activity of free HRP enzyme reported by Sigma Aldrich is 150 unitsmg However

the activity of HRP upon analysis was found to be 52158 unitsmg which is much lower

than the reported value The activity of HRP was found to be further lowered to 2133

unitsmg when it was immobilized on the PS-SiO2 Janus particles in the presence of a

linker The following formula is used to calculate enzyme activity

where ΔAmin is the change in absorbance per minute

22

Figure 2 10 Graph for the assay of solution of free enzyme containing 05mgL of HRP

ΔAmin = 01716 Unitsmg of free enzyme = 52158

During EDCNHS coupling 04 mg enzyme was added to 1 ml of 025 vv suspension

of Janus particles Thus initial concentration of enzyme in the mixture was 04 mgml

Assuming retained activity of the enzyme in supernatant solution the amount of enzyme

in supernatant was calculated to be 03868 mgml from the assay Amount of enzyme

functionalized on the Janus particles was calculated as the difference between enzyme

previously added for immobilization (04 mgml) and the enzyme present in supernatant

(03868 mgml) and was found to be 00132 mgml Enzyme assay was performed on the

enzyme immobilized Janus particles to estimate the activity of immobilized enzyme

(Figure 2 11) and was found to be 2133 unitsmg

y = 0002x + 00516

0

01

02

03

04

05

06

07

08

0 50 100 150 200 250 300 350 400

Ab

sorb

an

ce

51

0 n

m

Time (sec)

Assay of free enzyme HRP

23

Figure 2 11 Graph for the assay of of immobilized enzyme on 05 μm Janus particles

(~025 vv) ΔAmin = 01853 Unitsmg of free enzyme = 2133

232 Diffusion coefficient measurements

No significant increase was observed in diffusion coefficient of HRP-immobilized Janus

particles in the presence of H2O2 as the substrate Similar results were obtained for

diffusion coefficient measurement with and without linker moieties bridging the enzymes

and particles(Table 2 1 Table 2 2) Thus the linker did not seem to assist in increasing

the particle diffusion by improving or retaining the activity of the enzyme

y = 00015x + 00953

0

01

02

03

04

05

06

07

0 50 100 150 200 250 300 350 400

Ab

sorb

an

ce

510 n

m

Time (sec)

Assay of HRP immobilized 05 μm Janus particles

24

Table 2 1 Diffusion coefficient measurements of HRP immobilized Janus particles

without linker

HRP immobilized

Janus particles in

003 H2O2

Control - Janus

particles in

003 H2O2

Control - HRP

immobilized Janus

particles in DI water

Diffusion coefficient

(10-10

cm2s)

1359416 1051777 1308675

Standard deviation 4534801 4122075 4918597

Table 2 2 Diffusion coefficient measurements of HRP immobilized Janus particles in

the presence of linker

HRP immobilized

Janus particles in

003 H2O2

Control - Janus

particles in

003 H2O2

Control - HRP

immobilized Janus

particles in DI water

Diffusion coefficient

(10-10

cm2s)

13838 1339829 1407891

Standard deviation 5644692 5649364 5797218

25

Figure 2 12 Diffusion coefficient measurements of PS-SiO2 Janus particles without and

with 6-aminohexanoic acid linker

24 Discussion

To compare the diffusion coefficients observed during the experiments theoretical

diffusion coefficient was calculated using following equation33

r

kTD altraslation

6 Equation 21

where r is the hydrodynamic radius of particle Dtranslational is translational diffusion

coefficient of the particle T is temperature of the system η is the viscosity of fluid

through which the particles are moving and k is the Boltzmann constant From FE-SEM

images the size of the particles used was determined to be 450 nm For 450 nm diameter

0

20

40

60

80

100

120

140

160

Without linker With linker

Dif

fusio

n c

oeff

icie

nt

(10

-10 c

m2s

)

HRP immobilized 05 μm Janus particles - Diffusion coefficient measurements

HRP immobilized Janus particles in 003 H2O2

Control - Janus particles in 003 H2O2

Control - HRP immobilized Janus particles in DI water

26

particle Dtranslational is equal to 109 μm2s which is comparable to the diffusion

coefficient measured for the enzyme immobilized particles

HRP was found to be active on the Janus particles however the activity of the enzyme

was reduced upon immobilization Lower enzyme activity can be due to the coupling

method used which in this case was EDCNHS covalent binding However EDCNHS

coupling method has been shown to significantly retain the enzyme activity when the

enzyme is immobilized on carboxylated multi-walled carbon nanotubes under appropriate

conditions34

Hence EDCNHS binding method by itself is not believed to be

responsible for reduction of enzyme activity upon immobilization

Direct binding of enzyme to the Janus particle may lead to reduction in the enzyme

activity because of space constraints for the enzyme and difficulty for the passage of

substrate to the active site of the enzyme due to stearic hindrance Hence a linker was

inserted between the enzyme and Janus particle during enzyme immobilization However

inserting a linker between the enzyme and the particle did not lead to enhanced diffusion

of Janus particles or increase in enzyme activity of immobilized enzyme

Lack of enhanced diffusion can be firstly attributed to the reduction in enzyme activity

upon immobilization Enzyme activity may be retained by modifying the conditions

during EDCNHS coupling or changing the binding method altogether The degree of

immobilization of HRP on carboxylated Janus particles can possibly be affected by

several factors such as temperature the concentration of the coupling reagent EDC

27

concentration HRP concentration in solution and incubation time34

Different

combinations of these factors can be tested to find optimum conditions for

immobilization of HRP Alternate binding method can involve thiolation of HRP by the

covalent conjugation of mercaptopropionic acid and then the thiolated HRP can then be

attached on the gold surface of a polystyrene-gold Janus particle35

Thus further

modifications can be made to the system in order to retain enzyme activity on the

immobilized particles and thereby power nanomotors using enzymes

28

Chapter 3

Predator-prey system of enzyme immobilized particles

31 Introduction

Predator-prey systems are widely found in nature and play a major role in biological

processes3637

Predator-prey systems include all the interactions in which one organism

pursues andor consumes another organism One familiar predator-prey system at the

macroscale is that of a tiger chasing and consuming a deer Another not so familiar one

includes Baleen whales which eat millions of microscopic planktons at once38

At the

microscale such kind of interactions can be observed in the case of phagocytes in the

body which are capable of engulfing foreign microorganisms and killing them with a

combination of microbicidal systems39

In the case of cancer the tumor is the prey and

the mature myeloid cells in the body are the predator However the tumor here has a

defense mechanism against the predator in which the tumor produces cytokines which

inhibits the maturation of the myeloid cells and thereby prevents predation40

One similarity that can be observed in all these systems is that the prey possesses some

attribute which is desired by the predator which makes the predator chase the prey A

similar relationship is seen in case of enzymes which are involved in cascade reactions41

In cascade reactions product synthesized by one enzyme is the reactant required by

another enzyme to carry out its own catalytic reaction As such there is a predator-prey

29

kind of dependence between the two enzymes which may cause one enzyme to chase the

other enzyme when placed in suitable substrate solutions However the mechanism

hypothesized for such a system is totally different than the mechanism involved in

biological systems In biological systems such as bacterial chemotaxis or phagocytes

chasing foreign particles in human body complex biochemical reactions are responsible

for the predator-prey behavior42

In the proposed enzyme cascade system there are no

complex biochemical reactions involved The principle hypothesized for predator-prey

behavior of enzyme cascade reactions can be explained as follows Motion of synthetic

motors at nanoscale involves two components translational diffusion and rotational

Brownian diffusion At very short time scales of less than 1Dr where Dr (s-1

) is

rotational Brownian diffusion coefficient the motion of particles is unidirectional For

longer time scales of greater than 1Dr the direction of motion is random giving

poweredactive diffusion43

If the chemical homogeneity is broken that is when there is

higher concentration of a substrate present in one direction of the enzyme the enzyme

might acquire a subtle bias which may lead to a spatial aggregation of the enzymes based

on the substrate concentration gradient The bias can be a result of the relationship

between the speed of enzyme diffusion and substrate concentration where higher

substrate concentration lead to higher enzyme diffusion As a result in an enzyme

cascade system one enzyme will migrate towards higher concentration of substrate

which is present near the other enzyme thus giving rise to a predator-prey behavior

Such enzyme cascades are widely found in signaling networks and metabolic pathways in

living cells and can be used to produce predator-prey like nanomotors systems44

The

30

enzymes being used in the present study are horseradish peroxidase and glucose oxidase

Both enzymes are extremely stable4546

Glucose is the substrate for glucose oxidase

which is widely available and highly stable47

Horseradish peroxidase acts on hydrogen

peroxide It is involved in a cascade reaction with glucose oxidase which produces

hydrogen peroxide by converting glucose into gluconic acid (Figure 3 1)48

Figure 3 1 Concept of particle aggregation due to cascade reactions of immobilized

enzymes

In this experiment glucose oxidase (GOx) and horseradish peroxidase (HRP) were

immobilized on polystyrene carboxylate beads of different sizes using EDCNHS

coupling method Glucose and 3355-Tetramethylbenzidine (TMB) were dissolved

together in water and this solution was used as the substrate solution All the components

were present in a single system in DI water Aggregation of particles and precipitation of

oxidized TMB from the solution was expected to be observed when all these components

were present in such a system

Properties of horseradish peroxidase have been discussed in the previous chapter (Section

211 Horseradish peroxidase)

GOxGOx HRPHRP

Glucose + O2Gluconic acid + H2O2

TMB TMB oxidizedBlue precipitate

H2O

Aggregation

31

311 Glucose oxidase

Figure 3 2 Glucose oxidase

Glucose oxidase (GOx) belongs to the group of oxidoreductases and is also called

glucose aerodehydrogenase49

It is obtained from fungi and has been identified as an

antimicrobial enzyme50

GOx binds to β-D-glucopyranose and catalyzes the oxidation

reaction to from gluconolactone while simultaneously reducing oxygen to hydrogen

peroxide51

GOx has high substrate affinity and specificity high stability and high

turnover number which makes it a suitable biocatalyst for industrial applications52

Figure 3 3 Redox reaction of glucose catalyzed by glucose oxidase53

32

GOx is a glycoprotein with molecular weight of 155 kDa and consists of two identical

polypeptide subunits linked together by a disulfide bond The optimum pH for GOx is

55 though it is stable in a broad pH range of 4 to 7 and can be stored for a long period of

time51

The functional parameters for glucose oxidase include Km of 151 mM and kcat of

337 s-1

at temperature of 20 ordmC and pH of 754

Some of the inhibitors of GOx are 2-

Deoxy-D-glucose adenine nucleotides and NaNO3

32 Experimental

321 Materials and methods

Uniform carboxyl modified polystyrene microspheres 05 microm in diameter were obtained

from Polysciences Inc The size of the purchased microspheres was verified by FESEM

Peroxidase Type I from horseradish glucose oxidase Type II-S from Aspergillus niger

N-(3-dimethyl-aminopropyl)-Nrsquo-ethylcarbodiimide (EDC) and Hydrogen peroxide

solution 50 wt in water were purchased from Sigma-Aldrich N-hydroxysuccinimide

(NHS) was purchased from Fluka D-Glucose (99) was purchased from VWR

International Secure-Seal imaging spacers (9mm diameter) for glass slides were obtained

from Sigma Aldrich All chemicals were of reagent grade and used without further

purification Nanopure deionized water was used for all experiments and preparation of

solutions All measurements were made at room temperature (25 oC) unless mentioned

otherwise The UV-Visible measurements were performed using Agilent 8453A diode-

array UV-Visible Spectrophotometer with ChemStation UV-Vis Software Optical

33

microscopy experiments were performed using Axiovert 200 MAT microscope by Carl

Zeiss Diffusion coefficient measurements were obtained using Nanosight LM10

322 Optical microscopy experiments

The enzyme substrate system was observed under optical microscope to check for

aggregation and precipitation due to cascade reactions Several such systems were

studied Each system in general consisted of GOx and HRP immobilized on different

polystyrene particles The substrate solutions consisted of 01 M glucose and different

concentrations of TMB solution The systems were studied in the presence of all the

constituents and in the absence of glucose GOxHRP and TMB Systems consisting of

different sized HRP immobilized particles in substrate solution of 003 hydrogen

peroxide and TMB were also studied Videos were recorded over a period of 24 hours to

observe changes in the system Each experiment was performed atleast thrice to assess

reproducibility of results

323 Diffusion coefficient measurements using Nanosight LM10

Diffusion coefficient of GOx and HRP immobilized particles were measured using

Nanosight LM10 nanoparticle tracking instrument with Nanoparticle Tracking Analysis

(NTA) 20 software Diffusion coefficients were measured for different systems namely

GOx particles in 01 M glucose and oxygen both GOx and HRP functionalized particles

in 01 M glucose and oxygen along with controls for the individual systems Videos were

34

recorded for 30 seconds at the rate of 30 frames per second Each experiment was carried

out in triplicate to assess reproducibility

33 Results

331 Optical microscopy

According to the enzyme cascade model precipitation of TMB was possible only upon

the interaction of GOx and HRP immobilized particles in presence of appropriate

substrate solution Hence in the first set of experiments precipitation of TMB was used

as an indication of the type of interaction between GOx and HRP immobilized particles

Fan-shaped structures were observed when both HRP and GOx immobilized particles

were present in a substrate solution consisting of glucose and TMB These structures

started forming as a single strand and then branched out and intertwined to form the fan-

shaped structures (Figure 3 4)

Table 3 1 Observations for formation of fan-shaped structures in enzyme cascade

system in presence of different components

GOx HRP Glucose TMB Observation

Yes Yes Yes Yes

Fan-shaped structures observed particles associated with

the fan-shaped structures

No Yes Yes Yes No fan-shaped structures

Yes No Yes Yes No fan-shaped structures

Yes Yes No Yes No fan-shaped structures

Yes Yes Yes No No fan-shaped structures

35

Fan-shaped structures can also be observed in the system consisting of only HRP

immobilized particles in substrate solution of hydrogen peroxide and TMB The size of

the fan-shaped structures depends on the size of the enzyme immobilized particles Free

enzymes show extremely tiny structures which are difficult to view under the optical

microscope

Figure 3 4 Formation of fan-shaped structures when both HRP and GOx

immobilized particles were present in a substrate solution consisting of glucose and

TMB A In the initial stages single strands form on the surface of liquid (5Ox) B

Single strands branch out and intertwine to form fan-shaped structures (50x) C Many

such structures form and settle down after 20 minutes (20x)

A

C

B

36

Experiments were performed in the absence of TMB that is the system consisted of GOx

and HRP immobilized particles in glucose substrate solution In the absence of TMB the

particles were expected to move closer to each other without any precipitation due to the

cascade reaction However there was no conclusive aggregation of particles observed in

the system

Free Enzyme 005 μm HRP - immobilized particle

10 μm HRP - immobilized particle 05 μm HRP - immobilized particle

Figure 3 5 Size dependence of fan-shaped structures on the size of HRP -

immobilized particles (20x)

37

Table 3 2 Observations for particle aggregation in enzyme cascade system in presence

and absence of TMB

Glucose GOx HRP TMB Observation

Present Present Present Present Aggregation and precipitate

Present Present Present Absent No conclusive aggregation

Present Absent Present Present No aggregation or ppt

Absent Present Present Present No aggregation or ppt

Present Present Absent Present No aggregation or ppt

332 Diffusion coefficient measurements

Diffusion coefficients were measured for GOx immobilized particles of 05 μm diameter

in substrate solution of 01 M glucose and oxygen Diffusion coefficients of particles

were also measured for system consisting of GOx and HRP functionalized particles in

glucose and oxygen Each experiment was done in triplicate to assess reproducibility

Nanosight visualization chamber being a closed chamber oxygen was purged into all the

solutions before starting the experiments to ensure sufficient supply of oxygen for

catalytic reaction of GOx

38

Table 3 3 Diffusion coefficients for 05 μm GOx immobilized particles in 01 M

glucose purged with O2

Mean

GOx

particles in

01 M

glucose

Control - GOx

particles in DI

water

Control - GOx

particles in 01 M

glucose without

oxygen purging

Control -

particles in 01

M Glucose

Diffusion

coefficient

(x10-10

cm2s)

13316 12888 13827 13702

Standard

deviation 6307 4926 4424 7468

Table 3 4 Diffusion coefficients for GOx and HRP immobilized particles in 01 M

glucose and purged with O2

Mean

GOx- HRP-

functionalized

particles in 01 M

glucose

Control ndash Gox-

HRP- functionalized

particles in DI water

Control - particles

in 01 M glucose

Diffusion coefficient

(x10-10

cm2s)

13005 14434 13702

Standard deviation 5263 6622 7468

No significant increase was observed in diffusion coefficient of enzyme immobilized

particles in any of the systems

39

34 Discussion

Formation of fan-shaped structures due to patterned deposition of oxidized TMB

indicates some kind of interaction between GOx and HRP immobilized particles Particle

diameter dependence of the size of structures can be explained with a simple model in

Figure 3 6 A larger particle will have a higher surface area as compared to a smaller

particle Hence more enzyme is immobilized on the larger particle As a result higher

quantity of TMB is oxidized and precipitated by the larger particle as compared to the

smaller particle This leads to thicker strands of fan-shaped structures eventually scaling

up the whole system to give bigger fan-shaped structures

The exact reason for the formation of such fan-shaped structures which are dependent on

particle size could not be determined It is hypothesized that hydrogen peroxide andor

Figure 3 6 Model for size dependence of fan-shaped structures on the particle

diameter of enzyme immobilized particles Larger the particle more surface area is

available for precipitation of TMB which eventually leads to bigger fan-shaped

structures

Larger particle Smaller particle

40

TMB gradient in the substrate solution might be responsible for such structures If there

is higher concentration of H2O2 on one side of the particle and assuming an even

concentration distribution of TMB there will be selective precipitation in the direction of

H2O2 When several particles come in the vicinity of higher concentration of H2O2 the

precipitation lines will converge to give fan-shaped structures (Figure 3 7)

Figure 3 7 Hypothesis for formation of fan-shaped structures

In the system consisting of GOx immobilized particles HRP immobilized particles

glucose and TMB it was observed that the fan-shaped structures formed at the liquid-air

interface and eventually settled down to the bottom of the glass slide This makes sense

as oxygen is necessary for the catalytic reaction of conversion of glucose to gluconic acid

and hydrogen peroxide which takes place on the surface of GOx immobilized particle As

a result H2O2 is primarily formed at the liquid-air interface and is then used by HRP for

precipitation of TMB

Following the observations that precipitation of TMB occurs at the surface GOx and

HRP immobilized particles are also expected to interact at the surface in the absence of

TMB Due the cascade reaction it was expected that HRP immobilized particles will be

41

attracted to GOx immobilized particles in appropriate substrate solution Experiments

were carried out to visualize inter-particle interactions at the liquid-air interface

However liquid-air interface not being a stable system to image the precise interaction

of GOx and HRP immobilized particles could not be determined Significant external

disturbances were involved at the interface To enable the cascade reaction in the bulk of

the liquid oxygen was purged into the reaction system before starting the experiment

However this did not lead to significant reaction in the bulk of the liquid and the reaction

prominently took place at the liquid-air interface where the oxygen concentration was

higher

In order to observe the interaction of GOx and HRP immobilized particles better imaging

techniques need to be used to observe diffusion of particles in absence of external

disturbances at the liquid-air interface Large quantity of oxygen needs to be purged into

the system and retained to make the reaction more pronounced in the bulk of the substrate

solution Higher concentration of oxygen in water may also be achieved by using oxygen

enriched water which is commercially available55

Alternatively a different enzyme cascade system may be used which does not require a

gaseous substrate56

For example glycogen synthase synthesizes glycogen from glucose-

1-phosphate and glycogen phosphorylase breaks down glycogen to glucose-1-

phosphate57

This system does not require a gaseous substrate and might be able to

overcome the deficiencies of GOx and HRP enzyme cascade system

42

Chapter 4

Silver-titania nanomotors

41 Introduction

Bimetallic nanowires made of platinum and gold have been developed by WPaxton

etal1 The rods move autonomously in a solution of hydrogen peroxide In the present

study silver-titania (Ag-TiO2) particles are being synthesized and investigated for motion

in presence of hydrogen peroxide and UV light With proper control of conditions and

UV light trigger it can be possible to control the speed and direction of motion of these

nanoparticles

Hydrogen peroxide is produced in the human body by phagocytes as an immune response

to foreign particles and pathogens5859

Also silver nanoparticles have been shown to be

biocompatible and capable of interacting with cells in human body without disrupting

cellular function60

Similarly it has been demonstrated that titania is compatible with

different cells DNA and proteins in human body and does not cause toxic effects Hence

silver-titania nanomotors which swim in hydrogen peroxide may open interesting

avenues in applications like targeted drug delivery in those parts of the body where

hydrogen peroxide is present

43

42 Experimental

421 Materials and methods

Silver nitrate (999995) was purchased from Alfa Aesar Gum arabic was purchased

from MP Biomedicals Hydrogen peroxide solution 50 wt in water and ascorbic acid

were purchased from Sigma-Aldrich Nanopure deionized water was used for all

experiments and preparation of all solutions Secure-Seal imaging spacers (9 mm

diameter) were obtained from Sigma Aldrich All experiments were carried out at room

temperature of 25ordmC unless otherwise mentioned Ag-TiO2 particles were synthesized by

evaporation of titania in Semicore electron beam evaporator Particles were characterized

using field-emission scanning electron microscopy Optical microscopy experiments

were performed using Axiovert 200 MAT microscope by Carl Zeiss Particles were

tracked using Physvis particle tracking software (Figure 4 1) in which the position

information of a particle on screen is stored on a frame by frame basis The data is stored

in a dat file as X and Y co-ordinates of particles tracked on the screen This information

can be converted to diffusion coefficients of the particles by further manipulation using

MathWorks-MATLABreg software

44

Figure 4 1 Physvis particle tracking software

422 Synthesis of silver colloidal particles

Silver particles were synthesized by a nucleationgrowth type reaction developed by

Goya and Matijevic61

and modified by Velikov62

Silver nitrate was used as precursor

metal salt for synthesis of silver particles Solution A was 75 ml of 50 mM AgNO3 and

035 by weight gum arabic Solution B was 75 mL of 100 mM 1-ascorbic acid and

035 by weight gum arabic Solution B was rapidly added to solution A in a 250 mL

reaction flask The reaction flask was covered and particles were stirred for 48 hrs After

48 hours particles were allowed to settle for 2-3 hours in refrigerator and supernatant

was separated from the particles Particles were then rinsed with DI water and centrifuged

thrice to obtain pure silver particles Zeta potential of these particles was reported to be

45

around -50 to -60 mV and the size was around 1μm The size of the particles can be

varied by varying the amounts of ascorbic acid and silver nitrate Using molar ratio of 31

of ascorbic acid silver nitrate can give particles of 200 nm diameter whereas ratio of

151 can give particles of upto 5 μm diameter

423 Preparation of silver-titania (Ag-TiO2) particles

A monolayer of silver particles was prepared according to the procedure by Goldenberg

et al63

Glass slides were placed on the bottom of a petridish Water was put into the

petridish so that the entire slide was immersed in water A thin layer of hexane was then

added on top of water in the petridish A suspension of silver particles in ethanol (5

ww) was slowly introduced to the water-hexane interface in the dish After the

monolayer was formed the glass slide was lifted up through the monolayer with tweezers

The silver particles-covered glass slides were left at room temperature to dry overnight

Following drying a thin film of titania was evaporated on the glass slides in Semicore

evaporator (Figure 2 7) The spheres were partially coated with a 10 nm film of titania

Ag-TiO2 particles were removed from the glass slide by gentle sonication FE-SEM was

used to characterize the particles (Figure 4 2) The thin titania layer was not easily

distinguishable due to roughness of the particles

46

Figure 4 2 FESEM image of Ag-TiO2 particles

424 Optical microscopy experiments

Imaging spacers were placed on a glass slide Suspension of Ag-TiO2 particles was

placed on the glass slide and the particles were allowed to settle Movement of particles

was observed in DI water and videos were recorded at 50x magnification of optical

microscope for 30 sec 05 H2O2 was added to the particle suspension on the slide

Videos were recorded at 50x ranging from 30 sec to 10 min to study the movement of

silver titania particles in hydrogen peroxide solution Particles in both DI water and

hydrogen peroxide were subjected to UV light and videos were recorded to study particle

dynamics in presence of UV radiation Particle tracking was performed using Physvis

particle tracking software Atleast 30 particles in every system were tracked for 10

seconds each Particle tracking data was obtained in terms of X and Y coordinates of

particles on screen This data was manipulated using MathWorks-Matlab software and

graphs of mean square displacement of particles versus time were plotted Diffusion

coefficients of particles were calculated from these graphs

47

43 Results and Discussion

Ag-TiO2 particles demonstrated autonomous motion in the presence of H2O2 No UV

light was required for this motion Motion of the particles was accelerated by addition of

fresh H2O2 to the system When subjected to UV radiation the particles displayed

momentary acceleration for 4-5 seconds

Figure 4 3 Mechanism of motion of Ag-TiO2 nanomotors

In the presence of H2O2 silver part of the Ag-TiO2 particle converts H2O2 to H2O and O2

This may lead to propulsion of the Ag-TiO2 particle due to bubble propulsion andor self-

diffusiophoresis In the presence of UV light additional reaction takes place on the

titania part of Ag-TiO2 particle which increases the velocity of the Ag-TiO2 particles

(Figure 4 4) This UV triggered photoactivity of titania is due to hole-electron separation

caused by photons having energy equal to or greater than the band gap of titania646566

Most of the holes-electrons recombine but a small fraction move to the surface and react

with the available redox species like water and oxygen to form protons (H+) superoxide

(O2-) and hydroxyl (OH) radicals

6566 This may lead to propulsion by diffusiophoresis

as well as electrophoresis

48

Figure 4 4 Silver-titania motors move faster when subjected to UV radiation

In addition to linear motion rotational motion is also observed in several particles and

particle aggregates Aggregation of particles can lead to a net circular propulsive force

which can rotate the aggregate as shown in Figure 4 5

Figure 4 5 Rotation due to particle aggregation of Ag-TiO2 particles

Particle tracking by Physvis software revealed a significant increase in diffusion

coefficient of Ag-TiO2 particles in hydrogen peroxide solution as compared to DI water

49

The Ag-TiO2 particles have a diffusion coefficient of 1491 μm2s in hydrogen peroxide

versus only 180 μm2s in DI water Thus the Ag-TiO2 particles move around more than

10 times their body length per second in hydrogen peroxide (Figure 4 6) When

subjected to UV light the particles experience a significant acceleration in hydrogen

peroxide which lasts only for a short duration of time (4-5 sec) after which the particles

return to their original velocity The diffusion coefficient of the particles during this UV

light trigger is found to be around 5093 μm2s UV light trigger does not have any visible

effect on the Ag-TiO2 particles in DI water

Table 4 1 Translational diffusion coefficients of Ag-TiO2 particles in hydrogen

peroxide and water

Mean 05 H2O2 solution DI water

Diffusion coefficient of Ag-TiO2 (μm2s)

without UV light

1491 180

Diffusion coefficient of Ag-TiO2 (μm2s)

in the presence of UV light

5093 113

50

Figure 4 6 Mean square displacement curves of Ag-TiO2 Janus particles in hydrogen

peroxide and DI water

When higher concentration (~30 vv) of Ag-TiO2 particles in hydrogen peroxide is

subjected to UV radiation the particles exhibit pattern formation (Figure 4 7)

Aggregation of Ag-TiO2 Janus particles is observed when UV light is turned on and the

particles disaggregated when UV light is switched off This can be due to predator-prey

kind of interaction between silver and titania half of the Janus particles in the presence of

UV light Oxygen and water that are produced at the silver end by decomposition of

hydrogen peroxide are used up by titania when subjected to UV radiation This can lead

to attraction between titania and silver parts of neighboring Janus particles causing

aggregation

y = 20373x

y = 59994x

y = 71848x

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10 12

Mean

sq

uare

dis

pla

cem

en

t (μ

m2s

)

Time (sec)

Mean square displacement vs Time for Ag-TiO2

particles

05 Hydrogen peroxide and UV05 Hydrogen peroxide

DI Water

51

A UV OFF B UV ON

C UV OFF

Figure 4 7 Higher concentrations of Ag-TiO2 particles showing aggregation when UV

is turned on

52

Chapter 5

Summary and Conclusion

In summary no significant enhancement in diffusion was observed in case of enzyme

immobilized Janus particles in substrate solution Presence of a linker between enzyme

and particle did not increase the diffusion coefficient of particles Alternative enzyme

immobilization methods need to be tested like thiolation of enzyme and subsequent

binding to gold surface of a Janus particle35

or expressing the enzyme with polyhistidine

or avidin tag67

This can result in higher enzyme activity upon immobilization and may

lead to enhanced diffusion of Janus particles

Predator-prey system of GOx and HRP immobilized particles showed some promising

results Aggregation of particles and formation of fan-shaped structures by TMB

oxidation were observed under optical microscope when GOx and HRP immobilized

particles glucose and TMB were present in the system The size of the fan-shaped

structures was dependent on the size of enzyme immobilized particles This can be

indicative of some kind of interaction between the GOx and HRP immobilized particles

No conclusive aggregation of GOx and HRP immobilized particles was observed in

absence of TMB Better imaging techniques are required to study the interaction of GOx

and HRP immobilized particles at the unstable liquid-air interface where majority of the

reaction takes place Other enzyme cascade systems not requiring gaseous reactants like

glycogen synthase and glycogen phosphorylase should be tested for similar activity

53

Ag-TiO2 particles were found to exhibit autonomous motion in the presence of hydrogen

peroxide solution The motion was momentarily accelerated upon exposure to UV

radiation The particles showed a diffusion coefficient of 1491 μm2s in hydrogen

peroxide versus only 180 μm2s in DI water Upon subjecting particles to UV radiation in

presence of H2O2 diffusion coefficient increased to 5093 μm2s for a period of 4-5

seconds In addition to linear motion rotating motion could also be observed in some

particle aggregates Further development of these aggregate structures can lead to

formation of rotors at nanoscale Higher concentration of silver-titania particles in

hydrogen peroxide displayed aggregation in presence of UV light and disaggregated

when UV light was switched off This may be attributed to the predator-prey kind of

interaction between silver and titania where oxygen and water that are produced by

silver by decomposition of hydrogen peroxide are used up by titania when subjected to

UV radiation This promises to be an interesting system and needs to be further

investigated to explore its properties

54

References

1 Paxton WF Sundararajan S Mallouk TE Sen A Angew Chem Int Ed Engl

2006 45 5420-5429

2 Golestanian R Liverpool TB Adjari A Phys Rev Lett 2005 94 220801

3 httpwwwsilvermedicineorgh2o2html

4 Rami Pedahzur Ovadia Lev Badri Fattal Hillel Shuval Water Science and Technology

1995 31(5-6) 123-129

5 W Went F American Scientist 1968 56

6 Adriano Cavalcanti Tad Hogg Bijan Shirinzadeh IEEE MHS 2006 International

Symposium on Micro-NanoMechatronics and Human Science 2006 226-232

7 EM Purcell American Journal of Physics 1977 45 3-11

8 Golestanian R Phys Rev Lett 2010 105 018103(1)-018103(4)

9 S S Dukhin Z R Ulberg G L Dvornichenko B V Deryagin Russian Chemical

Bulletin 1983 31(8) 1535-1544

10 Dukhin AS Goetz PJ Ultrasound for characterizing colloids Particle sizing zeta

potential rheology Elsevier 2002 59

11 HJKeh HJTu Trends in Colloid and Interface Science XIV Progress in Colloid and

Polymer Science 2000 115 174-180

12 D Zinemanas A Nir International Journal of Multiphase Flow 1995 21(5) 787-800

55

13 Stephen Ebbens Jonathan Howse Soft matter 2010 6 726-739

14 J Vicario R Eelkema W R Browne A Meetsma R M La Crois B L Feringa

Chem Commun 2005 31 3936

15 Davide Pantarotto Wesley R Browne Ben L Feringa Chem Commun 2008 1533 -

1535

16 Lisa A Cameron Matthew J Footer Alexander van Oudenaarden Julie A Theriot

PNAS 1999 96(9) 4908-4913

17 Ali Najafi Ramin Golestanian Phys Rev E 2004 69 062901

18 Jinseok Kim Jungyul Park Sungwook Yang Jeongeun Baek Byungkyu Kim Sang

Ho Lee Eui-Sung Yoon Kukjin Chun Sukho Park Lab Chip 2007 7 1504 - 1508

19 Kurt M Dubowski Clinical Chemistry 2008 5411 1919ndash1920

20 Spector DA Yang Q Wade JB Am J Physiol Renal Physiol 2007 292(1) 467-474

21 Rhee SG Chang TS Bae YS Lee SR Kang SW J Am Soc Nephrol 2003 14(8-3)

211-215

22 Sakaue T Kapral R Mikhailov A S Eur Phys J B 2010 75 381ndash387

23 Muddana H S Sengupta S Mallouk T E Sen A Butler P J J Am Chem Soc

2010 132 (7) 2110ndash2111

24 Baker CJ Deahl K Domek J Orlandi EW Arch Biochem Biophys 2000 382(2) 232-

237

25 V Vojinovic RH Carvalho F Lemos JMS Cabral LP Fonseca BS Ferreira

Biochemical Engineering Journal 2007 35 126-135

56

26

OV Lebedeva NN Ugarova Russian Chemical Bulletin 1996 45(1) 18-25

27 Nigel C Veitch Phytochemistry 2004 65(3) 249-259

28 Kenneth H Petersen Journal of Immunological Methods 2009 340(1) 86-89

29 httpwwwbrenda-enzymesorgphpresult_flatphp4ecno=11117

30 Goldenberg L M Wagner J Stumpe J Paulke B-R Goumlrnitz E Langmuir 2002 18

5627-5629

31 httpwwwgoldbiocompdf3761-Protocol201pdf

32 Av Rovisco Pais Lisboa Portugal T amp D Technology West Yorkshire UK Journal

of Molecular Catalysis B Enzymatic 2004 28(2-3) 129-135

33 httpwwwphysicsnyuedugrierlabmethodsnode11html

34 Yoon-Mee Lee1 O-Yul Kwon Yeo-Joon Yoon Keungarp Ryu Biotechnology Letters

2006 28 39ndash43

35 JC Pyun SD Kim JW Chung Analytical Biochemistry 2005 347(2) 227-233

36 George H Pimbley Jr Mathematical Biosciences 1974 20(1-2) 27-51

37 Andy Fenton Sarah E Perkins Parasitology 2010 137 1027-1038

38 Mead James G Brownell Robert L Mammal Species of the World A Taxonomic and

Geographic Reference (3rd ed) Johns Hopkins University Press 2005 2 2142

57

39

Krause KH Schweiz Med Wochenschr 2000 130(4) 97-100

40 Irina Kareva Faina Berezovskaya Carlos Castillo-Chavez Journal of Biological

Dynamics 2010 4(4)315 - 327

41 Delaittre G Reynhout I Cornelissen J Nolte R Chemistry ndash A European Journal

2009 15 12600ndash12603

42 C Buller DH Johnson RC Tschumper Investigative Ophthalmology amp Visual Science

1990 31 2156-2163

43 Robert M Mazo Brownian Motion Fluctuations Dynamics and Applications Oxford

Science Publications 2009 57

44 Vivek K Mutalik KV Venkatesh Theoretical Biology and Medical Modelling 2005 2

19

45 Satoshi Nakamura Kunimasa Koga Biochemical and Biophysical Research

Communications 1977 78(2) 806-810

46 Krishnananda Chattopadhyay Shyamalava Mazumdar Biochemistry 2000 39 (1)

263ndash270

47 Satoshi Nakamura Yasuyuki Ogura J Biochem 1968 63 (3) 308-316

48 van Dongen SF Nallani M Cornelissen JJ Nolte RJ van Hest JC Chemistry 2009

15(5) 1107-1114

49 Ronald Bentley Methods in Enzymology 1955 1 340-345

50 Vartiainen J Raumlttouml M Paulussen S Packaging Technology and Science 2005

18 243ndash251

58

51 GA Sherbeny AA Shindia YMMM Sheriff International Journal of Agriculture

and Biology 2005 7

52 D Rando G-W Kohring F Giffhorn Appl Microbiol Biotechnol 1997 48

53 Worthington enzyme manual Worthington Biochemical Corporation

httpworthington-biochemcomgopdefaulthtml

54 httpwwwbrenda-enzymesorgphpresult_flatphp4ecno=11117

55 httpwwwoxygenphwatercom

56 P Boon Chock Sue Goo Rhee and Earl R Stadtman Annu Rev Biochem 1980 49

813-841

57 Sybil Golden P A Wals Joseph Katz Analytical Biochemistry 1977 77(2) 436-445

58 R L Baehner S K Murrmann J Davis R B Johnston Jr J Clin Invest 1975 56(3)

571ndash576

59 I Bejarano M P Terroacuten S D Paredes C Barriga A B Rodriacuteguez J A Pariente

Molecular and Cellular Biochemistry 2007 296(1-2) 77-84

60 Michael C Moulton Laura K Braydich-Stolle Mallikarjuna N Nadagouda Samantha

Kunzelman Saber M Hussain Rajender S Varma Nanoscale 2010 2 763-770

61 Goya DV Matijevic E Colloids and Surfaces A 1999 146 139

62 Velikov KP Zegers GE van Blaanderen A Langmuir 2003 19 1384

63 Goldenberg L M Wagner J Stumpe J Paulke B-R Goumlrnitz E Langmuir 2002 18

5627-5629

59

64

Banerjee S Gopal J Muraleedharan P Tyagi A K Raj B Curr Sci 2006

901378ndash1383

65 Bogomolov V N Kudinov E K Firsov Y A Sov Phys Solid State 1968 35 555

66 Kudo A Miseki Y Chem Soc Rev 2009 38 253ndash278

67 Gaj T Meyer SC Ghosh I Protein expression and Purification 2007 56 54-61

15

temperature (25 oC) unless mentioned otherwise The UV-Visible spectroscopy

measurements were performed using Agilent 8453A diode-array UV-Visible

Spectrophotometer with ChemStation UV-Vis Software Diffusion coefficient

measurements were performed using Nanosight LM10

222 Synthesis of Janus particles

A monolayer of particles was prepared according to the procedure (Figure 2 5) by

Goldenberg L et al30

Glass slides were placed on the bottom of a petridish Water was

put into the petridish so that the entire slide was immersed in water A thin layer of

hexane was added on top of water in the petridish A suspension of microspheres in

ethanol (5 ww) was introduced to the water-hexane interface After the monolayer was

formed the glass slide was lifted up through the monolayer with tweezers The

microsphere-covered glass slides were left to dry overnight at room temperature

Following drying a thin film of silica was evaporated on the glass slides in Semicore

evaporator The spheres were partially coated with 30 nm of silica Particles were

removed from the glass slide by gentle sonication The polystyrene-silica (PS-SiO2)

Janus particles thus obtained were washed in deionized water several times before

further immobilization steps were performed FESEM was used to confirm synthesis of

Janus particles

16

Figure 2 5 Preparation of PS-SiO2 Janus particles by evaporation

223 Immobilization of enzymes using EDCNHS coupling method

1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide and N-hydroxysulfosuccinimide

(EDCNHS) coupling procedure was followed for immobilizing horseradish peroxidase

type I on 05 μm PS-SiO2 Janus particles (Figure 2 6) In this process EDC reacts with a

carboxyl groups on the Janus particles to form an amine-reactive O-acylisourea

intermediate If this intermediate does not encounter an amine it can hydrolyze and

regenerate the carboxyl group In the presence of NHS EDC can be used to convert

carboxyl groups to amine-reactive sulfo-NHS esters which are much more stable and do

not readily hydrolyze to regenerate carboxyl groups In the current experiment

immobilization was performed in the presence as well as absence of a long spacer

17

molecule (linker) between Janus particles and enzymes The chemical 6-aminohexanoic

acid was used as the linker (Figure 2 7) HRP immobilized PS-SiO2 Janus particles were

stored in deionized water until required for further experiments

Figure 2 6 EDCNHS coupling method for immobilization of enzyme on carboxylated

polystyrene particles

Figure 2 7 Immobilization of enzyme on Janus particles with and without 6-

aminohexanoic acid linker

18

224 Enzyme assay

Assay for activity of HRP was performed using a spectrophotometric method which

involved using 4-aminoantipyrene as hydrogen donor3132

The reaction rate was

determined by measuring increase in absorbance at 510 nm resulting from the

decomposition of hydrogen peroxide One unit represents the decomposition of 1 μM of

hydrogen peroxide per minute at 25 degC and pH 70 under the specified conditions

The spectrophotometer was set to the wavelength of 510 nm and temperature of 25 degC

Phenol4-aminoantipyrene solution (14 mL) and 00017 M H2O2 solution (15 mL) were

pipetted into each cuvette The cuvette was incubated for 3-4 mins at 25degC and blank was

established A 01 mL solution of diluted enzyme was added to the above mixture and

absorbance was recorded for 4-5 mins Standard graphs of absorbance versus time were

drawn for known concentration of the free enzyme and compared to that of the

immobilized enzyme on the Janus particles The estimate of the amount of enzyme

present on the Janus particles was deduced from the difference between the quantity of

the free enzyme previously added for immobilization and the amount that was measured

to be in the supernatant after some of the enzyme became attached to the Janus particles

The experiment was carried out in triplicate to assess reproducibility

19

The formula used to calculate the activity of enzyme is

where ∆Amin denotes the change in absorbance per minute

225 Diffusion coefficient measurements using Nanosight LM10

Diffusion coefficients for HRP immobilized particles were measured using Nanosight

LM 10 nanoparticle tracking instrument with Nanoparticle Tracking Analysis (NTA) 20

software in the presence of 003 H2O2 solution as the substrate and also in the presence

of only deionized water The videos were recorded for 30 seconds at the rate of 30 frames

per second Each experiment was carried out in triplicate to assess reproducibility

Particles

visualization

zone

Microscope with

LM 10 unit

LM 10 unit

Figure 2 8 Nanosight LM 10 instrumentation

20

Nanosight LM 10 (Figure 2 8) generates videos of population of moving nanoparticles in

a liquid when illuminated by laser light The particles which pass through the laser beam

path are seen by the instrument as small points of light undergoing Brownian motion

(Figure 2 9a)

The videos are further analyzed by NTA (Nanoparticle tracking analysis) software

(Figure 2 9b) which identifies and tracks each particle independently on a frame-by-

frame basis The software also calculates and subtracts any bulk flow in the system The

mean square displacement is calculated for each particle as long as it is visible If the

particle blinks that is if it disappears momentarily and then reappears on the screen the

software will consider the same particle as two separate ones All analysis data is

exported to the excel files in terms of diffusion coefficients of the tracked particles

B A

Figure 2 9 NTA video analysis (a) Particles as they are seen on the screen during

experimentation in Nanosight LM10 (b) NTA screen as the particles are being analyzed for

their diffusion coefficients

21

23 Results

231 Enzyme assay

The activity of free HRP enzyme reported by Sigma Aldrich is 150 unitsmg However

the activity of HRP upon analysis was found to be 52158 unitsmg which is much lower

than the reported value The activity of HRP was found to be further lowered to 2133

unitsmg when it was immobilized on the PS-SiO2 Janus particles in the presence of a

linker The following formula is used to calculate enzyme activity

where ΔAmin is the change in absorbance per minute

22

Figure 2 10 Graph for the assay of solution of free enzyme containing 05mgL of HRP

ΔAmin = 01716 Unitsmg of free enzyme = 52158

During EDCNHS coupling 04 mg enzyme was added to 1 ml of 025 vv suspension

of Janus particles Thus initial concentration of enzyme in the mixture was 04 mgml

Assuming retained activity of the enzyme in supernatant solution the amount of enzyme

in supernatant was calculated to be 03868 mgml from the assay Amount of enzyme

functionalized on the Janus particles was calculated as the difference between enzyme

previously added for immobilization (04 mgml) and the enzyme present in supernatant

(03868 mgml) and was found to be 00132 mgml Enzyme assay was performed on the

enzyme immobilized Janus particles to estimate the activity of immobilized enzyme

(Figure 2 11) and was found to be 2133 unitsmg

y = 0002x + 00516

0

01

02

03

04

05

06

07

08

0 50 100 150 200 250 300 350 400

Ab

sorb

an

ce

51

0 n

m

Time (sec)

Assay of free enzyme HRP

23

Figure 2 11 Graph for the assay of of immobilized enzyme on 05 μm Janus particles

(~025 vv) ΔAmin = 01853 Unitsmg of free enzyme = 2133

232 Diffusion coefficient measurements

No significant increase was observed in diffusion coefficient of HRP-immobilized Janus

particles in the presence of H2O2 as the substrate Similar results were obtained for

diffusion coefficient measurement with and without linker moieties bridging the enzymes

and particles(Table 2 1 Table 2 2) Thus the linker did not seem to assist in increasing

the particle diffusion by improving or retaining the activity of the enzyme

y = 00015x + 00953

0

01

02

03

04

05

06

07

0 50 100 150 200 250 300 350 400

Ab

sorb

an

ce

510 n

m

Time (sec)

Assay of HRP immobilized 05 μm Janus particles

24

Table 2 1 Diffusion coefficient measurements of HRP immobilized Janus particles

without linker

HRP immobilized

Janus particles in

003 H2O2

Control - Janus

particles in

003 H2O2

Control - HRP

immobilized Janus

particles in DI water

Diffusion coefficient

(10-10

cm2s)

1359416 1051777 1308675

Standard deviation 4534801 4122075 4918597

Table 2 2 Diffusion coefficient measurements of HRP immobilized Janus particles in

the presence of linker

HRP immobilized

Janus particles in

003 H2O2

Control - Janus

particles in

003 H2O2

Control - HRP

immobilized Janus

particles in DI water

Diffusion coefficient

(10-10

cm2s)

13838 1339829 1407891

Standard deviation 5644692 5649364 5797218

25

Figure 2 12 Diffusion coefficient measurements of PS-SiO2 Janus particles without and

with 6-aminohexanoic acid linker

24 Discussion

To compare the diffusion coefficients observed during the experiments theoretical

diffusion coefficient was calculated using following equation33

r

kTD altraslation

6 Equation 21

where r is the hydrodynamic radius of particle Dtranslational is translational diffusion

coefficient of the particle T is temperature of the system η is the viscosity of fluid

through which the particles are moving and k is the Boltzmann constant From FE-SEM

images the size of the particles used was determined to be 450 nm For 450 nm diameter

0

20

40

60

80

100

120

140

160

Without linker With linker

Dif

fusio

n c

oeff

icie

nt

(10

-10 c

m2s

)

HRP immobilized 05 μm Janus particles - Diffusion coefficient measurements

HRP immobilized Janus particles in 003 H2O2

Control - Janus particles in 003 H2O2

Control - HRP immobilized Janus particles in DI water

26

particle Dtranslational is equal to 109 μm2s which is comparable to the diffusion

coefficient measured for the enzyme immobilized particles

HRP was found to be active on the Janus particles however the activity of the enzyme

was reduced upon immobilization Lower enzyme activity can be due to the coupling

method used which in this case was EDCNHS covalent binding However EDCNHS

coupling method has been shown to significantly retain the enzyme activity when the

enzyme is immobilized on carboxylated multi-walled carbon nanotubes under appropriate

conditions34

Hence EDCNHS binding method by itself is not believed to be

responsible for reduction of enzyme activity upon immobilization

Direct binding of enzyme to the Janus particle may lead to reduction in the enzyme

activity because of space constraints for the enzyme and difficulty for the passage of

substrate to the active site of the enzyme due to stearic hindrance Hence a linker was

inserted between the enzyme and Janus particle during enzyme immobilization However

inserting a linker between the enzyme and the particle did not lead to enhanced diffusion

of Janus particles or increase in enzyme activity of immobilized enzyme

Lack of enhanced diffusion can be firstly attributed to the reduction in enzyme activity

upon immobilization Enzyme activity may be retained by modifying the conditions

during EDCNHS coupling or changing the binding method altogether The degree of

immobilization of HRP on carboxylated Janus particles can possibly be affected by

several factors such as temperature the concentration of the coupling reagent EDC

27

concentration HRP concentration in solution and incubation time34

Different

combinations of these factors can be tested to find optimum conditions for

immobilization of HRP Alternate binding method can involve thiolation of HRP by the

covalent conjugation of mercaptopropionic acid and then the thiolated HRP can then be

attached on the gold surface of a polystyrene-gold Janus particle35

Thus further

modifications can be made to the system in order to retain enzyme activity on the

immobilized particles and thereby power nanomotors using enzymes

28

Chapter 3

Predator-prey system of enzyme immobilized particles

31 Introduction

Predator-prey systems are widely found in nature and play a major role in biological

processes3637

Predator-prey systems include all the interactions in which one organism

pursues andor consumes another organism One familiar predator-prey system at the

macroscale is that of a tiger chasing and consuming a deer Another not so familiar one

includes Baleen whales which eat millions of microscopic planktons at once38

At the

microscale such kind of interactions can be observed in the case of phagocytes in the

body which are capable of engulfing foreign microorganisms and killing them with a

combination of microbicidal systems39

In the case of cancer the tumor is the prey and

the mature myeloid cells in the body are the predator However the tumor here has a

defense mechanism against the predator in which the tumor produces cytokines which

inhibits the maturation of the myeloid cells and thereby prevents predation40

One similarity that can be observed in all these systems is that the prey possesses some

attribute which is desired by the predator which makes the predator chase the prey A

similar relationship is seen in case of enzymes which are involved in cascade reactions41

In cascade reactions product synthesized by one enzyme is the reactant required by

another enzyme to carry out its own catalytic reaction As such there is a predator-prey

29

kind of dependence between the two enzymes which may cause one enzyme to chase the

other enzyme when placed in suitable substrate solutions However the mechanism

hypothesized for such a system is totally different than the mechanism involved in

biological systems In biological systems such as bacterial chemotaxis or phagocytes

chasing foreign particles in human body complex biochemical reactions are responsible

for the predator-prey behavior42

In the proposed enzyme cascade system there are no

complex biochemical reactions involved The principle hypothesized for predator-prey

behavior of enzyme cascade reactions can be explained as follows Motion of synthetic

motors at nanoscale involves two components translational diffusion and rotational

Brownian diffusion At very short time scales of less than 1Dr where Dr (s-1

) is

rotational Brownian diffusion coefficient the motion of particles is unidirectional For

longer time scales of greater than 1Dr the direction of motion is random giving

poweredactive diffusion43

If the chemical homogeneity is broken that is when there is

higher concentration of a substrate present in one direction of the enzyme the enzyme

might acquire a subtle bias which may lead to a spatial aggregation of the enzymes based

on the substrate concentration gradient The bias can be a result of the relationship

between the speed of enzyme diffusion and substrate concentration where higher

substrate concentration lead to higher enzyme diffusion As a result in an enzyme

cascade system one enzyme will migrate towards higher concentration of substrate

which is present near the other enzyme thus giving rise to a predator-prey behavior

Such enzyme cascades are widely found in signaling networks and metabolic pathways in

living cells and can be used to produce predator-prey like nanomotors systems44

The

30

enzymes being used in the present study are horseradish peroxidase and glucose oxidase

Both enzymes are extremely stable4546

Glucose is the substrate for glucose oxidase

which is widely available and highly stable47

Horseradish peroxidase acts on hydrogen

peroxide It is involved in a cascade reaction with glucose oxidase which produces

hydrogen peroxide by converting glucose into gluconic acid (Figure 3 1)48

Figure 3 1 Concept of particle aggregation due to cascade reactions of immobilized

enzymes

In this experiment glucose oxidase (GOx) and horseradish peroxidase (HRP) were

immobilized on polystyrene carboxylate beads of different sizes using EDCNHS

coupling method Glucose and 3355-Tetramethylbenzidine (TMB) were dissolved

together in water and this solution was used as the substrate solution All the components

were present in a single system in DI water Aggregation of particles and precipitation of

oxidized TMB from the solution was expected to be observed when all these components

were present in such a system

Properties of horseradish peroxidase have been discussed in the previous chapter (Section

211 Horseradish peroxidase)

GOxGOx HRPHRP

Glucose + O2Gluconic acid + H2O2

TMB TMB oxidizedBlue precipitate

H2O

Aggregation

31

311 Glucose oxidase

Figure 3 2 Glucose oxidase

Glucose oxidase (GOx) belongs to the group of oxidoreductases and is also called

glucose aerodehydrogenase49

It is obtained from fungi and has been identified as an

antimicrobial enzyme50

GOx binds to β-D-glucopyranose and catalyzes the oxidation

reaction to from gluconolactone while simultaneously reducing oxygen to hydrogen

peroxide51

GOx has high substrate affinity and specificity high stability and high

turnover number which makes it a suitable biocatalyst for industrial applications52

Figure 3 3 Redox reaction of glucose catalyzed by glucose oxidase53

32

GOx is a glycoprotein with molecular weight of 155 kDa and consists of two identical

polypeptide subunits linked together by a disulfide bond The optimum pH for GOx is

55 though it is stable in a broad pH range of 4 to 7 and can be stored for a long period of

time51

The functional parameters for glucose oxidase include Km of 151 mM and kcat of

337 s-1

at temperature of 20 ordmC and pH of 754

Some of the inhibitors of GOx are 2-

Deoxy-D-glucose adenine nucleotides and NaNO3

32 Experimental

321 Materials and methods

Uniform carboxyl modified polystyrene microspheres 05 microm in diameter were obtained

from Polysciences Inc The size of the purchased microspheres was verified by FESEM

Peroxidase Type I from horseradish glucose oxidase Type II-S from Aspergillus niger

N-(3-dimethyl-aminopropyl)-Nrsquo-ethylcarbodiimide (EDC) and Hydrogen peroxide

solution 50 wt in water were purchased from Sigma-Aldrich N-hydroxysuccinimide

(NHS) was purchased from Fluka D-Glucose (99) was purchased from VWR

International Secure-Seal imaging spacers (9mm diameter) for glass slides were obtained

from Sigma Aldrich All chemicals were of reagent grade and used without further

purification Nanopure deionized water was used for all experiments and preparation of

solutions All measurements were made at room temperature (25 oC) unless mentioned

otherwise The UV-Visible measurements were performed using Agilent 8453A diode-

array UV-Visible Spectrophotometer with ChemStation UV-Vis Software Optical

33

microscopy experiments were performed using Axiovert 200 MAT microscope by Carl

Zeiss Diffusion coefficient measurements were obtained using Nanosight LM10

322 Optical microscopy experiments

The enzyme substrate system was observed under optical microscope to check for

aggregation and precipitation due to cascade reactions Several such systems were

studied Each system in general consisted of GOx and HRP immobilized on different

polystyrene particles The substrate solutions consisted of 01 M glucose and different

concentrations of TMB solution The systems were studied in the presence of all the

constituents and in the absence of glucose GOxHRP and TMB Systems consisting of

different sized HRP immobilized particles in substrate solution of 003 hydrogen

peroxide and TMB were also studied Videos were recorded over a period of 24 hours to

observe changes in the system Each experiment was performed atleast thrice to assess

reproducibility of results

323 Diffusion coefficient measurements using Nanosight LM10

Diffusion coefficient of GOx and HRP immobilized particles were measured using

Nanosight LM10 nanoparticle tracking instrument with Nanoparticle Tracking Analysis

(NTA) 20 software Diffusion coefficients were measured for different systems namely

GOx particles in 01 M glucose and oxygen both GOx and HRP functionalized particles

in 01 M glucose and oxygen along with controls for the individual systems Videos were

34

recorded for 30 seconds at the rate of 30 frames per second Each experiment was carried

out in triplicate to assess reproducibility

33 Results

331 Optical microscopy

According to the enzyme cascade model precipitation of TMB was possible only upon

the interaction of GOx and HRP immobilized particles in presence of appropriate

substrate solution Hence in the first set of experiments precipitation of TMB was used

as an indication of the type of interaction between GOx and HRP immobilized particles

Fan-shaped structures were observed when both HRP and GOx immobilized particles

were present in a substrate solution consisting of glucose and TMB These structures

started forming as a single strand and then branched out and intertwined to form the fan-

shaped structures (Figure 3 4)

Table 3 1 Observations for formation of fan-shaped structures in enzyme cascade

system in presence of different components

GOx HRP Glucose TMB Observation

Yes Yes Yes Yes

Fan-shaped structures observed particles associated with

the fan-shaped structures

No Yes Yes Yes No fan-shaped structures

Yes No Yes Yes No fan-shaped structures

Yes Yes No Yes No fan-shaped structures

Yes Yes Yes No No fan-shaped structures

35

Fan-shaped structures can also be observed in the system consisting of only HRP

immobilized particles in substrate solution of hydrogen peroxide and TMB The size of

the fan-shaped structures depends on the size of the enzyme immobilized particles Free

enzymes show extremely tiny structures which are difficult to view under the optical

microscope

Figure 3 4 Formation of fan-shaped structures when both HRP and GOx

immobilized particles were present in a substrate solution consisting of glucose and

TMB A In the initial stages single strands form on the surface of liquid (5Ox) B

Single strands branch out and intertwine to form fan-shaped structures (50x) C Many

such structures form and settle down after 20 minutes (20x)

A

C

B

36

Experiments were performed in the absence of TMB that is the system consisted of GOx

and HRP immobilized particles in glucose substrate solution In the absence of TMB the

particles were expected to move closer to each other without any precipitation due to the

cascade reaction However there was no conclusive aggregation of particles observed in

the system

Free Enzyme 005 μm HRP - immobilized particle

10 μm HRP - immobilized particle 05 μm HRP - immobilized particle

Figure 3 5 Size dependence of fan-shaped structures on the size of HRP -

immobilized particles (20x)

37

Table 3 2 Observations for particle aggregation in enzyme cascade system in presence

and absence of TMB

Glucose GOx HRP TMB Observation

Present Present Present Present Aggregation and precipitate

Present Present Present Absent No conclusive aggregation

Present Absent Present Present No aggregation or ppt

Absent Present Present Present No aggregation or ppt

Present Present Absent Present No aggregation or ppt

332 Diffusion coefficient measurements

Diffusion coefficients were measured for GOx immobilized particles of 05 μm diameter

in substrate solution of 01 M glucose and oxygen Diffusion coefficients of particles

were also measured for system consisting of GOx and HRP functionalized particles in

glucose and oxygen Each experiment was done in triplicate to assess reproducibility

Nanosight visualization chamber being a closed chamber oxygen was purged into all the

solutions before starting the experiments to ensure sufficient supply of oxygen for

catalytic reaction of GOx

38

Table 3 3 Diffusion coefficients for 05 μm GOx immobilized particles in 01 M

glucose purged with O2

Mean

GOx

particles in

01 M

glucose

Control - GOx

particles in DI

water

Control - GOx

particles in 01 M

glucose without

oxygen purging

Control -

particles in 01

M Glucose

Diffusion

coefficient

(x10-10

cm2s)

13316 12888 13827 13702

Standard

deviation 6307 4926 4424 7468

Table 3 4 Diffusion coefficients for GOx and HRP immobilized particles in 01 M

glucose and purged with O2

Mean

GOx- HRP-

functionalized

particles in 01 M

glucose

Control ndash Gox-

HRP- functionalized

particles in DI water

Control - particles

in 01 M glucose

Diffusion coefficient

(x10-10

cm2s)

13005 14434 13702

Standard deviation 5263 6622 7468

No significant increase was observed in diffusion coefficient of enzyme immobilized

particles in any of the systems

39

34 Discussion

Formation of fan-shaped structures due to patterned deposition of oxidized TMB

indicates some kind of interaction between GOx and HRP immobilized particles Particle

diameter dependence of the size of structures can be explained with a simple model in

Figure 3 6 A larger particle will have a higher surface area as compared to a smaller

particle Hence more enzyme is immobilized on the larger particle As a result higher

quantity of TMB is oxidized and precipitated by the larger particle as compared to the

smaller particle This leads to thicker strands of fan-shaped structures eventually scaling

up the whole system to give bigger fan-shaped structures

The exact reason for the formation of such fan-shaped structures which are dependent on

particle size could not be determined It is hypothesized that hydrogen peroxide andor

Figure 3 6 Model for size dependence of fan-shaped structures on the particle

diameter of enzyme immobilized particles Larger the particle more surface area is

available for precipitation of TMB which eventually leads to bigger fan-shaped

structures

Larger particle Smaller particle

40

TMB gradient in the substrate solution might be responsible for such structures If there

is higher concentration of H2O2 on one side of the particle and assuming an even

concentration distribution of TMB there will be selective precipitation in the direction of

H2O2 When several particles come in the vicinity of higher concentration of H2O2 the

precipitation lines will converge to give fan-shaped structures (Figure 3 7)

Figure 3 7 Hypothesis for formation of fan-shaped structures

In the system consisting of GOx immobilized particles HRP immobilized particles

glucose and TMB it was observed that the fan-shaped structures formed at the liquid-air

interface and eventually settled down to the bottom of the glass slide This makes sense

as oxygen is necessary for the catalytic reaction of conversion of glucose to gluconic acid

and hydrogen peroxide which takes place on the surface of GOx immobilized particle As

a result H2O2 is primarily formed at the liquid-air interface and is then used by HRP for

precipitation of TMB

Following the observations that precipitation of TMB occurs at the surface GOx and

HRP immobilized particles are also expected to interact at the surface in the absence of

TMB Due the cascade reaction it was expected that HRP immobilized particles will be

41

attracted to GOx immobilized particles in appropriate substrate solution Experiments

were carried out to visualize inter-particle interactions at the liquid-air interface

However liquid-air interface not being a stable system to image the precise interaction

of GOx and HRP immobilized particles could not be determined Significant external

disturbances were involved at the interface To enable the cascade reaction in the bulk of

the liquid oxygen was purged into the reaction system before starting the experiment

However this did not lead to significant reaction in the bulk of the liquid and the reaction

prominently took place at the liquid-air interface where the oxygen concentration was

higher

In order to observe the interaction of GOx and HRP immobilized particles better imaging

techniques need to be used to observe diffusion of particles in absence of external

disturbances at the liquid-air interface Large quantity of oxygen needs to be purged into

the system and retained to make the reaction more pronounced in the bulk of the substrate

solution Higher concentration of oxygen in water may also be achieved by using oxygen

enriched water which is commercially available55

Alternatively a different enzyme cascade system may be used which does not require a

gaseous substrate56

For example glycogen synthase synthesizes glycogen from glucose-

1-phosphate and glycogen phosphorylase breaks down glycogen to glucose-1-

phosphate57

This system does not require a gaseous substrate and might be able to

overcome the deficiencies of GOx and HRP enzyme cascade system

42

Chapter 4

Silver-titania nanomotors

41 Introduction

Bimetallic nanowires made of platinum and gold have been developed by WPaxton

etal1 The rods move autonomously in a solution of hydrogen peroxide In the present

study silver-titania (Ag-TiO2) particles are being synthesized and investigated for motion

in presence of hydrogen peroxide and UV light With proper control of conditions and

UV light trigger it can be possible to control the speed and direction of motion of these

nanoparticles

Hydrogen peroxide is produced in the human body by phagocytes as an immune response

to foreign particles and pathogens5859

Also silver nanoparticles have been shown to be

biocompatible and capable of interacting with cells in human body without disrupting

cellular function60

Similarly it has been demonstrated that titania is compatible with

different cells DNA and proteins in human body and does not cause toxic effects Hence

silver-titania nanomotors which swim in hydrogen peroxide may open interesting

avenues in applications like targeted drug delivery in those parts of the body where

hydrogen peroxide is present

43

42 Experimental

421 Materials and methods

Silver nitrate (999995) was purchased from Alfa Aesar Gum arabic was purchased

from MP Biomedicals Hydrogen peroxide solution 50 wt in water and ascorbic acid

were purchased from Sigma-Aldrich Nanopure deionized water was used for all

experiments and preparation of all solutions Secure-Seal imaging spacers (9 mm

diameter) were obtained from Sigma Aldrich All experiments were carried out at room

temperature of 25ordmC unless otherwise mentioned Ag-TiO2 particles were synthesized by

evaporation of titania in Semicore electron beam evaporator Particles were characterized

using field-emission scanning electron microscopy Optical microscopy experiments

were performed using Axiovert 200 MAT microscope by Carl Zeiss Particles were

tracked using Physvis particle tracking software (Figure 4 1) in which the position

information of a particle on screen is stored on a frame by frame basis The data is stored

in a dat file as X and Y co-ordinates of particles tracked on the screen This information

can be converted to diffusion coefficients of the particles by further manipulation using

MathWorks-MATLABreg software

44

Figure 4 1 Physvis particle tracking software

422 Synthesis of silver colloidal particles

Silver particles were synthesized by a nucleationgrowth type reaction developed by

Goya and Matijevic61

and modified by Velikov62

Silver nitrate was used as precursor

metal salt for synthesis of silver particles Solution A was 75 ml of 50 mM AgNO3 and

035 by weight gum arabic Solution B was 75 mL of 100 mM 1-ascorbic acid and

035 by weight gum arabic Solution B was rapidly added to solution A in a 250 mL

reaction flask The reaction flask was covered and particles were stirred for 48 hrs After

48 hours particles were allowed to settle for 2-3 hours in refrigerator and supernatant

was separated from the particles Particles were then rinsed with DI water and centrifuged

thrice to obtain pure silver particles Zeta potential of these particles was reported to be

45

around -50 to -60 mV and the size was around 1μm The size of the particles can be

varied by varying the amounts of ascorbic acid and silver nitrate Using molar ratio of 31

of ascorbic acid silver nitrate can give particles of 200 nm diameter whereas ratio of

151 can give particles of upto 5 μm diameter

423 Preparation of silver-titania (Ag-TiO2) particles

A monolayer of silver particles was prepared according to the procedure by Goldenberg

et al63

Glass slides were placed on the bottom of a petridish Water was put into the

petridish so that the entire slide was immersed in water A thin layer of hexane was then

added on top of water in the petridish A suspension of silver particles in ethanol (5

ww) was slowly introduced to the water-hexane interface in the dish After the

monolayer was formed the glass slide was lifted up through the monolayer with tweezers

The silver particles-covered glass slides were left at room temperature to dry overnight

Following drying a thin film of titania was evaporated on the glass slides in Semicore

evaporator (Figure 2 7) The spheres were partially coated with a 10 nm film of titania

Ag-TiO2 particles were removed from the glass slide by gentle sonication FE-SEM was

used to characterize the particles (Figure 4 2) The thin titania layer was not easily

distinguishable due to roughness of the particles

46

Figure 4 2 FESEM image of Ag-TiO2 particles

424 Optical microscopy experiments

Imaging spacers were placed on a glass slide Suspension of Ag-TiO2 particles was

placed on the glass slide and the particles were allowed to settle Movement of particles

was observed in DI water and videos were recorded at 50x magnification of optical

microscope for 30 sec 05 H2O2 was added to the particle suspension on the slide

Videos were recorded at 50x ranging from 30 sec to 10 min to study the movement of

silver titania particles in hydrogen peroxide solution Particles in both DI water and

hydrogen peroxide were subjected to UV light and videos were recorded to study particle

dynamics in presence of UV radiation Particle tracking was performed using Physvis

particle tracking software Atleast 30 particles in every system were tracked for 10

seconds each Particle tracking data was obtained in terms of X and Y coordinates of

particles on screen This data was manipulated using MathWorks-Matlab software and

graphs of mean square displacement of particles versus time were plotted Diffusion

coefficients of particles were calculated from these graphs

47

43 Results and Discussion

Ag-TiO2 particles demonstrated autonomous motion in the presence of H2O2 No UV

light was required for this motion Motion of the particles was accelerated by addition of

fresh H2O2 to the system When subjected to UV radiation the particles displayed

momentary acceleration for 4-5 seconds

Figure 4 3 Mechanism of motion of Ag-TiO2 nanomotors

In the presence of H2O2 silver part of the Ag-TiO2 particle converts H2O2 to H2O and O2

This may lead to propulsion of the Ag-TiO2 particle due to bubble propulsion andor self-

diffusiophoresis In the presence of UV light additional reaction takes place on the

titania part of Ag-TiO2 particle which increases the velocity of the Ag-TiO2 particles

(Figure 4 4) This UV triggered photoactivity of titania is due to hole-electron separation

caused by photons having energy equal to or greater than the band gap of titania646566

Most of the holes-electrons recombine but a small fraction move to the surface and react

with the available redox species like water and oxygen to form protons (H+) superoxide

(O2-) and hydroxyl (OH) radicals

6566 This may lead to propulsion by diffusiophoresis

as well as electrophoresis

48

Figure 4 4 Silver-titania motors move faster when subjected to UV radiation

In addition to linear motion rotational motion is also observed in several particles and

particle aggregates Aggregation of particles can lead to a net circular propulsive force

which can rotate the aggregate as shown in Figure 4 5

Figure 4 5 Rotation due to particle aggregation of Ag-TiO2 particles

Particle tracking by Physvis software revealed a significant increase in diffusion

coefficient of Ag-TiO2 particles in hydrogen peroxide solution as compared to DI water

49

The Ag-TiO2 particles have a diffusion coefficient of 1491 μm2s in hydrogen peroxide

versus only 180 μm2s in DI water Thus the Ag-TiO2 particles move around more than

10 times their body length per second in hydrogen peroxide (Figure 4 6) When

subjected to UV light the particles experience a significant acceleration in hydrogen

peroxide which lasts only for a short duration of time (4-5 sec) after which the particles

return to their original velocity The diffusion coefficient of the particles during this UV

light trigger is found to be around 5093 μm2s UV light trigger does not have any visible

effect on the Ag-TiO2 particles in DI water

Table 4 1 Translational diffusion coefficients of Ag-TiO2 particles in hydrogen

peroxide and water

Mean 05 H2O2 solution DI water

Diffusion coefficient of Ag-TiO2 (μm2s)

without UV light

1491 180

Diffusion coefficient of Ag-TiO2 (μm2s)

in the presence of UV light

5093 113

50

Figure 4 6 Mean square displacement curves of Ag-TiO2 Janus particles in hydrogen

peroxide and DI water

When higher concentration (~30 vv) of Ag-TiO2 particles in hydrogen peroxide is

subjected to UV radiation the particles exhibit pattern formation (Figure 4 7)

Aggregation of Ag-TiO2 Janus particles is observed when UV light is turned on and the

particles disaggregated when UV light is switched off This can be due to predator-prey

kind of interaction between silver and titania half of the Janus particles in the presence of

UV light Oxygen and water that are produced at the silver end by decomposition of

hydrogen peroxide are used up by titania when subjected to UV radiation This can lead

to attraction between titania and silver parts of neighboring Janus particles causing

aggregation

y = 20373x

y = 59994x

y = 71848x

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10 12

Mean

sq

uare

dis

pla

cem

en

t (μ

m2s

)

Time (sec)

Mean square displacement vs Time for Ag-TiO2

particles

05 Hydrogen peroxide and UV05 Hydrogen peroxide

DI Water

51

A UV OFF B UV ON

C UV OFF

Figure 4 7 Higher concentrations of Ag-TiO2 particles showing aggregation when UV

is turned on

52

Chapter 5

Summary and Conclusion

In summary no significant enhancement in diffusion was observed in case of enzyme

immobilized Janus particles in substrate solution Presence of a linker between enzyme

and particle did not increase the diffusion coefficient of particles Alternative enzyme

immobilization methods need to be tested like thiolation of enzyme and subsequent

binding to gold surface of a Janus particle35

or expressing the enzyme with polyhistidine

or avidin tag67

This can result in higher enzyme activity upon immobilization and may

lead to enhanced diffusion of Janus particles

Predator-prey system of GOx and HRP immobilized particles showed some promising

results Aggregation of particles and formation of fan-shaped structures by TMB

oxidation were observed under optical microscope when GOx and HRP immobilized

particles glucose and TMB were present in the system The size of the fan-shaped

structures was dependent on the size of enzyme immobilized particles This can be

indicative of some kind of interaction between the GOx and HRP immobilized particles

No conclusive aggregation of GOx and HRP immobilized particles was observed in

absence of TMB Better imaging techniques are required to study the interaction of GOx

and HRP immobilized particles at the unstable liquid-air interface where majority of the

reaction takes place Other enzyme cascade systems not requiring gaseous reactants like

glycogen synthase and glycogen phosphorylase should be tested for similar activity

53

Ag-TiO2 particles were found to exhibit autonomous motion in the presence of hydrogen

peroxide solution The motion was momentarily accelerated upon exposure to UV

radiation The particles showed a diffusion coefficient of 1491 μm2s in hydrogen

peroxide versus only 180 μm2s in DI water Upon subjecting particles to UV radiation in

presence of H2O2 diffusion coefficient increased to 5093 μm2s for a period of 4-5

seconds In addition to linear motion rotating motion could also be observed in some

particle aggregates Further development of these aggregate structures can lead to

formation of rotors at nanoscale Higher concentration of silver-titania particles in

hydrogen peroxide displayed aggregation in presence of UV light and disaggregated

when UV light was switched off This may be attributed to the predator-prey kind of

interaction between silver and titania where oxygen and water that are produced by

silver by decomposition of hydrogen peroxide are used up by titania when subjected to

UV radiation This promises to be an interesting system and needs to be further

investigated to explore its properties

54

References

1 Paxton WF Sundararajan S Mallouk TE Sen A Angew Chem Int Ed Engl

2006 45 5420-5429

2 Golestanian R Liverpool TB Adjari A Phys Rev Lett 2005 94 220801

3 httpwwwsilvermedicineorgh2o2html

4 Rami Pedahzur Ovadia Lev Badri Fattal Hillel Shuval Water Science and Technology

1995 31(5-6) 123-129

5 W Went F American Scientist 1968 56

6 Adriano Cavalcanti Tad Hogg Bijan Shirinzadeh IEEE MHS 2006 International

Symposium on Micro-NanoMechatronics and Human Science 2006 226-232

7 EM Purcell American Journal of Physics 1977 45 3-11

8 Golestanian R Phys Rev Lett 2010 105 018103(1)-018103(4)

9 S S Dukhin Z R Ulberg G L Dvornichenko B V Deryagin Russian Chemical

Bulletin 1983 31(8) 1535-1544

10 Dukhin AS Goetz PJ Ultrasound for characterizing colloids Particle sizing zeta

potential rheology Elsevier 2002 59

11 HJKeh HJTu Trends in Colloid and Interface Science XIV Progress in Colloid and

Polymer Science 2000 115 174-180

12 D Zinemanas A Nir International Journal of Multiphase Flow 1995 21(5) 787-800

55

13 Stephen Ebbens Jonathan Howse Soft matter 2010 6 726-739

14 J Vicario R Eelkema W R Browne A Meetsma R M La Crois B L Feringa

Chem Commun 2005 31 3936

15 Davide Pantarotto Wesley R Browne Ben L Feringa Chem Commun 2008 1533 -

1535

16 Lisa A Cameron Matthew J Footer Alexander van Oudenaarden Julie A Theriot

PNAS 1999 96(9) 4908-4913

17 Ali Najafi Ramin Golestanian Phys Rev E 2004 69 062901

18 Jinseok Kim Jungyul Park Sungwook Yang Jeongeun Baek Byungkyu Kim Sang

Ho Lee Eui-Sung Yoon Kukjin Chun Sukho Park Lab Chip 2007 7 1504 - 1508

19 Kurt M Dubowski Clinical Chemistry 2008 5411 1919ndash1920

20 Spector DA Yang Q Wade JB Am J Physiol Renal Physiol 2007 292(1) 467-474

21 Rhee SG Chang TS Bae YS Lee SR Kang SW J Am Soc Nephrol 2003 14(8-3)

211-215

22 Sakaue T Kapral R Mikhailov A S Eur Phys J B 2010 75 381ndash387

23 Muddana H S Sengupta S Mallouk T E Sen A Butler P J J Am Chem Soc

2010 132 (7) 2110ndash2111

24 Baker CJ Deahl K Domek J Orlandi EW Arch Biochem Biophys 2000 382(2) 232-

237

25 V Vojinovic RH Carvalho F Lemos JMS Cabral LP Fonseca BS Ferreira

Biochemical Engineering Journal 2007 35 126-135

56

26

OV Lebedeva NN Ugarova Russian Chemical Bulletin 1996 45(1) 18-25

27 Nigel C Veitch Phytochemistry 2004 65(3) 249-259

28 Kenneth H Petersen Journal of Immunological Methods 2009 340(1) 86-89

29 httpwwwbrenda-enzymesorgphpresult_flatphp4ecno=11117

30 Goldenberg L M Wagner J Stumpe J Paulke B-R Goumlrnitz E Langmuir 2002 18

5627-5629

31 httpwwwgoldbiocompdf3761-Protocol201pdf

32 Av Rovisco Pais Lisboa Portugal T amp D Technology West Yorkshire UK Journal

of Molecular Catalysis B Enzymatic 2004 28(2-3) 129-135

33 httpwwwphysicsnyuedugrierlabmethodsnode11html

34 Yoon-Mee Lee1 O-Yul Kwon Yeo-Joon Yoon Keungarp Ryu Biotechnology Letters

2006 28 39ndash43

35 JC Pyun SD Kim JW Chung Analytical Biochemistry 2005 347(2) 227-233

36 George H Pimbley Jr Mathematical Biosciences 1974 20(1-2) 27-51

37 Andy Fenton Sarah E Perkins Parasitology 2010 137 1027-1038

38 Mead James G Brownell Robert L Mammal Species of the World A Taxonomic and

Geographic Reference (3rd ed) Johns Hopkins University Press 2005 2 2142

57

39

Krause KH Schweiz Med Wochenschr 2000 130(4) 97-100

40 Irina Kareva Faina Berezovskaya Carlos Castillo-Chavez Journal of Biological

Dynamics 2010 4(4)315 - 327

41 Delaittre G Reynhout I Cornelissen J Nolte R Chemistry ndash A European Journal

2009 15 12600ndash12603

42 C Buller DH Johnson RC Tschumper Investigative Ophthalmology amp Visual Science

1990 31 2156-2163

43 Robert M Mazo Brownian Motion Fluctuations Dynamics and Applications Oxford

Science Publications 2009 57

44 Vivek K Mutalik KV Venkatesh Theoretical Biology and Medical Modelling 2005 2

19

45 Satoshi Nakamura Kunimasa Koga Biochemical and Biophysical Research

Communications 1977 78(2) 806-810

46 Krishnananda Chattopadhyay Shyamalava Mazumdar Biochemistry 2000 39 (1)

263ndash270

47 Satoshi Nakamura Yasuyuki Ogura J Biochem 1968 63 (3) 308-316

48 van Dongen SF Nallani M Cornelissen JJ Nolte RJ van Hest JC Chemistry 2009

15(5) 1107-1114

49 Ronald Bentley Methods in Enzymology 1955 1 340-345

50 Vartiainen J Raumlttouml M Paulussen S Packaging Technology and Science 2005

18 243ndash251

58

51 GA Sherbeny AA Shindia YMMM Sheriff International Journal of Agriculture

and Biology 2005 7

52 D Rando G-W Kohring F Giffhorn Appl Microbiol Biotechnol 1997 48

53 Worthington enzyme manual Worthington Biochemical Corporation

httpworthington-biochemcomgopdefaulthtml

54 httpwwwbrenda-enzymesorgphpresult_flatphp4ecno=11117

55 httpwwwoxygenphwatercom

56 P Boon Chock Sue Goo Rhee and Earl R Stadtman Annu Rev Biochem 1980 49

813-841

57 Sybil Golden P A Wals Joseph Katz Analytical Biochemistry 1977 77(2) 436-445

58 R L Baehner S K Murrmann J Davis R B Johnston Jr J Clin Invest 1975 56(3)

571ndash576

59 I Bejarano M P Terroacuten S D Paredes C Barriga A B Rodriacuteguez J A Pariente

Molecular and Cellular Biochemistry 2007 296(1-2) 77-84

60 Michael C Moulton Laura K Braydich-Stolle Mallikarjuna N Nadagouda Samantha

Kunzelman Saber M Hussain Rajender S Varma Nanoscale 2010 2 763-770

61 Goya DV Matijevic E Colloids and Surfaces A 1999 146 139

62 Velikov KP Zegers GE van Blaanderen A Langmuir 2003 19 1384

63 Goldenberg L M Wagner J Stumpe J Paulke B-R Goumlrnitz E Langmuir 2002 18

5627-5629

59

64

Banerjee S Gopal J Muraleedharan P Tyagi A K Raj B Curr Sci 2006

901378ndash1383

65 Bogomolov V N Kudinov E K Firsov Y A Sov Phys Solid State 1968 35 555

66 Kudo A Miseki Y Chem Soc Rev 2009 38 253ndash278

67 Gaj T Meyer SC Ghosh I Protein expression and Purification 2007 56 54-61

16

Figure 2 5 Preparation of PS-SiO2 Janus particles by evaporation

223 Immobilization of enzymes using EDCNHS coupling method

1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide and N-hydroxysulfosuccinimide

(EDCNHS) coupling procedure was followed for immobilizing horseradish peroxidase

type I on 05 μm PS-SiO2 Janus particles (Figure 2 6) In this process EDC reacts with a

carboxyl groups on the Janus particles to form an amine-reactive O-acylisourea

intermediate If this intermediate does not encounter an amine it can hydrolyze and

regenerate the carboxyl group In the presence of NHS EDC can be used to convert

carboxyl groups to amine-reactive sulfo-NHS esters which are much more stable and do

not readily hydrolyze to regenerate carboxyl groups In the current experiment

immobilization was performed in the presence as well as absence of a long spacer

17

molecule (linker) between Janus particles and enzymes The chemical 6-aminohexanoic

acid was used as the linker (Figure 2 7) HRP immobilized PS-SiO2 Janus particles were

stored in deionized water until required for further experiments

Figure 2 6 EDCNHS coupling method for immobilization of enzyme on carboxylated

polystyrene particles

Figure 2 7 Immobilization of enzyme on Janus particles with and without 6-

aminohexanoic acid linker

18

224 Enzyme assay

Assay for activity of HRP was performed using a spectrophotometric method which

involved using 4-aminoantipyrene as hydrogen donor3132

The reaction rate was

determined by measuring increase in absorbance at 510 nm resulting from the

decomposition of hydrogen peroxide One unit represents the decomposition of 1 μM of

hydrogen peroxide per minute at 25 degC and pH 70 under the specified conditions

The spectrophotometer was set to the wavelength of 510 nm and temperature of 25 degC

Phenol4-aminoantipyrene solution (14 mL) and 00017 M H2O2 solution (15 mL) were

pipetted into each cuvette The cuvette was incubated for 3-4 mins at 25degC and blank was

established A 01 mL solution of diluted enzyme was added to the above mixture and

absorbance was recorded for 4-5 mins Standard graphs of absorbance versus time were

drawn for known concentration of the free enzyme and compared to that of the

immobilized enzyme on the Janus particles The estimate of the amount of enzyme

present on the Janus particles was deduced from the difference between the quantity of

the free enzyme previously added for immobilization and the amount that was measured

to be in the supernatant after some of the enzyme became attached to the Janus particles

The experiment was carried out in triplicate to assess reproducibility

19

The formula used to calculate the activity of enzyme is

where ∆Amin denotes the change in absorbance per minute

225 Diffusion coefficient measurements using Nanosight LM10

Diffusion coefficients for HRP immobilized particles were measured using Nanosight

LM 10 nanoparticle tracking instrument with Nanoparticle Tracking Analysis (NTA) 20

software in the presence of 003 H2O2 solution as the substrate and also in the presence

of only deionized water The videos were recorded for 30 seconds at the rate of 30 frames

per second Each experiment was carried out in triplicate to assess reproducibility

Particles

visualization

zone

Microscope with

LM 10 unit

LM 10 unit

Figure 2 8 Nanosight LM 10 instrumentation

20

Nanosight LM 10 (Figure 2 8) generates videos of population of moving nanoparticles in

a liquid when illuminated by laser light The particles which pass through the laser beam

path are seen by the instrument as small points of light undergoing Brownian motion

(Figure 2 9a)

The videos are further analyzed by NTA (Nanoparticle tracking analysis) software

(Figure 2 9b) which identifies and tracks each particle independently on a frame-by-

frame basis The software also calculates and subtracts any bulk flow in the system The

mean square displacement is calculated for each particle as long as it is visible If the

particle blinks that is if it disappears momentarily and then reappears on the screen the

software will consider the same particle as two separate ones All analysis data is

exported to the excel files in terms of diffusion coefficients of the tracked particles

B A

Figure 2 9 NTA video analysis (a) Particles as they are seen on the screen during

experimentation in Nanosight LM10 (b) NTA screen as the particles are being analyzed for

their diffusion coefficients

21

23 Results

231 Enzyme assay

The activity of free HRP enzyme reported by Sigma Aldrich is 150 unitsmg However

the activity of HRP upon analysis was found to be 52158 unitsmg which is much lower

than the reported value The activity of HRP was found to be further lowered to 2133

unitsmg when it was immobilized on the PS-SiO2 Janus particles in the presence of a

linker The following formula is used to calculate enzyme activity

where ΔAmin is the change in absorbance per minute

22

Figure 2 10 Graph for the assay of solution of free enzyme containing 05mgL of HRP

ΔAmin = 01716 Unitsmg of free enzyme = 52158

During EDCNHS coupling 04 mg enzyme was added to 1 ml of 025 vv suspension

of Janus particles Thus initial concentration of enzyme in the mixture was 04 mgml

Assuming retained activity of the enzyme in supernatant solution the amount of enzyme

in supernatant was calculated to be 03868 mgml from the assay Amount of enzyme

functionalized on the Janus particles was calculated as the difference between enzyme

previously added for immobilization (04 mgml) and the enzyme present in supernatant

(03868 mgml) and was found to be 00132 mgml Enzyme assay was performed on the

enzyme immobilized Janus particles to estimate the activity of immobilized enzyme

(Figure 2 11) and was found to be 2133 unitsmg

y = 0002x + 00516

0

01

02

03

04

05

06

07

08

0 50 100 150 200 250 300 350 400

Ab

sorb

an

ce

51

0 n

m

Time (sec)

Assay of free enzyme HRP

23

Figure 2 11 Graph for the assay of of immobilized enzyme on 05 μm Janus particles

(~025 vv) ΔAmin = 01853 Unitsmg of free enzyme = 2133

232 Diffusion coefficient measurements

No significant increase was observed in diffusion coefficient of HRP-immobilized Janus

particles in the presence of H2O2 as the substrate Similar results were obtained for

diffusion coefficient measurement with and without linker moieties bridging the enzymes

and particles(Table 2 1 Table 2 2) Thus the linker did not seem to assist in increasing

the particle diffusion by improving or retaining the activity of the enzyme

y = 00015x + 00953

0

01

02

03

04

05

06

07

0 50 100 150 200 250 300 350 400

Ab

sorb

an

ce

510 n

m

Time (sec)

Assay of HRP immobilized 05 μm Janus particles

24

Table 2 1 Diffusion coefficient measurements of HRP immobilized Janus particles

without linker

HRP immobilized

Janus particles in

003 H2O2

Control - Janus

particles in

003 H2O2

Control - HRP

immobilized Janus

particles in DI water

Diffusion coefficient

(10-10

cm2s)

1359416 1051777 1308675

Standard deviation 4534801 4122075 4918597

Table 2 2 Diffusion coefficient measurements of HRP immobilized Janus particles in

the presence of linker

HRP immobilized

Janus particles in

003 H2O2

Control - Janus

particles in

003 H2O2

Control - HRP

immobilized Janus

particles in DI water

Diffusion coefficient

(10-10

cm2s)

13838 1339829 1407891

Standard deviation 5644692 5649364 5797218

25

Figure 2 12 Diffusion coefficient measurements of PS-SiO2 Janus particles without and

with 6-aminohexanoic acid linker

24 Discussion

To compare the diffusion coefficients observed during the experiments theoretical

diffusion coefficient was calculated using following equation33

r

kTD altraslation

6 Equation 21

where r is the hydrodynamic radius of particle Dtranslational is translational diffusion

coefficient of the particle T is temperature of the system η is the viscosity of fluid

through which the particles are moving and k is the Boltzmann constant From FE-SEM

images the size of the particles used was determined to be 450 nm For 450 nm diameter

0

20

40

60

80

100

120

140

160

Without linker With linker

Dif

fusio

n c

oeff

icie

nt

(10

-10 c

m2s

)

HRP immobilized 05 μm Janus particles - Diffusion coefficient measurements

HRP immobilized Janus particles in 003 H2O2

Control - Janus particles in 003 H2O2

Control - HRP immobilized Janus particles in DI water

26

particle Dtranslational is equal to 109 μm2s which is comparable to the diffusion

coefficient measured for the enzyme immobilized particles

HRP was found to be active on the Janus particles however the activity of the enzyme

was reduced upon immobilization Lower enzyme activity can be due to the coupling

method used which in this case was EDCNHS covalent binding However EDCNHS

coupling method has been shown to significantly retain the enzyme activity when the

enzyme is immobilized on carboxylated multi-walled carbon nanotubes under appropriate

conditions34

Hence EDCNHS binding method by itself is not believed to be

responsible for reduction of enzyme activity upon immobilization

Direct binding of enzyme to the Janus particle may lead to reduction in the enzyme

activity because of space constraints for the enzyme and difficulty for the passage of

substrate to the active site of the enzyme due to stearic hindrance Hence a linker was

inserted between the enzyme and Janus particle during enzyme immobilization However

inserting a linker between the enzyme and the particle did not lead to enhanced diffusion

of Janus particles or increase in enzyme activity of immobilized enzyme

Lack of enhanced diffusion can be firstly attributed to the reduction in enzyme activity

upon immobilization Enzyme activity may be retained by modifying the conditions

during EDCNHS coupling or changing the binding method altogether The degree of

immobilization of HRP on carboxylated Janus particles can possibly be affected by

several factors such as temperature the concentration of the coupling reagent EDC

27

concentration HRP concentration in solution and incubation time34

Different

combinations of these factors can be tested to find optimum conditions for

immobilization of HRP Alternate binding method can involve thiolation of HRP by the

covalent conjugation of mercaptopropionic acid and then the thiolated HRP can then be

attached on the gold surface of a polystyrene-gold Janus particle35

Thus further

modifications can be made to the system in order to retain enzyme activity on the

immobilized particles and thereby power nanomotors using enzymes

28

Chapter 3

Predator-prey system of enzyme immobilized particles

31 Introduction

Predator-prey systems are widely found in nature and play a major role in biological

processes3637

Predator-prey systems include all the interactions in which one organism

pursues andor consumes another organism One familiar predator-prey system at the

macroscale is that of a tiger chasing and consuming a deer Another not so familiar one

includes Baleen whales which eat millions of microscopic planktons at once38

At the

microscale such kind of interactions can be observed in the case of phagocytes in the

body which are capable of engulfing foreign microorganisms and killing them with a

combination of microbicidal systems39

In the case of cancer the tumor is the prey and

the mature myeloid cells in the body are the predator However the tumor here has a

defense mechanism against the predator in which the tumor produces cytokines which

inhibits the maturation of the myeloid cells and thereby prevents predation40

One similarity that can be observed in all these systems is that the prey possesses some

attribute which is desired by the predator which makes the predator chase the prey A

similar relationship is seen in case of enzymes which are involved in cascade reactions41

In cascade reactions product synthesized by one enzyme is the reactant required by

another enzyme to carry out its own catalytic reaction As such there is a predator-prey

29

kind of dependence between the two enzymes which may cause one enzyme to chase the

other enzyme when placed in suitable substrate solutions However the mechanism

hypothesized for such a system is totally different than the mechanism involved in

biological systems In biological systems such as bacterial chemotaxis or phagocytes

chasing foreign particles in human body complex biochemical reactions are responsible

for the predator-prey behavior42

In the proposed enzyme cascade system there are no

complex biochemical reactions involved The principle hypothesized for predator-prey

behavior of enzyme cascade reactions can be explained as follows Motion of synthetic

motors at nanoscale involves two components translational diffusion and rotational

Brownian diffusion At very short time scales of less than 1Dr where Dr (s-1

) is

rotational Brownian diffusion coefficient the motion of particles is unidirectional For

longer time scales of greater than 1Dr the direction of motion is random giving

poweredactive diffusion43

If the chemical homogeneity is broken that is when there is

higher concentration of a substrate present in one direction of the enzyme the enzyme

might acquire a subtle bias which may lead to a spatial aggregation of the enzymes based

on the substrate concentration gradient The bias can be a result of the relationship

between the speed of enzyme diffusion and substrate concentration where higher

substrate concentration lead to higher enzyme diffusion As a result in an enzyme

cascade system one enzyme will migrate towards higher concentration of substrate

which is present near the other enzyme thus giving rise to a predator-prey behavior

Such enzyme cascades are widely found in signaling networks and metabolic pathways in

living cells and can be used to produce predator-prey like nanomotors systems44

The

30

enzymes being used in the present study are horseradish peroxidase and glucose oxidase

Both enzymes are extremely stable4546

Glucose is the substrate for glucose oxidase

which is widely available and highly stable47

Horseradish peroxidase acts on hydrogen

peroxide It is involved in a cascade reaction with glucose oxidase which produces

hydrogen peroxide by converting glucose into gluconic acid (Figure 3 1)48

Figure 3 1 Concept of particle aggregation due to cascade reactions of immobilized

enzymes

In this experiment glucose oxidase (GOx) and horseradish peroxidase (HRP) were

immobilized on polystyrene carboxylate beads of different sizes using EDCNHS

coupling method Glucose and 3355-Tetramethylbenzidine (TMB) were dissolved

together in water and this solution was used as the substrate solution All the components

were present in a single system in DI water Aggregation of particles and precipitation of

oxidized TMB from the solution was expected to be observed when all these components

were present in such a system

Properties of horseradish peroxidase have been discussed in the previous chapter (Section

211 Horseradish peroxidase)

GOxGOx HRPHRP

Glucose + O2Gluconic acid + H2O2

TMB TMB oxidizedBlue precipitate

H2O

Aggregation

31

311 Glucose oxidase

Figure 3 2 Glucose oxidase

Glucose oxidase (GOx) belongs to the group of oxidoreductases and is also called

glucose aerodehydrogenase49

It is obtained from fungi and has been identified as an

antimicrobial enzyme50

GOx binds to β-D-glucopyranose and catalyzes the oxidation

reaction to from gluconolactone while simultaneously reducing oxygen to hydrogen

peroxide51

GOx has high substrate affinity and specificity high stability and high

turnover number which makes it a suitable biocatalyst for industrial applications52

Figure 3 3 Redox reaction of glucose catalyzed by glucose oxidase53

32

GOx is a glycoprotein with molecular weight of 155 kDa and consists of two identical

polypeptide subunits linked together by a disulfide bond The optimum pH for GOx is

55 though it is stable in a broad pH range of 4 to 7 and can be stored for a long period of

time51

The functional parameters for glucose oxidase include Km of 151 mM and kcat of

337 s-1

at temperature of 20 ordmC and pH of 754

Some of the inhibitors of GOx are 2-

Deoxy-D-glucose adenine nucleotides and NaNO3

32 Experimental

321 Materials and methods

Uniform carboxyl modified polystyrene microspheres 05 microm in diameter were obtained

from Polysciences Inc The size of the purchased microspheres was verified by FESEM

Peroxidase Type I from horseradish glucose oxidase Type II-S from Aspergillus niger

N-(3-dimethyl-aminopropyl)-Nrsquo-ethylcarbodiimide (EDC) and Hydrogen peroxide

solution 50 wt in water were purchased from Sigma-Aldrich N-hydroxysuccinimide

(NHS) was purchased from Fluka D-Glucose (99) was purchased from VWR

International Secure-Seal imaging spacers (9mm diameter) for glass slides were obtained

from Sigma Aldrich All chemicals were of reagent grade and used without further

purification Nanopure deionized water was used for all experiments and preparation of

solutions All measurements were made at room temperature (25 oC) unless mentioned

otherwise The UV-Visible measurements were performed using Agilent 8453A diode-

array UV-Visible Spectrophotometer with ChemStation UV-Vis Software Optical

33

microscopy experiments were performed using Axiovert 200 MAT microscope by Carl

Zeiss Diffusion coefficient measurements were obtained using Nanosight LM10

322 Optical microscopy experiments

The enzyme substrate system was observed under optical microscope to check for

aggregation and precipitation due to cascade reactions Several such systems were

studied Each system in general consisted of GOx and HRP immobilized on different

polystyrene particles The substrate solutions consisted of 01 M glucose and different

concentrations of TMB solution The systems were studied in the presence of all the

constituents and in the absence of glucose GOxHRP and TMB Systems consisting of

different sized HRP immobilized particles in substrate solution of 003 hydrogen

peroxide and TMB were also studied Videos were recorded over a period of 24 hours to

observe changes in the system Each experiment was performed atleast thrice to assess

reproducibility of results

323 Diffusion coefficient measurements using Nanosight LM10

Diffusion coefficient of GOx and HRP immobilized particles were measured using

Nanosight LM10 nanoparticle tracking instrument with Nanoparticle Tracking Analysis

(NTA) 20 software Diffusion coefficients were measured for different systems namely

GOx particles in 01 M glucose and oxygen both GOx and HRP functionalized particles

in 01 M glucose and oxygen along with controls for the individual systems Videos were

34

recorded for 30 seconds at the rate of 30 frames per second Each experiment was carried

out in triplicate to assess reproducibility

33 Results

331 Optical microscopy

According to the enzyme cascade model precipitation of TMB was possible only upon

the interaction of GOx and HRP immobilized particles in presence of appropriate

substrate solution Hence in the first set of experiments precipitation of TMB was used

as an indication of the type of interaction between GOx and HRP immobilized particles

Fan-shaped structures were observed when both HRP and GOx immobilized particles

were present in a substrate solution consisting of glucose and TMB These structures

started forming as a single strand and then branched out and intertwined to form the fan-

shaped structures (Figure 3 4)

Table 3 1 Observations for formation of fan-shaped structures in enzyme cascade

system in presence of different components

GOx HRP Glucose TMB Observation

Yes Yes Yes Yes

Fan-shaped structures observed particles associated with

the fan-shaped structures

No Yes Yes Yes No fan-shaped structures

Yes No Yes Yes No fan-shaped structures

Yes Yes No Yes No fan-shaped structures

Yes Yes Yes No No fan-shaped structures

35

Fan-shaped structures can also be observed in the system consisting of only HRP

immobilized particles in substrate solution of hydrogen peroxide and TMB The size of

the fan-shaped structures depends on the size of the enzyme immobilized particles Free

enzymes show extremely tiny structures which are difficult to view under the optical

microscope

Figure 3 4 Formation of fan-shaped structures when both HRP and GOx

immobilized particles were present in a substrate solution consisting of glucose and

TMB A In the initial stages single strands form on the surface of liquid (5Ox) B

Single strands branch out and intertwine to form fan-shaped structures (50x) C Many

such structures form and settle down after 20 minutes (20x)

A

C

B

36

Experiments were performed in the absence of TMB that is the system consisted of GOx

and HRP immobilized particles in glucose substrate solution In the absence of TMB the

particles were expected to move closer to each other without any precipitation due to the

cascade reaction However there was no conclusive aggregation of particles observed in

the system

Free Enzyme 005 μm HRP - immobilized particle

10 μm HRP - immobilized particle 05 μm HRP - immobilized particle

Figure 3 5 Size dependence of fan-shaped structures on the size of HRP -

immobilized particles (20x)

37

Table 3 2 Observations for particle aggregation in enzyme cascade system in presence

and absence of TMB

Glucose GOx HRP TMB Observation

Present Present Present Present Aggregation and precipitate

Present Present Present Absent No conclusive aggregation

Present Absent Present Present No aggregation or ppt

Absent Present Present Present No aggregation or ppt

Present Present Absent Present No aggregation or ppt

332 Diffusion coefficient measurements

Diffusion coefficients were measured for GOx immobilized particles of 05 μm diameter

in substrate solution of 01 M glucose and oxygen Diffusion coefficients of particles

were also measured for system consisting of GOx and HRP functionalized particles in

glucose and oxygen Each experiment was done in triplicate to assess reproducibility

Nanosight visualization chamber being a closed chamber oxygen was purged into all the

solutions before starting the experiments to ensure sufficient supply of oxygen for

catalytic reaction of GOx

38

Table 3 3 Diffusion coefficients for 05 μm GOx immobilized particles in 01 M

glucose purged with O2

Mean

GOx

particles in

01 M

glucose

Control - GOx

particles in DI

water

Control - GOx

particles in 01 M

glucose without

oxygen purging

Control -

particles in 01

M Glucose

Diffusion

coefficient

(x10-10

cm2s)

13316 12888 13827 13702

Standard

deviation 6307 4926 4424 7468

Table 3 4 Diffusion coefficients for GOx and HRP immobilized particles in 01 M

glucose and purged with O2

Mean

GOx- HRP-

functionalized

particles in 01 M

glucose

Control ndash Gox-

HRP- functionalized

particles in DI water

Control - particles

in 01 M glucose

Diffusion coefficient

(x10-10

cm2s)

13005 14434 13702

Standard deviation 5263 6622 7468

No significant increase was observed in diffusion coefficient of enzyme immobilized

particles in any of the systems

39

34 Discussion

Formation of fan-shaped structures due to patterned deposition of oxidized TMB

indicates some kind of interaction between GOx and HRP immobilized particles Particle

diameter dependence of the size of structures can be explained with a simple model in

Figure 3 6 A larger particle will have a higher surface area as compared to a smaller

particle Hence more enzyme is immobilized on the larger particle As a result higher

quantity of TMB is oxidized and precipitated by the larger particle as compared to the

smaller particle This leads to thicker strands of fan-shaped structures eventually scaling

up the whole system to give bigger fan-shaped structures

The exact reason for the formation of such fan-shaped structures which are dependent on

particle size could not be determined It is hypothesized that hydrogen peroxide andor

Figure 3 6 Model for size dependence of fan-shaped structures on the particle

diameter of enzyme immobilized particles Larger the particle more surface area is

available for precipitation of TMB which eventually leads to bigger fan-shaped

structures

Larger particle Smaller particle

40

TMB gradient in the substrate solution might be responsible for such structures If there

is higher concentration of H2O2 on one side of the particle and assuming an even

concentration distribution of TMB there will be selective precipitation in the direction of

H2O2 When several particles come in the vicinity of higher concentration of H2O2 the

precipitation lines will converge to give fan-shaped structures (Figure 3 7)

Figure 3 7 Hypothesis for formation of fan-shaped structures

In the system consisting of GOx immobilized particles HRP immobilized particles

glucose and TMB it was observed that the fan-shaped structures formed at the liquid-air

interface and eventually settled down to the bottom of the glass slide This makes sense

as oxygen is necessary for the catalytic reaction of conversion of glucose to gluconic acid

and hydrogen peroxide which takes place on the surface of GOx immobilized particle As

a result H2O2 is primarily formed at the liquid-air interface and is then used by HRP for

precipitation of TMB

Following the observations that precipitation of TMB occurs at the surface GOx and

HRP immobilized particles are also expected to interact at the surface in the absence of

TMB Due the cascade reaction it was expected that HRP immobilized particles will be

41

attracted to GOx immobilized particles in appropriate substrate solution Experiments

were carried out to visualize inter-particle interactions at the liquid-air interface

However liquid-air interface not being a stable system to image the precise interaction

of GOx and HRP immobilized particles could not be determined Significant external

disturbances were involved at the interface To enable the cascade reaction in the bulk of

the liquid oxygen was purged into the reaction system before starting the experiment

However this did not lead to significant reaction in the bulk of the liquid and the reaction

prominently took place at the liquid-air interface where the oxygen concentration was

higher

In order to observe the interaction of GOx and HRP immobilized particles better imaging

techniques need to be used to observe diffusion of particles in absence of external

disturbances at the liquid-air interface Large quantity of oxygen needs to be purged into

the system and retained to make the reaction more pronounced in the bulk of the substrate

solution Higher concentration of oxygen in water may also be achieved by using oxygen

enriched water which is commercially available55

Alternatively a different enzyme cascade system may be used which does not require a

gaseous substrate56

For example glycogen synthase synthesizes glycogen from glucose-

1-phosphate and glycogen phosphorylase breaks down glycogen to glucose-1-

phosphate57

This system does not require a gaseous substrate and might be able to

overcome the deficiencies of GOx and HRP enzyme cascade system

42

Chapter 4

Silver-titania nanomotors

41 Introduction

Bimetallic nanowires made of platinum and gold have been developed by WPaxton

etal1 The rods move autonomously in a solution of hydrogen peroxide In the present

study silver-titania (Ag-TiO2) particles are being synthesized and investigated for motion

in presence of hydrogen peroxide and UV light With proper control of conditions and

UV light trigger it can be possible to control the speed and direction of motion of these

nanoparticles

Hydrogen peroxide is produced in the human body by phagocytes as an immune response

to foreign particles and pathogens5859

Also silver nanoparticles have been shown to be

biocompatible and capable of interacting with cells in human body without disrupting

cellular function60

Similarly it has been demonstrated that titania is compatible with

different cells DNA and proteins in human body and does not cause toxic effects Hence

silver-titania nanomotors which swim in hydrogen peroxide may open interesting

avenues in applications like targeted drug delivery in those parts of the body where

hydrogen peroxide is present

43

42 Experimental

421 Materials and methods

Silver nitrate (999995) was purchased from Alfa Aesar Gum arabic was purchased

from MP Biomedicals Hydrogen peroxide solution 50 wt in water and ascorbic acid

were purchased from Sigma-Aldrich Nanopure deionized water was used for all

experiments and preparation of all solutions Secure-Seal imaging spacers (9 mm

diameter) were obtained from Sigma Aldrich All experiments were carried out at room

temperature of 25ordmC unless otherwise mentioned Ag-TiO2 particles were synthesized by

evaporation of titania in Semicore electron beam evaporator Particles were characterized

using field-emission scanning electron microscopy Optical microscopy experiments

were performed using Axiovert 200 MAT microscope by Carl Zeiss Particles were

tracked using Physvis particle tracking software (Figure 4 1) in which the position

information of a particle on screen is stored on a frame by frame basis The data is stored

in a dat file as X and Y co-ordinates of particles tracked on the screen This information

can be converted to diffusion coefficients of the particles by further manipulation using

MathWorks-MATLABreg software

44

Figure 4 1 Physvis particle tracking software

422 Synthesis of silver colloidal particles

Silver particles were synthesized by a nucleationgrowth type reaction developed by

Goya and Matijevic61

and modified by Velikov62

Silver nitrate was used as precursor

metal salt for synthesis of silver particles Solution A was 75 ml of 50 mM AgNO3 and

035 by weight gum arabic Solution B was 75 mL of 100 mM 1-ascorbic acid and

035 by weight gum arabic Solution B was rapidly added to solution A in a 250 mL

reaction flask The reaction flask was covered and particles were stirred for 48 hrs After

48 hours particles were allowed to settle for 2-3 hours in refrigerator and supernatant

was separated from the particles Particles were then rinsed with DI water and centrifuged

thrice to obtain pure silver particles Zeta potential of these particles was reported to be

45

around -50 to -60 mV and the size was around 1μm The size of the particles can be

varied by varying the amounts of ascorbic acid and silver nitrate Using molar ratio of 31

of ascorbic acid silver nitrate can give particles of 200 nm diameter whereas ratio of

151 can give particles of upto 5 μm diameter

423 Preparation of silver-titania (Ag-TiO2) particles

A monolayer of silver particles was prepared according to the procedure by Goldenberg

et al63

Glass slides were placed on the bottom of a petridish Water was put into the

petridish so that the entire slide was immersed in water A thin layer of hexane was then

added on top of water in the petridish A suspension of silver particles in ethanol (5

ww) was slowly introduced to the water-hexane interface in the dish After the

monolayer was formed the glass slide was lifted up through the monolayer with tweezers

The silver particles-covered glass slides were left at room temperature to dry overnight

Following drying a thin film of titania was evaporated on the glass slides in Semicore

evaporator (Figure 2 7) The spheres were partially coated with a 10 nm film of titania

Ag-TiO2 particles were removed from the glass slide by gentle sonication FE-SEM was

used to characterize the particles (Figure 4 2) The thin titania layer was not easily

distinguishable due to roughness of the particles

46

Figure 4 2 FESEM image of Ag-TiO2 particles

424 Optical microscopy experiments

Imaging spacers were placed on a glass slide Suspension of Ag-TiO2 particles was

placed on the glass slide and the particles were allowed to settle Movement of particles

was observed in DI water and videos were recorded at 50x magnification of optical

microscope for 30 sec 05 H2O2 was added to the particle suspension on the slide

Videos were recorded at 50x ranging from 30 sec to 10 min to study the movement of

silver titania particles in hydrogen peroxide solution Particles in both DI water and

hydrogen peroxide were subjected to UV light and videos were recorded to study particle

dynamics in presence of UV radiation Particle tracking was performed using Physvis

particle tracking software Atleast 30 particles in every system were tracked for 10

seconds each Particle tracking data was obtained in terms of X and Y coordinates of

particles on screen This data was manipulated using MathWorks-Matlab software and

graphs of mean square displacement of particles versus time were plotted Diffusion

coefficients of particles were calculated from these graphs

47

43 Results and Discussion

Ag-TiO2 particles demonstrated autonomous motion in the presence of H2O2 No UV

light was required for this motion Motion of the particles was accelerated by addition of

fresh H2O2 to the system When subjected to UV radiation the particles displayed

momentary acceleration for 4-5 seconds

Figure 4 3 Mechanism of motion of Ag-TiO2 nanomotors

In the presence of H2O2 silver part of the Ag-TiO2 particle converts H2O2 to H2O and O2

This may lead to propulsion of the Ag-TiO2 particle due to bubble propulsion andor self-

diffusiophoresis In the presence of UV light additional reaction takes place on the

titania part of Ag-TiO2 particle which increases the velocity of the Ag-TiO2 particles

(Figure 4 4) This UV triggered photoactivity of titania is due to hole-electron separation

caused by photons having energy equal to or greater than the band gap of titania646566

Most of the holes-electrons recombine but a small fraction move to the surface and react

with the available redox species like water and oxygen to form protons (H+) superoxide

(O2-) and hydroxyl (OH) radicals

6566 This may lead to propulsion by diffusiophoresis

as well as electrophoresis

48

Figure 4 4 Silver-titania motors move faster when subjected to UV radiation

In addition to linear motion rotational motion is also observed in several particles and

particle aggregates Aggregation of particles can lead to a net circular propulsive force

which can rotate the aggregate as shown in Figure 4 5

Figure 4 5 Rotation due to particle aggregation of Ag-TiO2 particles

Particle tracking by Physvis software revealed a significant increase in diffusion

coefficient of Ag-TiO2 particles in hydrogen peroxide solution as compared to DI water

49

The Ag-TiO2 particles have a diffusion coefficient of 1491 μm2s in hydrogen peroxide

versus only 180 μm2s in DI water Thus the Ag-TiO2 particles move around more than

10 times their body length per second in hydrogen peroxide (Figure 4 6) When

subjected to UV light the particles experience a significant acceleration in hydrogen

peroxide which lasts only for a short duration of time (4-5 sec) after which the particles

return to their original velocity The diffusion coefficient of the particles during this UV

light trigger is found to be around 5093 μm2s UV light trigger does not have any visible

effect on the Ag-TiO2 particles in DI water

Table 4 1 Translational diffusion coefficients of Ag-TiO2 particles in hydrogen

peroxide and water

Mean 05 H2O2 solution DI water

Diffusion coefficient of Ag-TiO2 (μm2s)

without UV light

1491 180

Diffusion coefficient of Ag-TiO2 (μm2s)

in the presence of UV light

5093 113

50

Figure 4 6 Mean square displacement curves of Ag-TiO2 Janus particles in hydrogen

peroxide and DI water

When higher concentration (~30 vv) of Ag-TiO2 particles in hydrogen peroxide is

subjected to UV radiation the particles exhibit pattern formation (Figure 4 7)

Aggregation of Ag-TiO2 Janus particles is observed when UV light is turned on and the

particles disaggregated when UV light is switched off This can be due to predator-prey

kind of interaction between silver and titania half of the Janus particles in the presence of

UV light Oxygen and water that are produced at the silver end by decomposition of

hydrogen peroxide are used up by titania when subjected to UV radiation This can lead

to attraction between titania and silver parts of neighboring Janus particles causing

aggregation

y = 20373x

y = 59994x

y = 71848x

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10 12

Mean

sq

uare

dis

pla

cem

en

t (μ

m2s

)

Time (sec)

Mean square displacement vs Time for Ag-TiO2

particles

05 Hydrogen peroxide and UV05 Hydrogen peroxide

DI Water

51

A UV OFF B UV ON

C UV OFF

Figure 4 7 Higher concentrations of Ag-TiO2 particles showing aggregation when UV

is turned on

52

Chapter 5

Summary and Conclusion

In summary no significant enhancement in diffusion was observed in case of enzyme

immobilized Janus particles in substrate solution Presence of a linker between enzyme

and particle did not increase the diffusion coefficient of particles Alternative enzyme

immobilization methods need to be tested like thiolation of enzyme and subsequent

binding to gold surface of a Janus particle35

or expressing the enzyme with polyhistidine

or avidin tag67

This can result in higher enzyme activity upon immobilization and may

lead to enhanced diffusion of Janus particles

Predator-prey system of GOx and HRP immobilized particles showed some promising

results Aggregation of particles and formation of fan-shaped structures by TMB

oxidation were observed under optical microscope when GOx and HRP immobilized

particles glucose and TMB were present in the system The size of the fan-shaped

structures was dependent on the size of enzyme immobilized particles This can be

indicative of some kind of interaction between the GOx and HRP immobilized particles

No conclusive aggregation of GOx and HRP immobilized particles was observed in

absence of TMB Better imaging techniques are required to study the interaction of GOx

and HRP immobilized particles at the unstable liquid-air interface where majority of the

reaction takes place Other enzyme cascade systems not requiring gaseous reactants like

glycogen synthase and glycogen phosphorylase should be tested for similar activity

53

Ag-TiO2 particles were found to exhibit autonomous motion in the presence of hydrogen

peroxide solution The motion was momentarily accelerated upon exposure to UV

radiation The particles showed a diffusion coefficient of 1491 μm2s in hydrogen

peroxide versus only 180 μm2s in DI water Upon subjecting particles to UV radiation in

presence of H2O2 diffusion coefficient increased to 5093 μm2s for a period of 4-5

seconds In addition to linear motion rotating motion could also be observed in some

particle aggregates Further development of these aggregate structures can lead to

formation of rotors at nanoscale Higher concentration of silver-titania particles in

hydrogen peroxide displayed aggregation in presence of UV light and disaggregated

when UV light was switched off This may be attributed to the predator-prey kind of

interaction between silver and titania where oxygen and water that are produced by

silver by decomposition of hydrogen peroxide are used up by titania when subjected to

UV radiation This promises to be an interesting system and needs to be further

investigated to explore its properties

54

References

1 Paxton WF Sundararajan S Mallouk TE Sen A Angew Chem Int Ed Engl

2006 45 5420-5429

2 Golestanian R Liverpool TB Adjari A Phys Rev Lett 2005 94 220801

3 httpwwwsilvermedicineorgh2o2html

4 Rami Pedahzur Ovadia Lev Badri Fattal Hillel Shuval Water Science and Technology

1995 31(5-6) 123-129

5 W Went F American Scientist 1968 56

6 Adriano Cavalcanti Tad Hogg Bijan Shirinzadeh IEEE MHS 2006 International

Symposium on Micro-NanoMechatronics and Human Science 2006 226-232

7 EM Purcell American Journal of Physics 1977 45 3-11

8 Golestanian R Phys Rev Lett 2010 105 018103(1)-018103(4)

9 S S Dukhin Z R Ulberg G L Dvornichenko B V Deryagin Russian Chemical

Bulletin 1983 31(8) 1535-1544

10 Dukhin AS Goetz PJ Ultrasound for characterizing colloids Particle sizing zeta

potential rheology Elsevier 2002 59

11 HJKeh HJTu Trends in Colloid and Interface Science XIV Progress in Colloid and

Polymer Science 2000 115 174-180

12 D Zinemanas A Nir International Journal of Multiphase Flow 1995 21(5) 787-800

55

13 Stephen Ebbens Jonathan Howse Soft matter 2010 6 726-739

14 J Vicario R Eelkema W R Browne A Meetsma R M La Crois B L Feringa

Chem Commun 2005 31 3936

15 Davide Pantarotto Wesley R Browne Ben L Feringa Chem Commun 2008 1533 -

1535

16 Lisa A Cameron Matthew J Footer Alexander van Oudenaarden Julie A Theriot

PNAS 1999 96(9) 4908-4913

17 Ali Najafi Ramin Golestanian Phys Rev E 2004 69 062901

18 Jinseok Kim Jungyul Park Sungwook Yang Jeongeun Baek Byungkyu Kim Sang

Ho Lee Eui-Sung Yoon Kukjin Chun Sukho Park Lab Chip 2007 7 1504 - 1508

19 Kurt M Dubowski Clinical Chemistry 2008 5411 1919ndash1920

20 Spector DA Yang Q Wade JB Am J Physiol Renal Physiol 2007 292(1) 467-474

21 Rhee SG Chang TS Bae YS Lee SR Kang SW J Am Soc Nephrol 2003 14(8-3)

211-215

22 Sakaue T Kapral R Mikhailov A S Eur Phys J B 2010 75 381ndash387

23 Muddana H S Sengupta S Mallouk T E Sen A Butler P J J Am Chem Soc

2010 132 (7) 2110ndash2111

24 Baker CJ Deahl K Domek J Orlandi EW Arch Biochem Biophys 2000 382(2) 232-

237

25 V Vojinovic RH Carvalho F Lemos JMS Cabral LP Fonseca BS Ferreira

Biochemical Engineering Journal 2007 35 126-135

56

26

OV Lebedeva NN Ugarova Russian Chemical Bulletin 1996 45(1) 18-25

27 Nigel C Veitch Phytochemistry 2004 65(3) 249-259

28 Kenneth H Petersen Journal of Immunological Methods 2009 340(1) 86-89

29 httpwwwbrenda-enzymesorgphpresult_flatphp4ecno=11117

30 Goldenberg L M Wagner J Stumpe J Paulke B-R Goumlrnitz E Langmuir 2002 18

5627-5629

31 httpwwwgoldbiocompdf3761-Protocol201pdf

32 Av Rovisco Pais Lisboa Portugal T amp D Technology West Yorkshire UK Journal

of Molecular Catalysis B Enzymatic 2004 28(2-3) 129-135

33 httpwwwphysicsnyuedugrierlabmethodsnode11html

34 Yoon-Mee Lee1 O-Yul Kwon Yeo-Joon Yoon Keungarp Ryu Biotechnology Letters

2006 28 39ndash43

35 JC Pyun SD Kim JW Chung Analytical Biochemistry 2005 347(2) 227-233

36 George H Pimbley Jr Mathematical Biosciences 1974 20(1-2) 27-51

37 Andy Fenton Sarah E Perkins Parasitology 2010 137 1027-1038

38 Mead James G Brownell Robert L Mammal Species of the World A Taxonomic and

Geographic Reference (3rd ed) Johns Hopkins University Press 2005 2 2142

57

39

Krause KH Schweiz Med Wochenschr 2000 130(4) 97-100

40 Irina Kareva Faina Berezovskaya Carlos Castillo-Chavez Journal of Biological

Dynamics 2010 4(4)315 - 327

41 Delaittre G Reynhout I Cornelissen J Nolte R Chemistry ndash A European Journal

2009 15 12600ndash12603

42 C Buller DH Johnson RC Tschumper Investigative Ophthalmology amp Visual Science

1990 31 2156-2163

43 Robert M Mazo Brownian Motion Fluctuations Dynamics and Applications Oxford

Science Publications 2009 57

44 Vivek K Mutalik KV Venkatesh Theoretical Biology and Medical Modelling 2005 2

19

45 Satoshi Nakamura Kunimasa Koga Biochemical and Biophysical Research

Communications 1977 78(2) 806-810

46 Krishnananda Chattopadhyay Shyamalava Mazumdar Biochemistry 2000 39 (1)

263ndash270

47 Satoshi Nakamura Yasuyuki Ogura J Biochem 1968 63 (3) 308-316

48 van Dongen SF Nallani M Cornelissen JJ Nolte RJ van Hest JC Chemistry 2009

15(5) 1107-1114

49 Ronald Bentley Methods in Enzymology 1955 1 340-345

50 Vartiainen J Raumlttouml M Paulussen S Packaging Technology and Science 2005

18 243ndash251

58

51 GA Sherbeny AA Shindia YMMM Sheriff International Journal of Agriculture

and Biology 2005 7

52 D Rando G-W Kohring F Giffhorn Appl Microbiol Biotechnol 1997 48

53 Worthington enzyme manual Worthington Biochemical Corporation

httpworthington-biochemcomgopdefaulthtml

54 httpwwwbrenda-enzymesorgphpresult_flatphp4ecno=11117

55 httpwwwoxygenphwatercom

56 P Boon Chock Sue Goo Rhee and Earl R Stadtman Annu Rev Biochem 1980 49

813-841

57 Sybil Golden P A Wals Joseph Katz Analytical Biochemistry 1977 77(2) 436-445

58 R L Baehner S K Murrmann J Davis R B Johnston Jr J Clin Invest 1975 56(3)

571ndash576

59 I Bejarano M P Terroacuten S D Paredes C Barriga A B Rodriacuteguez J A Pariente

Molecular and Cellular Biochemistry 2007 296(1-2) 77-84

60 Michael C Moulton Laura K Braydich-Stolle Mallikarjuna N Nadagouda Samantha

Kunzelman Saber M Hussain Rajender S Varma Nanoscale 2010 2 763-770

61 Goya DV Matijevic E Colloids and Surfaces A 1999 146 139

62 Velikov KP Zegers GE van Blaanderen A Langmuir 2003 19 1384

63 Goldenberg L M Wagner J Stumpe J Paulke B-R Goumlrnitz E Langmuir 2002 18

5627-5629

59

64

Banerjee S Gopal J Muraleedharan P Tyagi A K Raj B Curr Sci 2006

901378ndash1383

65 Bogomolov V N Kudinov E K Firsov Y A Sov Phys Solid State 1968 35 555

66 Kudo A Miseki Y Chem Soc Rev 2009 38 253ndash278

67 Gaj T Meyer SC Ghosh I Protein expression and Purification 2007 56 54-61

17

molecule (linker) between Janus particles and enzymes The chemical 6-aminohexanoic

acid was used as the linker (Figure 2 7) HRP immobilized PS-SiO2 Janus particles were

stored in deionized water until required for further experiments

Figure 2 6 EDCNHS coupling method for immobilization of enzyme on carboxylated

polystyrene particles

Figure 2 7 Immobilization of enzyme on Janus particles with and without 6-

aminohexanoic acid linker

18

224 Enzyme assay

Assay for activity of HRP was performed using a spectrophotometric method which

involved using 4-aminoantipyrene as hydrogen donor3132

The reaction rate was

determined by measuring increase in absorbance at 510 nm resulting from the

decomposition of hydrogen peroxide One unit represents the decomposition of 1 μM of

hydrogen peroxide per minute at 25 degC and pH 70 under the specified conditions

The spectrophotometer was set to the wavelength of 510 nm and temperature of 25 degC

Phenol4-aminoantipyrene solution (14 mL) and 00017 M H2O2 solution (15 mL) were

pipetted into each cuvette The cuvette was incubated for 3-4 mins at 25degC and blank was

established A 01 mL solution of diluted enzyme was added to the above mixture and

absorbance was recorded for 4-5 mins Standard graphs of absorbance versus time were

drawn for known concentration of the free enzyme and compared to that of the

immobilized enzyme on the Janus particles The estimate of the amount of enzyme

present on the Janus particles was deduced from the difference between the quantity of

the free enzyme previously added for immobilization and the amount that was measured

to be in the supernatant after some of the enzyme became attached to the Janus particles

The experiment was carried out in triplicate to assess reproducibility

19

The formula used to calculate the activity of enzyme is

where ∆Amin denotes the change in absorbance per minute

225 Diffusion coefficient measurements using Nanosight LM10

Diffusion coefficients for HRP immobilized particles were measured using Nanosight

LM 10 nanoparticle tracking instrument with Nanoparticle Tracking Analysis (NTA) 20

software in the presence of 003 H2O2 solution as the substrate and also in the presence

of only deionized water The videos were recorded for 30 seconds at the rate of 30 frames

per second Each experiment was carried out in triplicate to assess reproducibility

Particles

visualization

zone

Microscope with

LM 10 unit

LM 10 unit

Figure 2 8 Nanosight LM 10 instrumentation

20

Nanosight LM 10 (Figure 2 8) generates videos of population of moving nanoparticles in

a liquid when illuminated by laser light The particles which pass through the laser beam

path are seen by the instrument as small points of light undergoing Brownian motion

(Figure 2 9a)

The videos are further analyzed by NTA (Nanoparticle tracking analysis) software

(Figure 2 9b) which identifies and tracks each particle independently on a frame-by-

frame basis The software also calculates and subtracts any bulk flow in the system The

mean square displacement is calculated for each particle as long as it is visible If the

particle blinks that is if it disappears momentarily and then reappears on the screen the

software will consider the same particle as two separate ones All analysis data is

exported to the excel files in terms of diffusion coefficients of the tracked particles

B A

Figure 2 9 NTA video analysis (a) Particles as they are seen on the screen during

experimentation in Nanosight LM10 (b) NTA screen as the particles are being analyzed for

their diffusion coefficients

21

23 Results

231 Enzyme assay

The activity of free HRP enzyme reported by Sigma Aldrich is 150 unitsmg However

the activity of HRP upon analysis was found to be 52158 unitsmg which is much lower

than the reported value The activity of HRP was found to be further lowered to 2133

unitsmg when it was immobilized on the PS-SiO2 Janus particles in the presence of a

linker The following formula is used to calculate enzyme activity

where ΔAmin is the change in absorbance per minute

22

Figure 2 10 Graph for the assay of solution of free enzyme containing 05mgL of HRP

ΔAmin = 01716 Unitsmg of free enzyme = 52158

During EDCNHS coupling 04 mg enzyme was added to 1 ml of 025 vv suspension

of Janus particles Thus initial concentration of enzyme in the mixture was 04 mgml

Assuming retained activity of the enzyme in supernatant solution the amount of enzyme

in supernatant was calculated to be 03868 mgml from the assay Amount of enzyme

functionalized on the Janus particles was calculated as the difference between enzyme

previously added for immobilization (04 mgml) and the enzyme present in supernatant

(03868 mgml) and was found to be 00132 mgml Enzyme assay was performed on the

enzyme immobilized Janus particles to estimate the activity of immobilized enzyme

(Figure 2 11) and was found to be 2133 unitsmg

y = 0002x + 00516

0

01

02

03

04

05

06

07

08

0 50 100 150 200 250 300 350 400

Ab

sorb

an

ce

51

0 n

m

Time (sec)

Assay of free enzyme HRP

23

Figure 2 11 Graph for the assay of of immobilized enzyme on 05 μm Janus particles

(~025 vv) ΔAmin = 01853 Unitsmg of free enzyme = 2133

232 Diffusion coefficient measurements

No significant increase was observed in diffusion coefficient of HRP-immobilized Janus

particles in the presence of H2O2 as the substrate Similar results were obtained for

diffusion coefficient measurement with and without linker moieties bridging the enzymes

and particles(Table 2 1 Table 2 2) Thus the linker did not seem to assist in increasing

the particle diffusion by improving or retaining the activity of the enzyme

y = 00015x + 00953

0

01

02

03

04

05

06

07

0 50 100 150 200 250 300 350 400

Ab

sorb

an

ce

510 n

m

Time (sec)

Assay of HRP immobilized 05 μm Janus particles

24

Table 2 1 Diffusion coefficient measurements of HRP immobilized Janus particles

without linker

HRP immobilized

Janus particles in

003 H2O2

Control - Janus

particles in

003 H2O2

Control - HRP

immobilized Janus

particles in DI water

Diffusion coefficient

(10-10

cm2s)

1359416 1051777 1308675

Standard deviation 4534801 4122075 4918597

Table 2 2 Diffusion coefficient measurements of HRP immobilized Janus particles in

the presence of linker

HRP immobilized

Janus particles in

003 H2O2

Control - Janus

particles in

003 H2O2

Control - HRP

immobilized Janus

particles in DI water

Diffusion coefficient

(10-10

cm2s)

13838 1339829 1407891

Standard deviation 5644692 5649364 5797218

25

Figure 2 12 Diffusion coefficient measurements of PS-SiO2 Janus particles without and

with 6-aminohexanoic acid linker

24 Discussion

To compare the diffusion coefficients observed during the experiments theoretical

diffusion coefficient was calculated using following equation33

r

kTD altraslation

6 Equation 21

where r is the hydrodynamic radius of particle Dtranslational is translational diffusion

coefficient of the particle T is temperature of the system η is the viscosity of fluid

through which the particles are moving and k is the Boltzmann constant From FE-SEM

images the size of the particles used was determined to be 450 nm For 450 nm diameter

0

20

40

60

80

100

120

140

160

Without linker With linker

Dif

fusio

n c

oeff

icie

nt

(10

-10 c

m2s

)

HRP immobilized 05 μm Janus particles - Diffusion coefficient measurements

HRP immobilized Janus particles in 003 H2O2

Control - Janus particles in 003 H2O2

Control - HRP immobilized Janus particles in DI water

26

particle Dtranslational is equal to 109 μm2s which is comparable to the diffusion

coefficient measured for the enzyme immobilized particles

HRP was found to be active on the Janus particles however the activity of the enzyme

was reduced upon immobilization Lower enzyme activity can be due to the coupling

method used which in this case was EDCNHS covalent binding However EDCNHS

coupling method has been shown to significantly retain the enzyme activity when the

enzyme is immobilized on carboxylated multi-walled carbon nanotubes under appropriate

conditions34

Hence EDCNHS binding method by itself is not believed to be

responsible for reduction of enzyme activity upon immobilization

Direct binding of enzyme to the Janus particle may lead to reduction in the enzyme

activity because of space constraints for the enzyme and difficulty for the passage of

substrate to the active site of the enzyme due to stearic hindrance Hence a linker was

inserted between the enzyme and Janus particle during enzyme immobilization However

inserting a linker between the enzyme and the particle did not lead to enhanced diffusion

of Janus particles or increase in enzyme activity of immobilized enzyme

Lack of enhanced diffusion can be firstly attributed to the reduction in enzyme activity

upon immobilization Enzyme activity may be retained by modifying the conditions

during EDCNHS coupling or changing the binding method altogether The degree of

immobilization of HRP on carboxylated Janus particles can possibly be affected by

several factors such as temperature the concentration of the coupling reagent EDC

27

concentration HRP concentration in solution and incubation time34

Different

combinations of these factors can be tested to find optimum conditions for

immobilization of HRP Alternate binding method can involve thiolation of HRP by the

covalent conjugation of mercaptopropionic acid and then the thiolated HRP can then be

attached on the gold surface of a polystyrene-gold Janus particle35

Thus further

modifications can be made to the system in order to retain enzyme activity on the

immobilized particles and thereby power nanomotors using enzymes

28

Chapter 3

Predator-prey system of enzyme immobilized particles

31 Introduction

Predator-prey systems are widely found in nature and play a major role in biological

processes3637

Predator-prey systems include all the interactions in which one organism

pursues andor consumes another organism One familiar predator-prey system at the

macroscale is that of a tiger chasing and consuming a deer Another not so familiar one

includes Baleen whales which eat millions of microscopic planktons at once38

At the

microscale such kind of interactions can be observed in the case of phagocytes in the

body which are capable of engulfing foreign microorganisms and killing them with a

combination of microbicidal systems39

In the case of cancer the tumor is the prey and

the mature myeloid cells in the body are the predator However the tumor here has a

defense mechanism against the predator in which the tumor produces cytokines which

inhibits the maturation of the myeloid cells and thereby prevents predation40

One similarity that can be observed in all these systems is that the prey possesses some

attribute which is desired by the predator which makes the predator chase the prey A

similar relationship is seen in case of enzymes which are involved in cascade reactions41

In cascade reactions product synthesized by one enzyme is the reactant required by

another enzyme to carry out its own catalytic reaction As such there is a predator-prey

29

kind of dependence between the two enzymes which may cause one enzyme to chase the

other enzyme when placed in suitable substrate solutions However the mechanism

hypothesized for such a system is totally different than the mechanism involved in

biological systems In biological systems such as bacterial chemotaxis or phagocytes

chasing foreign particles in human body complex biochemical reactions are responsible

for the predator-prey behavior42

In the proposed enzyme cascade system there are no

complex biochemical reactions involved The principle hypothesized for predator-prey

behavior of enzyme cascade reactions can be explained as follows Motion of synthetic

motors at nanoscale involves two components translational diffusion and rotational

Brownian diffusion At very short time scales of less than 1Dr where Dr (s-1

) is

rotational Brownian diffusion coefficient the motion of particles is unidirectional For

longer time scales of greater than 1Dr the direction of motion is random giving

poweredactive diffusion43

If the chemical homogeneity is broken that is when there is

higher concentration of a substrate present in one direction of the enzyme the enzyme

might acquire a subtle bias which may lead to a spatial aggregation of the enzymes based

on the substrate concentration gradient The bias can be a result of the relationship

between the speed of enzyme diffusion and substrate concentration where higher

substrate concentration lead to higher enzyme diffusion As a result in an enzyme

cascade system one enzyme will migrate towards higher concentration of substrate

which is present near the other enzyme thus giving rise to a predator-prey behavior

Such enzyme cascades are widely found in signaling networks and metabolic pathways in

living cells and can be used to produce predator-prey like nanomotors systems44

The

30

enzymes being used in the present study are horseradish peroxidase and glucose oxidase

Both enzymes are extremely stable4546

Glucose is the substrate for glucose oxidase

which is widely available and highly stable47

Horseradish peroxidase acts on hydrogen

peroxide It is involved in a cascade reaction with glucose oxidase which produces

hydrogen peroxide by converting glucose into gluconic acid (Figure 3 1)48

Figure 3 1 Concept of particle aggregation due to cascade reactions of immobilized

enzymes

In this experiment glucose oxidase (GOx) and horseradish peroxidase (HRP) were

immobilized on polystyrene carboxylate beads of different sizes using EDCNHS

coupling method Glucose and 3355-Tetramethylbenzidine (TMB) were dissolved

together in water and this solution was used as the substrate solution All the components

were present in a single system in DI water Aggregation of particles and precipitation of

oxidized TMB from the solution was expected to be observed when all these components

were present in such a system

Properties of horseradish peroxidase have been discussed in the previous chapter (Section

211 Horseradish peroxidase)

GOxGOx HRPHRP

Glucose + O2Gluconic acid + H2O2

TMB TMB oxidizedBlue precipitate

H2O

Aggregation

31

311 Glucose oxidase

Figure 3 2 Glucose oxidase

Glucose oxidase (GOx) belongs to the group of oxidoreductases and is also called

glucose aerodehydrogenase49

It is obtained from fungi and has been identified as an

antimicrobial enzyme50

GOx binds to β-D-glucopyranose and catalyzes the oxidation

reaction to from gluconolactone while simultaneously reducing oxygen to hydrogen

peroxide51

GOx has high substrate affinity and specificity high stability and high

turnover number which makes it a suitable biocatalyst for industrial applications52

Figure 3 3 Redox reaction of glucose catalyzed by glucose oxidase53

32

GOx is a glycoprotein with molecular weight of 155 kDa and consists of two identical

polypeptide subunits linked together by a disulfide bond The optimum pH for GOx is

55 though it is stable in a broad pH range of 4 to 7 and can be stored for a long period of

time51

The functional parameters for glucose oxidase include Km of 151 mM and kcat of

337 s-1

at temperature of 20 ordmC and pH of 754

Some of the inhibitors of GOx are 2-

Deoxy-D-glucose adenine nucleotides and NaNO3

32 Experimental

321 Materials and methods

Uniform carboxyl modified polystyrene microspheres 05 microm in diameter were obtained

from Polysciences Inc The size of the purchased microspheres was verified by FESEM

Peroxidase Type I from horseradish glucose oxidase Type II-S from Aspergillus niger

N-(3-dimethyl-aminopropyl)-Nrsquo-ethylcarbodiimide (EDC) and Hydrogen peroxide

solution 50 wt in water were purchased from Sigma-Aldrich N-hydroxysuccinimide

(NHS) was purchased from Fluka D-Glucose (99) was purchased from VWR

International Secure-Seal imaging spacers (9mm diameter) for glass slides were obtained

from Sigma Aldrich All chemicals were of reagent grade and used without further

purification Nanopure deionized water was used for all experiments and preparation of

solutions All measurements were made at room temperature (25 oC) unless mentioned

otherwise The UV-Visible measurements were performed using Agilent 8453A diode-

array UV-Visible Spectrophotometer with ChemStation UV-Vis Software Optical

33

microscopy experiments were performed using Axiovert 200 MAT microscope by Carl

Zeiss Diffusion coefficient measurements were obtained using Nanosight LM10

322 Optical microscopy experiments

The enzyme substrate system was observed under optical microscope to check for

aggregation and precipitation due to cascade reactions Several such systems were

studied Each system in general consisted of GOx and HRP immobilized on different

polystyrene particles The substrate solutions consisted of 01 M glucose and different

concentrations of TMB solution The systems were studied in the presence of all the

constituents and in the absence of glucose GOxHRP and TMB Systems consisting of

different sized HRP immobilized particles in substrate solution of 003 hydrogen

peroxide and TMB were also studied Videos were recorded over a period of 24 hours to

observe changes in the system Each experiment was performed atleast thrice to assess

reproducibility of results

323 Diffusion coefficient measurements using Nanosight LM10

Diffusion coefficient of GOx and HRP immobilized particles were measured using

Nanosight LM10 nanoparticle tracking instrument with Nanoparticle Tracking Analysis

(NTA) 20 software Diffusion coefficients were measured for different systems namely

GOx particles in 01 M glucose and oxygen both GOx and HRP functionalized particles

in 01 M glucose and oxygen along with controls for the individual systems Videos were

34

recorded for 30 seconds at the rate of 30 frames per second Each experiment was carried

out in triplicate to assess reproducibility

33 Results

331 Optical microscopy

According to the enzyme cascade model precipitation of TMB was possible only upon

the interaction of GOx and HRP immobilized particles in presence of appropriate

substrate solution Hence in the first set of experiments precipitation of TMB was used

as an indication of the type of interaction between GOx and HRP immobilized particles

Fan-shaped structures were observed when both HRP and GOx immobilized particles

were present in a substrate solution consisting of glucose and TMB These structures

started forming as a single strand and then branched out and intertwined to form the fan-

shaped structures (Figure 3 4)

Table 3 1 Observations for formation of fan-shaped structures in enzyme cascade

system in presence of different components

GOx HRP Glucose TMB Observation

Yes Yes Yes Yes

Fan-shaped structures observed particles associated with

the fan-shaped structures

No Yes Yes Yes No fan-shaped structures

Yes No Yes Yes No fan-shaped structures

Yes Yes No Yes No fan-shaped structures

Yes Yes Yes No No fan-shaped structures

35

Fan-shaped structures can also be observed in the system consisting of only HRP

immobilized particles in substrate solution of hydrogen peroxide and TMB The size of

the fan-shaped structures depends on the size of the enzyme immobilized particles Free

enzymes show extremely tiny structures which are difficult to view under the optical

microscope

Figure 3 4 Formation of fan-shaped structures when both HRP and GOx

immobilized particles were present in a substrate solution consisting of glucose and

TMB A In the initial stages single strands form on the surface of liquid (5Ox) B

Single strands branch out and intertwine to form fan-shaped structures (50x) C Many

such structures form and settle down after 20 minutes (20x)

A

C

B

36

Experiments were performed in the absence of TMB that is the system consisted of GOx

and HRP immobilized particles in glucose substrate solution In the absence of TMB the

particles were expected to move closer to each other without any precipitation due to the

cascade reaction However there was no conclusive aggregation of particles observed in

the system

Free Enzyme 005 μm HRP - immobilized particle

10 μm HRP - immobilized particle 05 μm HRP - immobilized particle

Figure 3 5 Size dependence of fan-shaped structures on the size of HRP -

immobilized particles (20x)

37

Table 3 2 Observations for particle aggregation in enzyme cascade system in presence

and absence of TMB

Glucose GOx HRP TMB Observation

Present Present Present Present Aggregation and precipitate

Present Present Present Absent No conclusive aggregation

Present Absent Present Present No aggregation or ppt

Absent Present Present Present No aggregation or ppt

Present Present Absent Present No aggregation or ppt

332 Diffusion coefficient measurements

Diffusion coefficients were measured for GOx immobilized particles of 05 μm diameter

in substrate solution of 01 M glucose and oxygen Diffusion coefficients of particles

were also measured for system consisting of GOx and HRP functionalized particles in

glucose and oxygen Each experiment was done in triplicate to assess reproducibility

Nanosight visualization chamber being a closed chamber oxygen was purged into all the

solutions before starting the experiments to ensure sufficient supply of oxygen for

catalytic reaction of GOx

38

Table 3 3 Diffusion coefficients for 05 μm GOx immobilized particles in 01 M

glucose purged with O2

Mean

GOx

particles in

01 M

glucose

Control - GOx

particles in DI

water

Control - GOx

particles in 01 M

glucose without

oxygen purging

Control -

particles in 01

M Glucose

Diffusion

coefficient

(x10-10

cm2s)

13316 12888 13827 13702

Standard

deviation 6307 4926 4424 7468

Table 3 4 Diffusion coefficients for GOx and HRP immobilized particles in 01 M

glucose and purged with O2

Mean

GOx- HRP-

functionalized

particles in 01 M

glucose

Control ndash Gox-

HRP- functionalized

particles in DI water

Control - particles

in 01 M glucose

Diffusion coefficient

(x10-10

cm2s)

13005 14434 13702

Standard deviation 5263 6622 7468

No significant increase was observed in diffusion coefficient of enzyme immobilized

particles in any of the systems

39

34 Discussion

Formation of fan-shaped structures due to patterned deposition of oxidized TMB

indicates some kind of interaction between GOx and HRP immobilized particles Particle

diameter dependence of the size of structures can be explained with a simple model in

Figure 3 6 A larger particle will have a higher surface area as compared to a smaller

particle Hence more enzyme is immobilized on the larger particle As a result higher

quantity of TMB is oxidized and precipitated by the larger particle as compared to the

smaller particle This leads to thicker strands of fan-shaped structures eventually scaling

up the whole system to give bigger fan-shaped structures

The exact reason for the formation of such fan-shaped structures which are dependent on

particle size could not be determined It is hypothesized that hydrogen peroxide andor

Figure 3 6 Model for size dependence of fan-shaped structures on the particle

diameter of enzyme immobilized particles Larger the particle more surface area is

available for precipitation of TMB which eventually leads to bigger fan-shaped

structures

Larger particle Smaller particle

40

TMB gradient in the substrate solution might be responsible for such structures If there

is higher concentration of H2O2 on one side of the particle and assuming an even

concentration distribution of TMB there will be selective precipitation in the direction of

H2O2 When several particles come in the vicinity of higher concentration of H2O2 the

precipitation lines will converge to give fan-shaped structures (Figure 3 7)

Figure 3 7 Hypothesis for formation of fan-shaped structures

In the system consisting of GOx immobilized particles HRP immobilized particles

glucose and TMB it was observed that the fan-shaped structures formed at the liquid-air

interface and eventually settled down to the bottom of the glass slide This makes sense

as oxygen is necessary for the catalytic reaction of conversion of glucose to gluconic acid

and hydrogen peroxide which takes place on the surface of GOx immobilized particle As

a result H2O2 is primarily formed at the liquid-air interface and is then used by HRP for

precipitation of TMB

Following the observations that precipitation of TMB occurs at the surface GOx and

HRP immobilized particles are also expected to interact at the surface in the absence of

TMB Due the cascade reaction it was expected that HRP immobilized particles will be

41

attracted to GOx immobilized particles in appropriate substrate solution Experiments

were carried out to visualize inter-particle interactions at the liquid-air interface

However liquid-air interface not being a stable system to image the precise interaction

of GOx and HRP immobilized particles could not be determined Significant external

disturbances were involved at the interface To enable the cascade reaction in the bulk of

the liquid oxygen was purged into the reaction system before starting the experiment

However this did not lead to significant reaction in the bulk of the liquid and the reaction

prominently took place at the liquid-air interface where the oxygen concentration was

higher

In order to observe the interaction of GOx and HRP immobilized particles better imaging

techniques need to be used to observe diffusion of particles in absence of external

disturbances at the liquid-air interface Large quantity of oxygen needs to be purged into

the system and retained to make the reaction more pronounced in the bulk of the substrate

solution Higher concentration of oxygen in water may also be achieved by using oxygen

enriched water which is commercially available55

Alternatively a different enzyme cascade system may be used which does not require a

gaseous substrate56

For example glycogen synthase synthesizes glycogen from glucose-

1-phosphate and glycogen phosphorylase breaks down glycogen to glucose-1-

phosphate57

This system does not require a gaseous substrate and might be able to

overcome the deficiencies of GOx and HRP enzyme cascade system

42

Chapter 4

Silver-titania nanomotors

41 Introduction

Bimetallic nanowires made of platinum and gold have been developed by WPaxton

etal1 The rods move autonomously in a solution of hydrogen peroxide In the present

study silver-titania (Ag-TiO2) particles are being synthesized and investigated for motion

in presence of hydrogen peroxide and UV light With proper control of conditions and

UV light trigger it can be possible to control the speed and direction of motion of these

nanoparticles

Hydrogen peroxide is produced in the human body by phagocytes as an immune response

to foreign particles and pathogens5859

Also silver nanoparticles have been shown to be

biocompatible and capable of interacting with cells in human body without disrupting

cellular function60

Similarly it has been demonstrated that titania is compatible with

different cells DNA and proteins in human body and does not cause toxic effects Hence

silver-titania nanomotors which swim in hydrogen peroxide may open interesting

avenues in applications like targeted drug delivery in those parts of the body where

hydrogen peroxide is present

43

42 Experimental

421 Materials and methods

Silver nitrate (999995) was purchased from Alfa Aesar Gum arabic was purchased

from MP Biomedicals Hydrogen peroxide solution 50 wt in water and ascorbic acid

were purchased from Sigma-Aldrich Nanopure deionized water was used for all

experiments and preparation of all solutions Secure-Seal imaging spacers (9 mm

diameter) were obtained from Sigma Aldrich All experiments were carried out at room

temperature of 25ordmC unless otherwise mentioned Ag-TiO2 particles were synthesized by

evaporation of titania in Semicore electron beam evaporator Particles were characterized

using field-emission scanning electron microscopy Optical microscopy experiments

were performed using Axiovert 200 MAT microscope by Carl Zeiss Particles were

tracked using Physvis particle tracking software (Figure 4 1) in which the position

information of a particle on screen is stored on a frame by frame basis The data is stored

in a dat file as X and Y co-ordinates of particles tracked on the screen This information

can be converted to diffusion coefficients of the particles by further manipulation using

MathWorks-MATLABreg software

44

Figure 4 1 Physvis particle tracking software

422 Synthesis of silver colloidal particles

Silver particles were synthesized by a nucleationgrowth type reaction developed by

Goya and Matijevic61

and modified by Velikov62

Silver nitrate was used as precursor

metal salt for synthesis of silver particles Solution A was 75 ml of 50 mM AgNO3 and

035 by weight gum arabic Solution B was 75 mL of 100 mM 1-ascorbic acid and

035 by weight gum arabic Solution B was rapidly added to solution A in a 250 mL

reaction flask The reaction flask was covered and particles were stirred for 48 hrs After

48 hours particles were allowed to settle for 2-3 hours in refrigerator and supernatant

was separated from the particles Particles were then rinsed with DI water and centrifuged

thrice to obtain pure silver particles Zeta potential of these particles was reported to be

45

around -50 to -60 mV and the size was around 1μm The size of the particles can be

varied by varying the amounts of ascorbic acid and silver nitrate Using molar ratio of 31

of ascorbic acid silver nitrate can give particles of 200 nm diameter whereas ratio of

151 can give particles of upto 5 μm diameter

423 Preparation of silver-titania (Ag-TiO2) particles

A monolayer of silver particles was prepared according to the procedure by Goldenberg

et al63

Glass slides were placed on the bottom of a petridish Water was put into the

petridish so that the entire slide was immersed in water A thin layer of hexane was then

added on top of water in the petridish A suspension of silver particles in ethanol (5

ww) was slowly introduced to the water-hexane interface in the dish After the

monolayer was formed the glass slide was lifted up through the monolayer with tweezers

The silver particles-covered glass slides were left at room temperature to dry overnight

Following drying a thin film of titania was evaporated on the glass slides in Semicore

evaporator (Figure 2 7) The spheres were partially coated with a 10 nm film of titania

Ag-TiO2 particles were removed from the glass slide by gentle sonication FE-SEM was

used to characterize the particles (Figure 4 2) The thin titania layer was not easily

distinguishable due to roughness of the particles

46

Figure 4 2 FESEM image of Ag-TiO2 particles

424 Optical microscopy experiments

Imaging spacers were placed on a glass slide Suspension of Ag-TiO2 particles was

placed on the glass slide and the particles were allowed to settle Movement of particles

was observed in DI water and videos were recorded at 50x magnification of optical

microscope for 30 sec 05 H2O2 was added to the particle suspension on the slide

Videos were recorded at 50x ranging from 30 sec to 10 min to study the movement of

silver titania particles in hydrogen peroxide solution Particles in both DI water and

hydrogen peroxide were subjected to UV light and videos were recorded to study particle

dynamics in presence of UV radiation Particle tracking was performed using Physvis

particle tracking software Atleast 30 particles in every system were tracked for 10

seconds each Particle tracking data was obtained in terms of X and Y coordinates of

particles on screen This data was manipulated using MathWorks-Matlab software and

graphs of mean square displacement of particles versus time were plotted Diffusion

coefficients of particles were calculated from these graphs

47

43 Results and Discussion

Ag-TiO2 particles demonstrated autonomous motion in the presence of H2O2 No UV

light was required for this motion Motion of the particles was accelerated by addition of

fresh H2O2 to the system When subjected to UV radiation the particles displayed

momentary acceleration for 4-5 seconds

Figure 4 3 Mechanism of motion of Ag-TiO2 nanomotors

In the presence of H2O2 silver part of the Ag-TiO2 particle converts H2O2 to H2O and O2

This may lead to propulsion of the Ag-TiO2 particle due to bubble propulsion andor self-

diffusiophoresis In the presence of UV light additional reaction takes place on the

titania part of Ag-TiO2 particle which increases the velocity of the Ag-TiO2 particles

(Figure 4 4) This UV triggered photoactivity of titania is due to hole-electron separation

caused by photons having energy equal to or greater than the band gap of titania646566

Most of the holes-electrons recombine but a small fraction move to the surface and react

with the available redox species like water and oxygen to form protons (H+) superoxide

(O2-) and hydroxyl (OH) radicals

6566 This may lead to propulsion by diffusiophoresis

as well as electrophoresis

48

Figure 4 4 Silver-titania motors move faster when subjected to UV radiation

In addition to linear motion rotational motion is also observed in several particles and

particle aggregates Aggregation of particles can lead to a net circular propulsive force

which can rotate the aggregate as shown in Figure 4 5

Figure 4 5 Rotation due to particle aggregation of Ag-TiO2 particles

Particle tracking by Physvis software revealed a significant increase in diffusion

coefficient of Ag-TiO2 particles in hydrogen peroxide solution as compared to DI water

49

The Ag-TiO2 particles have a diffusion coefficient of 1491 μm2s in hydrogen peroxide

versus only 180 μm2s in DI water Thus the Ag-TiO2 particles move around more than

10 times their body length per second in hydrogen peroxide (Figure 4 6) When

subjected to UV light the particles experience a significant acceleration in hydrogen

peroxide which lasts only for a short duration of time (4-5 sec) after which the particles

return to their original velocity The diffusion coefficient of the particles during this UV

light trigger is found to be around 5093 μm2s UV light trigger does not have any visible

effect on the Ag-TiO2 particles in DI water

Table 4 1 Translational diffusion coefficients of Ag-TiO2 particles in hydrogen

peroxide and water

Mean 05 H2O2 solution DI water

Diffusion coefficient of Ag-TiO2 (μm2s)

without UV light

1491 180

Diffusion coefficient of Ag-TiO2 (μm2s)

in the presence of UV light

5093 113

50

Figure 4 6 Mean square displacement curves of Ag-TiO2 Janus particles in hydrogen

peroxide and DI water

When higher concentration (~30 vv) of Ag-TiO2 particles in hydrogen peroxide is

subjected to UV radiation the particles exhibit pattern formation (Figure 4 7)

Aggregation of Ag-TiO2 Janus particles is observed when UV light is turned on and the

particles disaggregated when UV light is switched off This can be due to predator-prey

kind of interaction between silver and titania half of the Janus particles in the presence of

UV light Oxygen and water that are produced at the silver end by decomposition of

hydrogen peroxide are used up by titania when subjected to UV radiation This can lead

to attraction between titania and silver parts of neighboring Janus particles causing

aggregation

y = 20373x

y = 59994x

y = 71848x

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10 12

Mean

sq

uare

dis

pla

cem

en

t (μ

m2s

)

Time (sec)

Mean square displacement vs Time for Ag-TiO2

particles

05 Hydrogen peroxide and UV05 Hydrogen peroxide

DI Water

51

A UV OFF B UV ON

C UV OFF

Figure 4 7 Higher concentrations of Ag-TiO2 particles showing aggregation when UV

is turned on

52

Chapter 5

Summary and Conclusion

In summary no significant enhancement in diffusion was observed in case of enzyme

immobilized Janus particles in substrate solution Presence of a linker between enzyme

and particle did not increase the diffusion coefficient of particles Alternative enzyme

immobilization methods need to be tested like thiolation of enzyme and subsequent

binding to gold surface of a Janus particle35

or expressing the enzyme with polyhistidine

or avidin tag67

This can result in higher enzyme activity upon immobilization and may

lead to enhanced diffusion of Janus particles

Predator-prey system of GOx and HRP immobilized particles showed some promising

results Aggregation of particles and formation of fan-shaped structures by TMB

oxidation were observed under optical microscope when GOx and HRP immobilized

particles glucose and TMB were present in the system The size of the fan-shaped

structures was dependent on the size of enzyme immobilized particles This can be

indicative of some kind of interaction between the GOx and HRP immobilized particles

No conclusive aggregation of GOx and HRP immobilized particles was observed in

absence of TMB Better imaging techniques are required to study the interaction of GOx

and HRP immobilized particles at the unstable liquid-air interface where majority of the

reaction takes place Other enzyme cascade systems not requiring gaseous reactants like

glycogen synthase and glycogen phosphorylase should be tested for similar activity

53

Ag-TiO2 particles were found to exhibit autonomous motion in the presence of hydrogen

peroxide solution The motion was momentarily accelerated upon exposure to UV

radiation The particles showed a diffusion coefficient of 1491 μm2s in hydrogen

peroxide versus only 180 μm2s in DI water Upon subjecting particles to UV radiation in

presence of H2O2 diffusion coefficient increased to 5093 μm2s for a period of 4-5

seconds In addition to linear motion rotating motion could also be observed in some

particle aggregates Further development of these aggregate structures can lead to

formation of rotors at nanoscale Higher concentration of silver-titania particles in

hydrogen peroxide displayed aggregation in presence of UV light and disaggregated

when UV light was switched off This may be attributed to the predator-prey kind of

interaction between silver and titania where oxygen and water that are produced by

silver by decomposition of hydrogen peroxide are used up by titania when subjected to

UV radiation This promises to be an interesting system and needs to be further

investigated to explore its properties

54

References

1 Paxton WF Sundararajan S Mallouk TE Sen A Angew Chem Int Ed Engl

2006 45 5420-5429

2 Golestanian R Liverpool TB Adjari A Phys Rev Lett 2005 94 220801

3 httpwwwsilvermedicineorgh2o2html

4 Rami Pedahzur Ovadia Lev Badri Fattal Hillel Shuval Water Science and Technology

1995 31(5-6) 123-129

5 W Went F American Scientist 1968 56

6 Adriano Cavalcanti Tad Hogg Bijan Shirinzadeh IEEE MHS 2006 International

Symposium on Micro-NanoMechatronics and Human Science 2006 226-232

7 EM Purcell American Journal of Physics 1977 45 3-11

8 Golestanian R Phys Rev Lett 2010 105 018103(1)-018103(4)

9 S S Dukhin Z R Ulberg G L Dvornichenko B V Deryagin Russian Chemical

Bulletin 1983 31(8) 1535-1544

10 Dukhin AS Goetz PJ Ultrasound for characterizing colloids Particle sizing zeta

potential rheology Elsevier 2002 59

11 HJKeh HJTu Trends in Colloid and Interface Science XIV Progress in Colloid and

Polymer Science 2000 115 174-180

12 D Zinemanas A Nir International Journal of Multiphase Flow 1995 21(5) 787-800

55

13 Stephen Ebbens Jonathan Howse Soft matter 2010 6 726-739

14 J Vicario R Eelkema W R Browne A Meetsma R M La Crois B L Feringa

Chem Commun 2005 31 3936

15 Davide Pantarotto Wesley R Browne Ben L Feringa Chem Commun 2008 1533 -

1535

16 Lisa A Cameron Matthew J Footer Alexander van Oudenaarden Julie A Theriot

PNAS 1999 96(9) 4908-4913

17 Ali Najafi Ramin Golestanian Phys Rev E 2004 69 062901

18 Jinseok Kim Jungyul Park Sungwook Yang Jeongeun Baek Byungkyu Kim Sang

Ho Lee Eui-Sung Yoon Kukjin Chun Sukho Park Lab Chip 2007 7 1504 - 1508

19 Kurt M Dubowski Clinical Chemistry 2008 5411 1919ndash1920

20 Spector DA Yang Q Wade JB Am J Physiol Renal Physiol 2007 292(1) 467-474

21 Rhee SG Chang TS Bae YS Lee SR Kang SW J Am Soc Nephrol 2003 14(8-3)

211-215

22 Sakaue T Kapral R Mikhailov A S Eur Phys J B 2010 75 381ndash387

23 Muddana H S Sengupta S Mallouk T E Sen A Butler P J J Am Chem Soc

2010 132 (7) 2110ndash2111

24 Baker CJ Deahl K Domek J Orlandi EW Arch Biochem Biophys 2000 382(2) 232-

237

25 V Vojinovic RH Carvalho F Lemos JMS Cabral LP Fonseca BS Ferreira

Biochemical Engineering Journal 2007 35 126-135

56

26

OV Lebedeva NN Ugarova Russian Chemical Bulletin 1996 45(1) 18-25

27 Nigel C Veitch Phytochemistry 2004 65(3) 249-259

28 Kenneth H Petersen Journal of Immunological Methods 2009 340(1) 86-89

29 httpwwwbrenda-enzymesorgphpresult_flatphp4ecno=11117

30 Goldenberg L M Wagner J Stumpe J Paulke B-R Goumlrnitz E Langmuir 2002 18

5627-5629

31 httpwwwgoldbiocompdf3761-Protocol201pdf

32 Av Rovisco Pais Lisboa Portugal T amp D Technology West Yorkshire UK Journal

of Molecular Catalysis B Enzymatic 2004 28(2-3) 129-135

33 httpwwwphysicsnyuedugrierlabmethodsnode11html

34 Yoon-Mee Lee1 O-Yul Kwon Yeo-Joon Yoon Keungarp Ryu Biotechnology Letters

2006 28 39ndash43

35 JC Pyun SD Kim JW Chung Analytical Biochemistry 2005 347(2) 227-233

36 George H Pimbley Jr Mathematical Biosciences 1974 20(1-2) 27-51

37 Andy Fenton Sarah E Perkins Parasitology 2010 137 1027-1038

38 Mead James G Brownell Robert L Mammal Species of the World A Taxonomic and

Geographic Reference (3rd ed) Johns Hopkins University Press 2005 2 2142

57

39

Krause KH Schweiz Med Wochenschr 2000 130(4) 97-100

40 Irina Kareva Faina Berezovskaya Carlos Castillo-Chavez Journal of Biological

Dynamics 2010 4(4)315 - 327

41 Delaittre G Reynhout I Cornelissen J Nolte R Chemistry ndash A European Journal

2009 15 12600ndash12603

42 C Buller DH Johnson RC Tschumper Investigative Ophthalmology amp Visual Science

1990 31 2156-2163

43 Robert M Mazo Brownian Motion Fluctuations Dynamics and Applications Oxford

Science Publications 2009 57

44 Vivek K Mutalik KV Venkatesh Theoretical Biology and Medical Modelling 2005 2

19

45 Satoshi Nakamura Kunimasa Koga Biochemical and Biophysical Research

Communications 1977 78(2) 806-810

46 Krishnananda Chattopadhyay Shyamalava Mazumdar Biochemistry 2000 39 (1)

263ndash270

47 Satoshi Nakamura Yasuyuki Ogura J Biochem 1968 63 (3) 308-316

48 van Dongen SF Nallani M Cornelissen JJ Nolte RJ van Hest JC Chemistry 2009

15(5) 1107-1114

49 Ronald Bentley Methods in Enzymology 1955 1 340-345

50 Vartiainen J Raumlttouml M Paulussen S Packaging Technology and Science 2005

18 243ndash251

58

51 GA Sherbeny AA Shindia YMMM Sheriff International Journal of Agriculture

and Biology 2005 7

52 D Rando G-W Kohring F Giffhorn Appl Microbiol Biotechnol 1997 48

53 Worthington enzyme manual Worthington Biochemical Corporation

httpworthington-biochemcomgopdefaulthtml

54 httpwwwbrenda-enzymesorgphpresult_flatphp4ecno=11117

55 httpwwwoxygenphwatercom

56 P Boon Chock Sue Goo Rhee and Earl R Stadtman Annu Rev Biochem 1980 49

813-841

57 Sybil Golden P A Wals Joseph Katz Analytical Biochemistry 1977 77(2) 436-445

58 R L Baehner S K Murrmann J Davis R B Johnston Jr J Clin Invest 1975 56(3)

571ndash576

59 I Bejarano M P Terroacuten S D Paredes C Barriga A B Rodriacuteguez J A Pariente

Molecular and Cellular Biochemistry 2007 296(1-2) 77-84

60 Michael C Moulton Laura K Braydich-Stolle Mallikarjuna N Nadagouda Samantha

Kunzelman Saber M Hussain Rajender S Varma Nanoscale 2010 2 763-770

61 Goya DV Matijevic E Colloids and Surfaces A 1999 146 139

62 Velikov KP Zegers GE van Blaanderen A Langmuir 2003 19 1384

63 Goldenberg L M Wagner J Stumpe J Paulke B-R Goumlrnitz E Langmuir 2002 18

5627-5629

59

64

Banerjee S Gopal J Muraleedharan P Tyagi A K Raj B Curr Sci 2006

901378ndash1383

65 Bogomolov V N Kudinov E K Firsov Y A Sov Phys Solid State 1968 35 555

66 Kudo A Miseki Y Chem Soc Rev 2009 38 253ndash278

67 Gaj T Meyer SC Ghosh I Protein expression and Purification 2007 56 54-61

18

224 Enzyme assay

Assay for activity of HRP was performed using a spectrophotometric method which

involved using 4-aminoantipyrene as hydrogen donor3132

The reaction rate was

determined by measuring increase in absorbance at 510 nm resulting from the

decomposition of hydrogen peroxide One unit represents the decomposition of 1 μM of

hydrogen peroxide per minute at 25 degC and pH 70 under the specified conditions

The spectrophotometer was set to the wavelength of 510 nm and temperature of 25 degC

Phenol4-aminoantipyrene solution (14 mL) and 00017 M H2O2 solution (15 mL) were

pipetted into each cuvette The cuvette was incubated for 3-4 mins at 25degC and blank was

established A 01 mL solution of diluted enzyme was added to the above mixture and

absorbance was recorded for 4-5 mins Standard graphs of absorbance versus time were

drawn for known concentration of the free enzyme and compared to that of the

immobilized enzyme on the Janus particles The estimate of the amount of enzyme

present on the Janus particles was deduced from the difference between the quantity of

the free enzyme previously added for immobilization and the amount that was measured

to be in the supernatant after some of the enzyme became attached to the Janus particles

The experiment was carried out in triplicate to assess reproducibility

19

The formula used to calculate the activity of enzyme is

where ∆Amin denotes the change in absorbance per minute

225 Diffusion coefficient measurements using Nanosight LM10

Diffusion coefficients for HRP immobilized particles were measured using Nanosight

LM 10 nanoparticle tracking instrument with Nanoparticle Tracking Analysis (NTA) 20

software in the presence of 003 H2O2 solution as the substrate and also in the presence

of only deionized water The videos were recorded for 30 seconds at the rate of 30 frames

per second Each experiment was carried out in triplicate to assess reproducibility

Particles

visualization

zone

Microscope with

LM 10 unit

LM 10 unit

Figure 2 8 Nanosight LM 10 instrumentation

20

Nanosight LM 10 (Figure 2 8) generates videos of population of moving nanoparticles in

a liquid when illuminated by laser light The particles which pass through the laser beam

path are seen by the instrument as small points of light undergoing Brownian motion

(Figure 2 9a)

The videos are further analyzed by NTA (Nanoparticle tracking analysis) software

(Figure 2 9b) which identifies and tracks each particle independently on a frame-by-

frame basis The software also calculates and subtracts any bulk flow in the system The

mean square displacement is calculated for each particle as long as it is visible If the

particle blinks that is if it disappears momentarily and then reappears on the screen the

software will consider the same particle as two separate ones All analysis data is

exported to the excel files in terms of diffusion coefficients of the tracked particles

B A

Figure 2 9 NTA video analysis (a) Particles as they are seen on the screen during

experimentation in Nanosight LM10 (b) NTA screen as the particles are being analyzed for

their diffusion coefficients

21

23 Results

231 Enzyme assay

The activity of free HRP enzyme reported by Sigma Aldrich is 150 unitsmg However

the activity of HRP upon analysis was found to be 52158 unitsmg which is much lower

than the reported value The activity of HRP was found to be further lowered to 2133

unitsmg when it was immobilized on the PS-SiO2 Janus particles in the presence of a

linker The following formula is used to calculate enzyme activity

where ΔAmin is the change in absorbance per minute

22

Figure 2 10 Graph for the assay of solution of free enzyme containing 05mgL of HRP

ΔAmin = 01716 Unitsmg of free enzyme = 52158

During EDCNHS coupling 04 mg enzyme was added to 1 ml of 025 vv suspension

of Janus particles Thus initial concentration of enzyme in the mixture was 04 mgml

Assuming retained activity of the enzyme in supernatant solution the amount of enzyme

in supernatant was calculated to be 03868 mgml from the assay Amount of enzyme

functionalized on the Janus particles was calculated as the difference between enzyme

previously added for immobilization (04 mgml) and the enzyme present in supernatant

(03868 mgml) and was found to be 00132 mgml Enzyme assay was performed on the

enzyme immobilized Janus particles to estimate the activity of immobilized enzyme

(Figure 2 11) and was found to be 2133 unitsmg

y = 0002x + 00516

0

01

02

03

04

05

06

07

08

0 50 100 150 200 250 300 350 400

Ab

sorb

an

ce

51

0 n

m

Time (sec)

Assay of free enzyme HRP

23

Figure 2 11 Graph for the assay of of immobilized enzyme on 05 μm Janus particles

(~025 vv) ΔAmin = 01853 Unitsmg of free enzyme = 2133

232 Diffusion coefficient measurements

No significant increase was observed in diffusion coefficient of HRP-immobilized Janus

particles in the presence of H2O2 as the substrate Similar results were obtained for

diffusion coefficient measurement with and without linker moieties bridging the enzymes

and particles(Table 2 1 Table 2 2) Thus the linker did not seem to assist in increasing

the particle diffusion by improving or retaining the activity of the enzyme

y = 00015x + 00953

0

01

02

03

04

05

06

07

0 50 100 150 200 250 300 350 400

Ab

sorb

an

ce

510 n

m

Time (sec)

Assay of HRP immobilized 05 μm Janus particles

24

Table 2 1 Diffusion coefficient measurements of HRP immobilized Janus particles

without linker

HRP immobilized

Janus particles in

003 H2O2

Control - Janus

particles in

003 H2O2

Control - HRP

immobilized Janus

particles in DI water

Diffusion coefficient

(10-10

cm2s)

1359416 1051777 1308675

Standard deviation 4534801 4122075 4918597

Table 2 2 Diffusion coefficient measurements of HRP immobilized Janus particles in

the presence of linker

HRP immobilized

Janus particles in

003 H2O2

Control - Janus

particles in

003 H2O2

Control - HRP

immobilized Janus

particles in DI water

Diffusion coefficient

(10-10

cm2s)

13838 1339829 1407891

Standard deviation 5644692 5649364 5797218

25

Figure 2 12 Diffusion coefficient measurements of PS-SiO2 Janus particles without and

with 6-aminohexanoic acid linker

24 Discussion

To compare the diffusion coefficients observed during the experiments theoretical

diffusion coefficient was calculated using following equation33

r

kTD altraslation

6 Equation 21

where r is the hydrodynamic radius of particle Dtranslational is translational diffusion

coefficient of the particle T is temperature of the system η is the viscosity of fluid

through which the particles are moving and k is the Boltzmann constant From FE-SEM

images the size of the particles used was determined to be 450 nm For 450 nm diameter

0

20

40

60

80

100

120

140

160

Without linker With linker

Dif

fusio

n c

oeff

icie

nt

(10

-10 c

m2s

)

HRP immobilized 05 μm Janus particles - Diffusion coefficient measurements

HRP immobilized Janus particles in 003 H2O2

Control - Janus particles in 003 H2O2

Control - HRP immobilized Janus particles in DI water

26

particle Dtranslational is equal to 109 μm2s which is comparable to the diffusion

coefficient measured for the enzyme immobilized particles

HRP was found to be active on the Janus particles however the activity of the enzyme

was reduced upon immobilization Lower enzyme activity can be due to the coupling

method used which in this case was EDCNHS covalent binding However EDCNHS

coupling method has been shown to significantly retain the enzyme activity when the

enzyme is immobilized on carboxylated multi-walled carbon nanotubes under appropriate

conditions34

Hence EDCNHS binding method by itself is not believed to be

responsible for reduction of enzyme activity upon immobilization

Direct binding of enzyme to the Janus particle may lead to reduction in the enzyme

activity because of space constraints for the enzyme and difficulty for the passage of

substrate to the active site of the enzyme due to stearic hindrance Hence a linker was

inserted between the enzyme and Janus particle during enzyme immobilization However

inserting a linker between the enzyme and the particle did not lead to enhanced diffusion

of Janus particles or increase in enzyme activity of immobilized enzyme

Lack of enhanced diffusion can be firstly attributed to the reduction in enzyme activity

upon immobilization Enzyme activity may be retained by modifying the conditions

during EDCNHS coupling or changing the binding method altogether The degree of

immobilization of HRP on carboxylated Janus particles can possibly be affected by

several factors such as temperature the concentration of the coupling reagent EDC

27

concentration HRP concentration in solution and incubation time34

Different

combinations of these factors can be tested to find optimum conditions for

immobilization of HRP Alternate binding method can involve thiolation of HRP by the

covalent conjugation of mercaptopropionic acid and then the thiolated HRP can then be

attached on the gold surface of a polystyrene-gold Janus particle35

Thus further

modifications can be made to the system in order to retain enzyme activity on the

immobilized particles and thereby power nanomotors using enzymes

28

Chapter 3

Predator-prey system of enzyme immobilized particles

31 Introduction

Predator-prey systems are widely found in nature and play a major role in biological

processes3637

Predator-prey systems include all the interactions in which one organism

pursues andor consumes another organism One familiar predator-prey system at the

macroscale is that of a tiger chasing and consuming a deer Another not so familiar one

includes Baleen whales which eat millions of microscopic planktons at once38

At the

microscale such kind of interactions can be observed in the case of phagocytes in the

body which are capable of engulfing foreign microorganisms and killing them with a

combination of microbicidal systems39

In the case of cancer the tumor is the prey and

the mature myeloid cells in the body are the predator However the tumor here has a

defense mechanism against the predator in which the tumor produces cytokines which

inhibits the maturation of the myeloid cells and thereby prevents predation40

One similarity that can be observed in all these systems is that the prey possesses some

attribute which is desired by the predator which makes the predator chase the prey A

similar relationship is seen in case of enzymes which are involved in cascade reactions41

In cascade reactions product synthesized by one enzyme is the reactant required by

another enzyme to carry out its own catalytic reaction As such there is a predator-prey

29

kind of dependence between the two enzymes which may cause one enzyme to chase the

other enzyme when placed in suitable substrate solutions However the mechanism

hypothesized for such a system is totally different than the mechanism involved in

biological systems In biological systems such as bacterial chemotaxis or phagocytes

chasing foreign particles in human body complex biochemical reactions are responsible

for the predator-prey behavior42

In the proposed enzyme cascade system there are no

complex biochemical reactions involved The principle hypothesized for predator-prey

behavior of enzyme cascade reactions can be explained as follows Motion of synthetic

motors at nanoscale involves two components translational diffusion and rotational

Brownian diffusion At very short time scales of less than 1Dr where Dr (s-1

) is

rotational Brownian diffusion coefficient the motion of particles is unidirectional For

longer time scales of greater than 1Dr the direction of motion is random giving

poweredactive diffusion43

If the chemical homogeneity is broken that is when there is

higher concentration of a substrate present in one direction of the enzyme the enzyme

might acquire a subtle bias which may lead to a spatial aggregation of the enzymes based

on the substrate concentration gradient The bias can be a result of the relationship

between the speed of enzyme diffusion and substrate concentration where higher

substrate concentration lead to higher enzyme diffusion As a result in an enzyme

cascade system one enzyme will migrate towards higher concentration of substrate

which is present near the other enzyme thus giving rise to a predator-prey behavior

Such enzyme cascades are widely found in signaling networks and metabolic pathways in

living cells and can be used to produce predator-prey like nanomotors systems44

The

30

enzymes being used in the present study are horseradish peroxidase and glucose oxidase

Both enzymes are extremely stable4546

Glucose is the substrate for glucose oxidase

which is widely available and highly stable47

Horseradish peroxidase acts on hydrogen

peroxide It is involved in a cascade reaction with glucose oxidase which produces

hydrogen peroxide by converting glucose into gluconic acid (Figure 3 1)48

Figure 3 1 Concept of particle aggregation due to cascade reactions of immobilized

enzymes

In this experiment glucose oxidase (GOx) and horseradish peroxidase (HRP) were

immobilized on polystyrene carboxylate beads of different sizes using EDCNHS

coupling method Glucose and 3355-Tetramethylbenzidine (TMB) were dissolved

together in water and this solution was used as the substrate solution All the components

were present in a single system in DI water Aggregation of particles and precipitation of

oxidized TMB from the solution was expected to be observed when all these components

were present in such a system

Properties of horseradish peroxidase have been discussed in the previous chapter (Section

211 Horseradish peroxidase)

GOxGOx HRPHRP

Glucose + O2Gluconic acid + H2O2

TMB TMB oxidizedBlue precipitate

H2O

Aggregation

31

311 Glucose oxidase

Figure 3 2 Glucose oxidase

Glucose oxidase (GOx) belongs to the group of oxidoreductases and is also called

glucose aerodehydrogenase49

It is obtained from fungi and has been identified as an

antimicrobial enzyme50

GOx binds to β-D-glucopyranose and catalyzes the oxidation

reaction to from gluconolactone while simultaneously reducing oxygen to hydrogen

peroxide51

GOx has high substrate affinity and specificity high stability and high

turnover number which makes it a suitable biocatalyst for industrial applications52

Figure 3 3 Redox reaction of glucose catalyzed by glucose oxidase53

32

GOx is a glycoprotein with molecular weight of 155 kDa and consists of two identical

polypeptide subunits linked together by a disulfide bond The optimum pH for GOx is

55 though it is stable in a broad pH range of 4 to 7 and can be stored for a long period of

time51

The functional parameters for glucose oxidase include Km of 151 mM and kcat of

337 s-1

at temperature of 20 ordmC and pH of 754

Some of the inhibitors of GOx are 2-

Deoxy-D-glucose adenine nucleotides and NaNO3

32 Experimental

321 Materials and methods

Uniform carboxyl modified polystyrene microspheres 05 microm in diameter were obtained

from Polysciences Inc The size of the purchased microspheres was verified by FESEM

Peroxidase Type I from horseradish glucose oxidase Type II-S from Aspergillus niger

N-(3-dimethyl-aminopropyl)-Nrsquo-ethylcarbodiimide (EDC) and Hydrogen peroxide

solution 50 wt in water were purchased from Sigma-Aldrich N-hydroxysuccinimide

(NHS) was purchased from Fluka D-Glucose (99) was purchased from VWR

International Secure-Seal imaging spacers (9mm diameter) for glass slides were obtained

from Sigma Aldrich All chemicals were of reagent grade and used without further

purification Nanopure deionized water was used for all experiments and preparation of

solutions All measurements were made at room temperature (25 oC) unless mentioned

otherwise The UV-Visible measurements were performed using Agilent 8453A diode-

array UV-Visible Spectrophotometer with ChemStation UV-Vis Software Optical

33

microscopy experiments were performed using Axiovert 200 MAT microscope by Carl

Zeiss Diffusion coefficient measurements were obtained using Nanosight LM10

322 Optical microscopy experiments

The enzyme substrate system was observed under optical microscope to check for

aggregation and precipitation due to cascade reactions Several such systems were

studied Each system in general consisted of GOx and HRP immobilized on different

polystyrene particles The substrate solutions consisted of 01 M glucose and different

concentrations of TMB solution The systems were studied in the presence of all the

constituents and in the absence of glucose GOxHRP and TMB Systems consisting of

different sized HRP immobilized particles in substrate solution of 003 hydrogen

peroxide and TMB were also studied Videos were recorded over a period of 24 hours to

observe changes in the system Each experiment was performed atleast thrice to assess

reproducibility of results

323 Diffusion coefficient measurements using Nanosight LM10

Diffusion coefficient of GOx and HRP immobilized particles were measured using

Nanosight LM10 nanoparticle tracking instrument with Nanoparticle Tracking Analysis

(NTA) 20 software Diffusion coefficients were measured for different systems namely

GOx particles in 01 M glucose and oxygen both GOx and HRP functionalized particles

in 01 M glucose and oxygen along with controls for the individual systems Videos were

34

recorded for 30 seconds at the rate of 30 frames per second Each experiment was carried

out in triplicate to assess reproducibility

33 Results

331 Optical microscopy

According to the enzyme cascade model precipitation of TMB was possible only upon

the interaction of GOx and HRP immobilized particles in presence of appropriate

substrate solution Hence in the first set of experiments precipitation of TMB was used

as an indication of the type of interaction between GOx and HRP immobilized particles

Fan-shaped structures were observed when both HRP and GOx immobilized particles

were present in a substrate solution consisting of glucose and TMB These structures

started forming as a single strand and then branched out and intertwined to form the fan-

shaped structures (Figure 3 4)

Table 3 1 Observations for formation of fan-shaped structures in enzyme cascade

system in presence of different components

GOx HRP Glucose TMB Observation

Yes Yes Yes Yes

Fan-shaped structures observed particles associated with

the fan-shaped structures

No Yes Yes Yes No fan-shaped structures

Yes No Yes Yes No fan-shaped structures

Yes Yes No Yes No fan-shaped structures

Yes Yes Yes No No fan-shaped structures

35

Fan-shaped structures can also be observed in the system consisting of only HRP

immobilized particles in substrate solution of hydrogen peroxide and TMB The size of

the fan-shaped structures depends on the size of the enzyme immobilized particles Free

enzymes show extremely tiny structures which are difficult to view under the optical

microscope

Figure 3 4 Formation of fan-shaped structures when both HRP and GOx

immobilized particles were present in a substrate solution consisting of glucose and

TMB A In the initial stages single strands form on the surface of liquid (5Ox) B

Single strands branch out and intertwine to form fan-shaped structures (50x) C Many

such structures form and settle down after 20 minutes (20x)

A

C

B

36

Experiments were performed in the absence of TMB that is the system consisted of GOx

and HRP immobilized particles in glucose substrate solution In the absence of TMB the

particles were expected to move closer to each other without any precipitation due to the

cascade reaction However there was no conclusive aggregation of particles observed in

the system

Free Enzyme 005 μm HRP - immobilized particle

10 μm HRP - immobilized particle 05 μm HRP - immobilized particle

Figure 3 5 Size dependence of fan-shaped structures on the size of HRP -

immobilized particles (20x)

37

Table 3 2 Observations for particle aggregation in enzyme cascade system in presence

and absence of TMB

Glucose GOx HRP TMB Observation

Present Present Present Present Aggregation and precipitate

Present Present Present Absent No conclusive aggregation

Present Absent Present Present No aggregation or ppt

Absent Present Present Present No aggregation or ppt

Present Present Absent Present No aggregation or ppt

332 Diffusion coefficient measurements

Diffusion coefficients were measured for GOx immobilized particles of 05 μm diameter

in substrate solution of 01 M glucose and oxygen Diffusion coefficients of particles

were also measured for system consisting of GOx and HRP functionalized particles in

glucose and oxygen Each experiment was done in triplicate to assess reproducibility

Nanosight visualization chamber being a closed chamber oxygen was purged into all the

solutions before starting the experiments to ensure sufficient supply of oxygen for

catalytic reaction of GOx

38

Table 3 3 Diffusion coefficients for 05 μm GOx immobilized particles in 01 M

glucose purged with O2

Mean

GOx

particles in

01 M

glucose

Control - GOx

particles in DI

water

Control - GOx

particles in 01 M

glucose without

oxygen purging

Control -

particles in 01

M Glucose

Diffusion

coefficient

(x10-10

cm2s)

13316 12888 13827 13702

Standard

deviation 6307 4926 4424 7468

Table 3 4 Diffusion coefficients for GOx and HRP immobilized particles in 01 M

glucose and purged with O2

Mean

GOx- HRP-

functionalized

particles in 01 M

glucose

Control ndash Gox-

HRP- functionalized

particles in DI water

Control - particles

in 01 M glucose

Diffusion coefficient

(x10-10

cm2s)

13005 14434 13702

Standard deviation 5263 6622 7468

No significant increase was observed in diffusion coefficient of enzyme immobilized

particles in any of the systems

39

34 Discussion

Formation of fan-shaped structures due to patterned deposition of oxidized TMB

indicates some kind of interaction between GOx and HRP immobilized particles Particle

diameter dependence of the size of structures can be explained with a simple model in

Figure 3 6 A larger particle will have a higher surface area as compared to a smaller

particle Hence more enzyme is immobilized on the larger particle As a result higher

quantity of TMB is oxidized and precipitated by the larger particle as compared to the

smaller particle This leads to thicker strands of fan-shaped structures eventually scaling

up the whole system to give bigger fan-shaped structures

The exact reason for the formation of such fan-shaped structures which are dependent on

particle size could not be determined It is hypothesized that hydrogen peroxide andor

Figure 3 6 Model for size dependence of fan-shaped structures on the particle

diameter of enzyme immobilized particles Larger the particle more surface area is

available for precipitation of TMB which eventually leads to bigger fan-shaped

structures

Larger particle Smaller particle

40

TMB gradient in the substrate solution might be responsible for such structures If there

is higher concentration of H2O2 on one side of the particle and assuming an even

concentration distribution of TMB there will be selective precipitation in the direction of

H2O2 When several particles come in the vicinity of higher concentration of H2O2 the

precipitation lines will converge to give fan-shaped structures (Figure 3 7)

Figure 3 7 Hypothesis for formation of fan-shaped structures

In the system consisting of GOx immobilized particles HRP immobilized particles

glucose and TMB it was observed that the fan-shaped structures formed at the liquid-air

interface and eventually settled down to the bottom of the glass slide This makes sense

as oxygen is necessary for the catalytic reaction of conversion of glucose to gluconic acid

and hydrogen peroxide which takes place on the surface of GOx immobilized particle As

a result H2O2 is primarily formed at the liquid-air interface and is then used by HRP for

precipitation of TMB

Following the observations that precipitation of TMB occurs at the surface GOx and

HRP immobilized particles are also expected to interact at the surface in the absence of

TMB Due the cascade reaction it was expected that HRP immobilized particles will be

41

attracted to GOx immobilized particles in appropriate substrate solution Experiments

were carried out to visualize inter-particle interactions at the liquid-air interface

However liquid-air interface not being a stable system to image the precise interaction

of GOx and HRP immobilized particles could not be determined Significant external

disturbances were involved at the interface To enable the cascade reaction in the bulk of

the liquid oxygen was purged into the reaction system before starting the experiment

However this did not lead to significant reaction in the bulk of the liquid and the reaction

prominently took place at the liquid-air interface where the oxygen concentration was

higher

In order to observe the interaction of GOx and HRP immobilized particles better imaging

techniques need to be used to observe diffusion of particles in absence of external

disturbances at the liquid-air interface Large quantity of oxygen needs to be purged into

the system and retained to make the reaction more pronounced in the bulk of the substrate

solution Higher concentration of oxygen in water may also be achieved by using oxygen

enriched water which is commercially available55

Alternatively a different enzyme cascade system may be used which does not require a

gaseous substrate56

For example glycogen synthase synthesizes glycogen from glucose-

1-phosphate and glycogen phosphorylase breaks down glycogen to glucose-1-

phosphate57

This system does not require a gaseous substrate and might be able to

overcome the deficiencies of GOx and HRP enzyme cascade system

42

Chapter 4

Silver-titania nanomotors

41 Introduction

Bimetallic nanowires made of platinum and gold have been developed by WPaxton

etal1 The rods move autonomously in a solution of hydrogen peroxide In the present

study silver-titania (Ag-TiO2) particles are being synthesized and investigated for motion

in presence of hydrogen peroxide and UV light With proper control of conditions and

UV light trigger it can be possible to control the speed and direction of motion of these

nanoparticles

Hydrogen peroxide is produced in the human body by phagocytes as an immune response

to foreign particles and pathogens5859

Also silver nanoparticles have been shown to be

biocompatible and capable of interacting with cells in human body without disrupting

cellular function60

Similarly it has been demonstrated that titania is compatible with

different cells DNA and proteins in human body and does not cause toxic effects Hence

silver-titania nanomotors which swim in hydrogen peroxide may open interesting

avenues in applications like targeted drug delivery in those parts of the body where

hydrogen peroxide is present

43

42 Experimental

421 Materials and methods

Silver nitrate (999995) was purchased from Alfa Aesar Gum arabic was purchased

from MP Biomedicals Hydrogen peroxide solution 50 wt in water and ascorbic acid

were purchased from Sigma-Aldrich Nanopure deionized water was used for all

experiments and preparation of all solutions Secure-Seal imaging spacers (9 mm

diameter) were obtained from Sigma Aldrich All experiments were carried out at room

temperature of 25ordmC unless otherwise mentioned Ag-TiO2 particles were synthesized by

evaporation of titania in Semicore electron beam evaporator Particles were characterized

using field-emission scanning electron microscopy Optical microscopy experiments

were performed using Axiovert 200 MAT microscope by Carl Zeiss Particles were

tracked using Physvis particle tracking software (Figure 4 1) in which the position

information of a particle on screen is stored on a frame by frame basis The data is stored

in a dat file as X and Y co-ordinates of particles tracked on the screen This information

can be converted to diffusion coefficients of the particles by further manipulation using

MathWorks-MATLABreg software

44

Figure 4 1 Physvis particle tracking software

422 Synthesis of silver colloidal particles

Silver particles were synthesized by a nucleationgrowth type reaction developed by

Goya and Matijevic61

and modified by Velikov62

Silver nitrate was used as precursor

metal salt for synthesis of silver particles Solution A was 75 ml of 50 mM AgNO3 and

035 by weight gum arabic Solution B was 75 mL of 100 mM 1-ascorbic acid and

035 by weight gum arabic Solution B was rapidly added to solution A in a 250 mL

reaction flask The reaction flask was covered and particles were stirred for 48 hrs After

48 hours particles were allowed to settle for 2-3 hours in refrigerator and supernatant

was separated from the particles Particles were then rinsed with DI water and centrifuged

thrice to obtain pure silver particles Zeta potential of these particles was reported to be

45

around -50 to -60 mV and the size was around 1μm The size of the particles can be

varied by varying the amounts of ascorbic acid and silver nitrate Using molar ratio of 31

of ascorbic acid silver nitrate can give particles of 200 nm diameter whereas ratio of

151 can give particles of upto 5 μm diameter

423 Preparation of silver-titania (Ag-TiO2) particles

A monolayer of silver particles was prepared according to the procedure by Goldenberg

et al63

Glass slides were placed on the bottom of a petridish Water was put into the

petridish so that the entire slide was immersed in water A thin layer of hexane was then

added on top of water in the petridish A suspension of silver particles in ethanol (5

ww) was slowly introduced to the water-hexane interface in the dish After the

monolayer was formed the glass slide was lifted up through the monolayer with tweezers

The silver particles-covered glass slides were left at room temperature to dry overnight

Following drying a thin film of titania was evaporated on the glass slides in Semicore

evaporator (Figure 2 7) The spheres were partially coated with a 10 nm film of titania

Ag-TiO2 particles were removed from the glass slide by gentle sonication FE-SEM was

used to characterize the particles (Figure 4 2) The thin titania layer was not easily

distinguishable due to roughness of the particles

46

Figure 4 2 FESEM image of Ag-TiO2 particles

424 Optical microscopy experiments

Imaging spacers were placed on a glass slide Suspension of Ag-TiO2 particles was

placed on the glass slide and the particles were allowed to settle Movement of particles

was observed in DI water and videos were recorded at 50x magnification of optical

microscope for 30 sec 05 H2O2 was added to the particle suspension on the slide

Videos were recorded at 50x ranging from 30 sec to 10 min to study the movement of

silver titania particles in hydrogen peroxide solution Particles in both DI water and

hydrogen peroxide were subjected to UV light and videos were recorded to study particle

dynamics in presence of UV radiation Particle tracking was performed using Physvis

particle tracking software Atleast 30 particles in every system were tracked for 10

seconds each Particle tracking data was obtained in terms of X and Y coordinates of

particles on screen This data was manipulated using MathWorks-Matlab software and

graphs of mean square displacement of particles versus time were plotted Diffusion

coefficients of particles were calculated from these graphs

47

43 Results and Discussion

Ag-TiO2 particles demonstrated autonomous motion in the presence of H2O2 No UV

light was required for this motion Motion of the particles was accelerated by addition of

fresh H2O2 to the system When subjected to UV radiation the particles displayed

momentary acceleration for 4-5 seconds

Figure 4 3 Mechanism of motion of Ag-TiO2 nanomotors

In the presence of H2O2 silver part of the Ag-TiO2 particle converts H2O2 to H2O and O2

This may lead to propulsion of the Ag-TiO2 particle due to bubble propulsion andor self-

diffusiophoresis In the presence of UV light additional reaction takes place on the

titania part of Ag-TiO2 particle which increases the velocity of the Ag-TiO2 particles

(Figure 4 4) This UV triggered photoactivity of titania is due to hole-electron separation

caused by photons having energy equal to or greater than the band gap of titania646566

Most of the holes-electrons recombine but a small fraction move to the surface and react

with the available redox species like water and oxygen to form protons (H+) superoxide

(O2-) and hydroxyl (OH) radicals

6566 This may lead to propulsion by diffusiophoresis

as well as electrophoresis

48

Figure 4 4 Silver-titania motors move faster when subjected to UV radiation

In addition to linear motion rotational motion is also observed in several particles and

particle aggregates Aggregation of particles can lead to a net circular propulsive force

which can rotate the aggregate as shown in Figure 4 5

Figure 4 5 Rotation due to particle aggregation of Ag-TiO2 particles

Particle tracking by Physvis software revealed a significant increase in diffusion

coefficient of Ag-TiO2 particles in hydrogen peroxide solution as compared to DI water

49

The Ag-TiO2 particles have a diffusion coefficient of 1491 μm2s in hydrogen peroxide

versus only 180 μm2s in DI water Thus the Ag-TiO2 particles move around more than

10 times their body length per second in hydrogen peroxide (Figure 4 6) When

subjected to UV light the particles experience a significant acceleration in hydrogen

peroxide which lasts only for a short duration of time (4-5 sec) after which the particles

return to their original velocity The diffusion coefficient of the particles during this UV

light trigger is found to be around 5093 μm2s UV light trigger does not have any visible

effect on the Ag-TiO2 particles in DI water

Table 4 1 Translational diffusion coefficients of Ag-TiO2 particles in hydrogen

peroxide and water

Mean 05 H2O2 solution DI water

Diffusion coefficient of Ag-TiO2 (μm2s)

without UV light

1491 180

Diffusion coefficient of Ag-TiO2 (μm2s)

in the presence of UV light

5093 113

50

Figure 4 6 Mean square displacement curves of Ag-TiO2 Janus particles in hydrogen

peroxide and DI water

When higher concentration (~30 vv) of Ag-TiO2 particles in hydrogen peroxide is

subjected to UV radiation the particles exhibit pattern formation (Figure 4 7)

Aggregation of Ag-TiO2 Janus particles is observed when UV light is turned on and the

particles disaggregated when UV light is switched off This can be due to predator-prey

kind of interaction between silver and titania half of the Janus particles in the presence of

UV light Oxygen and water that are produced at the silver end by decomposition of

hydrogen peroxide are used up by titania when subjected to UV radiation This can lead

to attraction between titania and silver parts of neighboring Janus particles causing

aggregation

y = 20373x

y = 59994x

y = 71848x

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10 12

Mean

sq

uare

dis

pla

cem

en

t (μ

m2s

)

Time (sec)

Mean square displacement vs Time for Ag-TiO2

particles

05 Hydrogen peroxide and UV05 Hydrogen peroxide

DI Water

51

A UV OFF B UV ON

C UV OFF

Figure 4 7 Higher concentrations of Ag-TiO2 particles showing aggregation when UV

is turned on

52

Chapter 5

Summary and Conclusion

In summary no significant enhancement in diffusion was observed in case of enzyme

immobilized Janus particles in substrate solution Presence of a linker between enzyme

and particle did not increase the diffusion coefficient of particles Alternative enzyme

immobilization methods need to be tested like thiolation of enzyme and subsequent

binding to gold surface of a Janus particle35

or expressing the enzyme with polyhistidine

or avidin tag67

This can result in higher enzyme activity upon immobilization and may

lead to enhanced diffusion of Janus particles

Predator-prey system of GOx and HRP immobilized particles showed some promising

results Aggregation of particles and formation of fan-shaped structures by TMB

oxidation were observed under optical microscope when GOx and HRP immobilized

particles glucose and TMB were present in the system The size of the fan-shaped

structures was dependent on the size of enzyme immobilized particles This can be

indicative of some kind of interaction between the GOx and HRP immobilized particles

No conclusive aggregation of GOx and HRP immobilized particles was observed in

absence of TMB Better imaging techniques are required to study the interaction of GOx

and HRP immobilized particles at the unstable liquid-air interface where majority of the

reaction takes place Other enzyme cascade systems not requiring gaseous reactants like

glycogen synthase and glycogen phosphorylase should be tested for similar activity

53

Ag-TiO2 particles were found to exhibit autonomous motion in the presence of hydrogen

peroxide solution The motion was momentarily accelerated upon exposure to UV

radiation The particles showed a diffusion coefficient of 1491 μm2s in hydrogen

peroxide versus only 180 μm2s in DI water Upon subjecting particles to UV radiation in

presence of H2O2 diffusion coefficient increased to 5093 μm2s for a period of 4-5

seconds In addition to linear motion rotating motion could also be observed in some

particle aggregates Further development of these aggregate structures can lead to

formation of rotors at nanoscale Higher concentration of silver-titania particles in

hydrogen peroxide displayed aggregation in presence of UV light and disaggregated

when UV light was switched off This may be attributed to the predator-prey kind of

interaction between silver and titania where oxygen and water that are produced by

silver by decomposition of hydrogen peroxide are used up by titania when subjected to

UV radiation This promises to be an interesting system and needs to be further

investigated to explore its properties

54

References

1 Paxton WF Sundararajan S Mallouk TE Sen A Angew Chem Int Ed Engl

2006 45 5420-5429

2 Golestanian R Liverpool TB Adjari A Phys Rev Lett 2005 94 220801

3 httpwwwsilvermedicineorgh2o2html

4 Rami Pedahzur Ovadia Lev Badri Fattal Hillel Shuval Water Science and Technology

1995 31(5-6) 123-129

5 W Went F American Scientist 1968 56

6 Adriano Cavalcanti Tad Hogg Bijan Shirinzadeh IEEE MHS 2006 International

Symposium on Micro-NanoMechatronics and Human Science 2006 226-232

7 EM Purcell American Journal of Physics 1977 45 3-11

8 Golestanian R Phys Rev Lett 2010 105 018103(1)-018103(4)

9 S S Dukhin Z R Ulberg G L Dvornichenko B V Deryagin Russian Chemical

Bulletin 1983 31(8) 1535-1544

10 Dukhin AS Goetz PJ Ultrasound for characterizing colloids Particle sizing zeta

potential rheology Elsevier 2002 59

11 HJKeh HJTu Trends in Colloid and Interface Science XIV Progress in Colloid and

Polymer Science 2000 115 174-180

12 D Zinemanas A Nir International Journal of Multiphase Flow 1995 21(5) 787-800

55

13 Stephen Ebbens Jonathan Howse Soft matter 2010 6 726-739

14 J Vicario R Eelkema W R Browne A Meetsma R M La Crois B L Feringa

Chem Commun 2005 31 3936

15 Davide Pantarotto Wesley R Browne Ben L Feringa Chem Commun 2008 1533 -

1535

16 Lisa A Cameron Matthew J Footer Alexander van Oudenaarden Julie A Theriot

PNAS 1999 96(9) 4908-4913

17 Ali Najafi Ramin Golestanian Phys Rev E 2004 69 062901

18 Jinseok Kim Jungyul Park Sungwook Yang Jeongeun Baek Byungkyu Kim Sang

Ho Lee Eui-Sung Yoon Kukjin Chun Sukho Park Lab Chip 2007 7 1504 - 1508

19 Kurt M Dubowski Clinical Chemistry 2008 5411 1919ndash1920

20 Spector DA Yang Q Wade JB Am J Physiol Renal Physiol 2007 292(1) 467-474

21 Rhee SG Chang TS Bae YS Lee SR Kang SW J Am Soc Nephrol 2003 14(8-3)

211-215

22 Sakaue T Kapral R Mikhailov A S Eur Phys J B 2010 75 381ndash387

23 Muddana H S Sengupta S Mallouk T E Sen A Butler P J J Am Chem Soc

2010 132 (7) 2110ndash2111

24 Baker CJ Deahl K Domek J Orlandi EW Arch Biochem Biophys 2000 382(2) 232-

237

25 V Vojinovic RH Carvalho F Lemos JMS Cabral LP Fonseca BS Ferreira

Biochemical Engineering Journal 2007 35 126-135

56

26

OV Lebedeva NN Ugarova Russian Chemical Bulletin 1996 45(1) 18-25

27 Nigel C Veitch Phytochemistry 2004 65(3) 249-259

28 Kenneth H Petersen Journal of Immunological Methods 2009 340(1) 86-89

29 httpwwwbrenda-enzymesorgphpresult_flatphp4ecno=11117

30 Goldenberg L M Wagner J Stumpe J Paulke B-R Goumlrnitz E Langmuir 2002 18

5627-5629

31 httpwwwgoldbiocompdf3761-Protocol201pdf

32 Av Rovisco Pais Lisboa Portugal T amp D Technology West Yorkshire UK Journal

of Molecular Catalysis B Enzymatic 2004 28(2-3) 129-135

33 httpwwwphysicsnyuedugrierlabmethodsnode11html

34 Yoon-Mee Lee1 O-Yul Kwon Yeo-Joon Yoon Keungarp Ryu Biotechnology Letters

2006 28 39ndash43

35 JC Pyun SD Kim JW Chung Analytical Biochemistry 2005 347(2) 227-233

36 George H Pimbley Jr Mathematical Biosciences 1974 20(1-2) 27-51

37 Andy Fenton Sarah E Perkins Parasitology 2010 137 1027-1038

38 Mead James G Brownell Robert L Mammal Species of the World A Taxonomic and

Geographic Reference (3rd ed) Johns Hopkins University Press 2005 2 2142

57

39

Krause KH Schweiz Med Wochenschr 2000 130(4) 97-100

40 Irina Kareva Faina Berezovskaya Carlos Castillo-Chavez Journal of Biological

Dynamics 2010 4(4)315 - 327

41 Delaittre G Reynhout I Cornelissen J Nolte R Chemistry ndash A European Journal

2009 15 12600ndash12603

42 C Buller DH Johnson RC Tschumper Investigative Ophthalmology amp Visual Science

1990 31 2156-2163

43 Robert M Mazo Brownian Motion Fluctuations Dynamics and Applications Oxford

Science Publications 2009 57

44 Vivek K Mutalik KV Venkatesh Theoretical Biology and Medical Modelling 2005 2

19

45 Satoshi Nakamura Kunimasa Koga Biochemical and Biophysical Research

Communications 1977 78(2) 806-810

46 Krishnananda Chattopadhyay Shyamalava Mazumdar Biochemistry 2000 39 (1)

263ndash270

47 Satoshi Nakamura Yasuyuki Ogura J Biochem 1968 63 (3) 308-316

48 van Dongen SF Nallani M Cornelissen JJ Nolte RJ van Hest JC Chemistry 2009

15(5) 1107-1114

49 Ronald Bentley Methods in Enzymology 1955 1 340-345

50 Vartiainen J Raumlttouml M Paulussen S Packaging Technology and Science 2005

18 243ndash251

58

51 GA Sherbeny AA Shindia YMMM Sheriff International Journal of Agriculture

and Biology 2005 7

52 D Rando G-W Kohring F Giffhorn Appl Microbiol Biotechnol 1997 48

53 Worthington enzyme manual Worthington Biochemical Corporation

httpworthington-biochemcomgopdefaulthtml

54 httpwwwbrenda-enzymesorgphpresult_flatphp4ecno=11117

55 httpwwwoxygenphwatercom

56 P Boon Chock Sue Goo Rhee and Earl R Stadtman Annu Rev Biochem 1980 49

813-841

57 Sybil Golden P A Wals Joseph Katz Analytical Biochemistry 1977 77(2) 436-445

58 R L Baehner S K Murrmann J Davis R B Johnston Jr J Clin Invest 1975 56(3)

571ndash576

59 I Bejarano M P Terroacuten S D Paredes C Barriga A B Rodriacuteguez J A Pariente

Molecular and Cellular Biochemistry 2007 296(1-2) 77-84

60 Michael C Moulton Laura K Braydich-Stolle Mallikarjuna N Nadagouda Samantha

Kunzelman Saber M Hussain Rajender S Varma Nanoscale 2010 2 763-770

61 Goya DV Matijevic E Colloids and Surfaces A 1999 146 139

62 Velikov KP Zegers GE van Blaanderen A Langmuir 2003 19 1384

63 Goldenberg L M Wagner J Stumpe J Paulke B-R Goumlrnitz E Langmuir 2002 18

5627-5629

59

64

Banerjee S Gopal J Muraleedharan P Tyagi A K Raj B Curr Sci 2006

901378ndash1383

65 Bogomolov V N Kudinov E K Firsov Y A Sov Phys Solid State 1968 35 555

66 Kudo A Miseki Y Chem Soc Rev 2009 38 253ndash278

67 Gaj T Meyer SC Ghosh I Protein expression and Purification 2007 56 54-61

19

The formula used to calculate the activity of enzyme is

where ∆Amin denotes the change in absorbance per minute

225 Diffusion coefficient measurements using Nanosight LM10

Diffusion coefficients for HRP immobilized particles were measured using Nanosight

LM 10 nanoparticle tracking instrument with Nanoparticle Tracking Analysis (NTA) 20

software in the presence of 003 H2O2 solution as the substrate and also in the presence

of only deionized water The videos were recorded for 30 seconds at the rate of 30 frames

per second Each experiment was carried out in triplicate to assess reproducibility

Particles

visualization

zone

Microscope with

LM 10 unit

LM 10 unit

Figure 2 8 Nanosight LM 10 instrumentation

20

Nanosight LM 10 (Figure 2 8) generates videos of population of moving nanoparticles in

a liquid when illuminated by laser light The particles which pass through the laser beam

path are seen by the instrument as small points of light undergoing Brownian motion

(Figure 2 9a)

The videos are further analyzed by NTA (Nanoparticle tracking analysis) software

(Figure 2 9b) which identifies and tracks each particle independently on a frame-by-

frame basis The software also calculates and subtracts any bulk flow in the system The

mean square displacement is calculated for each particle as long as it is visible If the

particle blinks that is if it disappears momentarily and then reappears on the screen the

software will consider the same particle as two separate ones All analysis data is

exported to the excel files in terms of diffusion coefficients of the tracked particles

B A

Figure 2 9 NTA video analysis (a) Particles as they are seen on the screen during

experimentation in Nanosight LM10 (b) NTA screen as the particles are being analyzed for

their diffusion coefficients

21

23 Results

231 Enzyme assay

The activity of free HRP enzyme reported by Sigma Aldrich is 150 unitsmg However

the activity of HRP upon analysis was found to be 52158 unitsmg which is much lower

than the reported value The activity of HRP was found to be further lowered to 2133

unitsmg when it was immobilized on the PS-SiO2 Janus particles in the presence of a

linker The following formula is used to calculate enzyme activity

where ΔAmin is the change in absorbance per minute

22

Figure 2 10 Graph for the assay of solution of free enzyme containing 05mgL of HRP

ΔAmin = 01716 Unitsmg of free enzyme = 52158

During EDCNHS coupling 04 mg enzyme was added to 1 ml of 025 vv suspension

of Janus particles Thus initial concentration of enzyme in the mixture was 04 mgml

Assuming retained activity of the enzyme in supernatant solution the amount of enzyme

in supernatant was calculated to be 03868 mgml from the assay Amount of enzyme

functionalized on the Janus particles was calculated as the difference between enzyme

previously added for immobilization (04 mgml) and the enzyme present in supernatant

(03868 mgml) and was found to be 00132 mgml Enzyme assay was performed on the

enzyme immobilized Janus particles to estimate the activity of immobilized enzyme

(Figure 2 11) and was found to be 2133 unitsmg

y = 0002x + 00516

0

01

02

03

04

05

06

07

08

0 50 100 150 200 250 300 350 400

Ab

sorb

an

ce

51

0 n

m

Time (sec)

Assay of free enzyme HRP

23

Figure 2 11 Graph for the assay of of immobilized enzyme on 05 μm Janus particles

(~025 vv) ΔAmin = 01853 Unitsmg of free enzyme = 2133

232 Diffusion coefficient measurements

No significant increase was observed in diffusion coefficient of HRP-immobilized Janus

particles in the presence of H2O2 as the substrate Similar results were obtained for

diffusion coefficient measurement with and without linker moieties bridging the enzymes

and particles(Table 2 1 Table 2 2) Thus the linker did not seem to assist in increasing

the particle diffusion by improving or retaining the activity of the enzyme

y = 00015x + 00953

0

01

02

03

04

05

06

07

0 50 100 150 200 250 300 350 400

Ab

sorb

an

ce

510 n

m

Time (sec)

Assay of HRP immobilized 05 μm Janus particles

24

Table 2 1 Diffusion coefficient measurements of HRP immobilized Janus particles

without linker

HRP immobilized

Janus particles in

003 H2O2

Control - Janus

particles in

003 H2O2

Control - HRP

immobilized Janus

particles in DI water

Diffusion coefficient

(10-10

cm2s)

1359416 1051777 1308675

Standard deviation 4534801 4122075 4918597

Table 2 2 Diffusion coefficient measurements of HRP immobilized Janus particles in

the presence of linker

HRP immobilized

Janus particles in

003 H2O2

Control - Janus

particles in

003 H2O2

Control - HRP

immobilized Janus

particles in DI water

Diffusion coefficient

(10-10

cm2s)

13838 1339829 1407891

Standard deviation 5644692 5649364 5797218

25

Figure 2 12 Diffusion coefficient measurements of PS-SiO2 Janus particles without and

with 6-aminohexanoic acid linker

24 Discussion

To compare the diffusion coefficients observed during the experiments theoretical

diffusion coefficient was calculated using following equation33

r

kTD altraslation

6 Equation 21

where r is the hydrodynamic radius of particle Dtranslational is translational diffusion

coefficient of the particle T is temperature of the system η is the viscosity of fluid

through which the particles are moving and k is the Boltzmann constant From FE-SEM

images the size of the particles used was determined to be 450 nm For 450 nm diameter

0

20

40

60

80

100

120

140

160

Without linker With linker

Dif

fusio

n c

oeff

icie

nt

(10

-10 c

m2s

)

HRP immobilized 05 μm Janus particles - Diffusion coefficient measurements

HRP immobilized Janus particles in 003 H2O2

Control - Janus particles in 003 H2O2

Control - HRP immobilized Janus particles in DI water

26

particle Dtranslational is equal to 109 μm2s which is comparable to the diffusion

coefficient measured for the enzyme immobilized particles

HRP was found to be active on the Janus particles however the activity of the enzyme

was reduced upon immobilization Lower enzyme activity can be due to the coupling

method used which in this case was EDCNHS covalent binding However EDCNHS

coupling method has been shown to significantly retain the enzyme activity when the

enzyme is immobilized on carboxylated multi-walled carbon nanotubes under appropriate

conditions34

Hence EDCNHS binding method by itself is not believed to be

responsible for reduction of enzyme activity upon immobilization

Direct binding of enzyme to the Janus particle may lead to reduction in the enzyme

activity because of space constraints for the enzyme and difficulty for the passage of

substrate to the active site of the enzyme due to stearic hindrance Hence a linker was

inserted between the enzyme and Janus particle during enzyme immobilization However

inserting a linker between the enzyme and the particle did not lead to enhanced diffusion

of Janus particles or increase in enzyme activity of immobilized enzyme

Lack of enhanced diffusion can be firstly attributed to the reduction in enzyme activity

upon immobilization Enzyme activity may be retained by modifying the conditions

during EDCNHS coupling or changing the binding method altogether The degree of

immobilization of HRP on carboxylated Janus particles can possibly be affected by

several factors such as temperature the concentration of the coupling reagent EDC

27

concentration HRP concentration in solution and incubation time34

Different

combinations of these factors can be tested to find optimum conditions for

immobilization of HRP Alternate binding method can involve thiolation of HRP by the

covalent conjugation of mercaptopropionic acid and then the thiolated HRP can then be

attached on the gold surface of a polystyrene-gold Janus particle35

Thus further

modifications can be made to the system in order to retain enzyme activity on the

immobilized particles and thereby power nanomotors using enzymes

28

Chapter 3

Predator-prey system of enzyme immobilized particles

31 Introduction

Predator-prey systems are widely found in nature and play a major role in biological

processes3637

Predator-prey systems include all the interactions in which one organism

pursues andor consumes another organism One familiar predator-prey system at the

macroscale is that of a tiger chasing and consuming a deer Another not so familiar one

includes Baleen whales which eat millions of microscopic planktons at once38

At the

microscale such kind of interactions can be observed in the case of phagocytes in the

body which are capable of engulfing foreign microorganisms and killing them with a

combination of microbicidal systems39

In the case of cancer the tumor is the prey and

the mature myeloid cells in the body are the predator However the tumor here has a

defense mechanism against the predator in which the tumor produces cytokines which

inhibits the maturation of the myeloid cells and thereby prevents predation40

One similarity that can be observed in all these systems is that the prey possesses some

attribute which is desired by the predator which makes the predator chase the prey A

similar relationship is seen in case of enzymes which are involved in cascade reactions41

In cascade reactions product synthesized by one enzyme is the reactant required by

another enzyme to carry out its own catalytic reaction As such there is a predator-prey

29

kind of dependence between the two enzymes which may cause one enzyme to chase the

other enzyme when placed in suitable substrate solutions However the mechanism

hypothesized for such a system is totally different than the mechanism involved in

biological systems In biological systems such as bacterial chemotaxis or phagocytes

chasing foreign particles in human body complex biochemical reactions are responsible

for the predator-prey behavior42

In the proposed enzyme cascade system there are no

complex biochemical reactions involved The principle hypothesized for predator-prey

behavior of enzyme cascade reactions can be explained as follows Motion of synthetic

motors at nanoscale involves two components translational diffusion and rotational

Brownian diffusion At very short time scales of less than 1Dr where Dr (s-1

) is

rotational Brownian diffusion coefficient the motion of particles is unidirectional For

longer time scales of greater than 1Dr the direction of motion is random giving

poweredactive diffusion43

If the chemical homogeneity is broken that is when there is

higher concentration of a substrate present in one direction of the enzyme the enzyme

might acquire a subtle bias which may lead to a spatial aggregation of the enzymes based

on the substrate concentration gradient The bias can be a result of the relationship

between the speed of enzyme diffusion and substrate concentration where higher

substrate concentration lead to higher enzyme diffusion As a result in an enzyme

cascade system one enzyme will migrate towards higher concentration of substrate

which is present near the other enzyme thus giving rise to a predator-prey behavior

Such enzyme cascades are widely found in signaling networks and metabolic pathways in

living cells and can be used to produce predator-prey like nanomotors systems44

The

30

enzymes being used in the present study are horseradish peroxidase and glucose oxidase

Both enzymes are extremely stable4546

Glucose is the substrate for glucose oxidase

which is widely available and highly stable47

Horseradish peroxidase acts on hydrogen

peroxide It is involved in a cascade reaction with glucose oxidase which produces

hydrogen peroxide by converting glucose into gluconic acid (Figure 3 1)48

Figure 3 1 Concept of particle aggregation due to cascade reactions of immobilized

enzymes

In this experiment glucose oxidase (GOx) and horseradish peroxidase (HRP) were

immobilized on polystyrene carboxylate beads of different sizes using EDCNHS

coupling method Glucose and 3355-Tetramethylbenzidine (TMB) were dissolved

together in water and this solution was used as the substrate solution All the components

were present in a single system in DI water Aggregation of particles and precipitation of

oxidized TMB from the solution was expected to be observed when all these components

were present in such a system

Properties of horseradish peroxidase have been discussed in the previous chapter (Section

211 Horseradish peroxidase)

GOxGOx HRPHRP

Glucose + O2Gluconic acid + H2O2

TMB TMB oxidizedBlue precipitate

H2O

Aggregation

31

311 Glucose oxidase

Figure 3 2 Glucose oxidase

Glucose oxidase (GOx) belongs to the group of oxidoreductases and is also called

glucose aerodehydrogenase49

It is obtained from fungi and has been identified as an

antimicrobial enzyme50

GOx binds to β-D-glucopyranose and catalyzes the oxidation

reaction to from gluconolactone while simultaneously reducing oxygen to hydrogen

peroxide51

GOx has high substrate affinity and specificity high stability and high

turnover number which makes it a suitable biocatalyst for industrial applications52

Figure 3 3 Redox reaction of glucose catalyzed by glucose oxidase53

32

GOx is a glycoprotein with molecular weight of 155 kDa and consists of two identical

polypeptide subunits linked together by a disulfide bond The optimum pH for GOx is

55 though it is stable in a broad pH range of 4 to 7 and can be stored for a long period of

time51

The functional parameters for glucose oxidase include Km of 151 mM and kcat of

337 s-1

at temperature of 20 ordmC and pH of 754

Some of the inhibitors of GOx are 2-

Deoxy-D-glucose adenine nucleotides and NaNO3

32 Experimental

321 Materials and methods

Uniform carboxyl modified polystyrene microspheres 05 microm in diameter were obtained

from Polysciences Inc The size of the purchased microspheres was verified by FESEM

Peroxidase Type I from horseradish glucose oxidase Type II-S from Aspergillus niger

N-(3-dimethyl-aminopropyl)-Nrsquo-ethylcarbodiimide (EDC) and Hydrogen peroxide

solution 50 wt in water were purchased from Sigma-Aldrich N-hydroxysuccinimide

(NHS) was purchased from Fluka D-Glucose (99) was purchased from VWR

International Secure-Seal imaging spacers (9mm diameter) for glass slides were obtained

from Sigma Aldrich All chemicals were of reagent grade and used without further

purification Nanopure deionized water was used for all experiments and preparation of

solutions All measurements were made at room temperature (25 oC) unless mentioned

otherwise The UV-Visible measurements were performed using Agilent 8453A diode-

array UV-Visible Spectrophotometer with ChemStation UV-Vis Software Optical

33

microscopy experiments were performed using Axiovert 200 MAT microscope by Carl

Zeiss Diffusion coefficient measurements were obtained using Nanosight LM10

322 Optical microscopy experiments

The enzyme substrate system was observed under optical microscope to check for

aggregation and precipitation due to cascade reactions Several such systems were

studied Each system in general consisted of GOx and HRP immobilized on different

polystyrene particles The substrate solutions consisted of 01 M glucose and different

concentrations of TMB solution The systems were studied in the presence of all the

constituents and in the absence of glucose GOxHRP and TMB Systems consisting of

different sized HRP immobilized particles in substrate solution of 003 hydrogen

peroxide and TMB were also studied Videos were recorded over a period of 24 hours to

observe changes in the system Each experiment was performed atleast thrice to assess

reproducibility of results

323 Diffusion coefficient measurements using Nanosight LM10

Diffusion coefficient of GOx and HRP immobilized particles were measured using

Nanosight LM10 nanoparticle tracking instrument with Nanoparticle Tracking Analysis

(NTA) 20 software Diffusion coefficients were measured for different systems namely

GOx particles in 01 M glucose and oxygen both GOx and HRP functionalized particles

in 01 M glucose and oxygen along with controls for the individual systems Videos were

34

recorded for 30 seconds at the rate of 30 frames per second Each experiment was carried

out in triplicate to assess reproducibility

33 Results

331 Optical microscopy

According to the enzyme cascade model precipitation of TMB was possible only upon

the interaction of GOx and HRP immobilized particles in presence of appropriate

substrate solution Hence in the first set of experiments precipitation of TMB was used

as an indication of the type of interaction between GOx and HRP immobilized particles

Fan-shaped structures were observed when both HRP and GOx immobilized particles

were present in a substrate solution consisting of glucose and TMB These structures

started forming as a single strand and then branched out and intertwined to form the fan-

shaped structures (Figure 3 4)

Table 3 1 Observations for formation of fan-shaped structures in enzyme cascade

system in presence of different components

GOx HRP Glucose TMB Observation

Yes Yes Yes Yes

Fan-shaped structures observed particles associated with

the fan-shaped structures

No Yes Yes Yes No fan-shaped structures

Yes No Yes Yes No fan-shaped structures

Yes Yes No Yes No fan-shaped structures

Yes Yes Yes No No fan-shaped structures

35

Fan-shaped structures can also be observed in the system consisting of only HRP

immobilized particles in substrate solution of hydrogen peroxide and TMB The size of

the fan-shaped structures depends on the size of the enzyme immobilized particles Free

enzymes show extremely tiny structures which are difficult to view under the optical

microscope

Figure 3 4 Formation of fan-shaped structures when both HRP and GOx

immobilized particles were present in a substrate solution consisting of glucose and

TMB A In the initial stages single strands form on the surface of liquid (5Ox) B

Single strands branch out and intertwine to form fan-shaped structures (50x) C Many

such structures form and settle down after 20 minutes (20x)

A

C

B

36

Experiments were performed in the absence of TMB that is the system consisted of GOx

and HRP immobilized particles in glucose substrate solution In the absence of TMB the

particles were expected to move closer to each other without any precipitation due to the

cascade reaction However there was no conclusive aggregation of particles observed in

the system

Free Enzyme 005 μm HRP - immobilized particle

10 μm HRP - immobilized particle 05 μm HRP - immobilized particle

Figure 3 5 Size dependence of fan-shaped structures on the size of HRP -

immobilized particles (20x)

37

Table 3 2 Observations for particle aggregation in enzyme cascade system in presence

and absence of TMB

Glucose GOx HRP TMB Observation

Present Present Present Present Aggregation and precipitate

Present Present Present Absent No conclusive aggregation

Present Absent Present Present No aggregation or ppt

Absent Present Present Present No aggregation or ppt

Present Present Absent Present No aggregation or ppt

332 Diffusion coefficient measurements

Diffusion coefficients were measured for GOx immobilized particles of 05 μm diameter

in substrate solution of 01 M glucose and oxygen Diffusion coefficients of particles

were also measured for system consisting of GOx and HRP functionalized particles in

glucose and oxygen Each experiment was done in triplicate to assess reproducibility

Nanosight visualization chamber being a closed chamber oxygen was purged into all the

solutions before starting the experiments to ensure sufficient supply of oxygen for

catalytic reaction of GOx

38

Table 3 3 Diffusion coefficients for 05 μm GOx immobilized particles in 01 M

glucose purged with O2

Mean

GOx

particles in

01 M

glucose

Control - GOx

particles in DI

water

Control - GOx

particles in 01 M

glucose without

oxygen purging

Control -

particles in 01

M Glucose

Diffusion

coefficient

(x10-10

cm2s)

13316 12888 13827 13702

Standard

deviation 6307 4926 4424 7468

Table 3 4 Diffusion coefficients for GOx and HRP immobilized particles in 01 M

glucose and purged with O2

Mean

GOx- HRP-

functionalized

particles in 01 M

glucose

Control ndash Gox-

HRP- functionalized

particles in DI water

Control - particles

in 01 M glucose

Diffusion coefficient

(x10-10

cm2s)

13005 14434 13702

Standard deviation 5263 6622 7468

No significant increase was observed in diffusion coefficient of enzyme immobilized

particles in any of the systems

39

34 Discussion

Formation of fan-shaped structures due to patterned deposition of oxidized TMB

indicates some kind of interaction between GOx and HRP immobilized particles Particle

diameter dependence of the size of structures can be explained with a simple model in

Figure 3 6 A larger particle will have a higher surface area as compared to a smaller

particle Hence more enzyme is immobilized on the larger particle As a result higher

quantity of TMB is oxidized and precipitated by the larger particle as compared to the

smaller particle This leads to thicker strands of fan-shaped structures eventually scaling

up the whole system to give bigger fan-shaped structures

The exact reason for the formation of such fan-shaped structures which are dependent on

particle size could not be determined It is hypothesized that hydrogen peroxide andor

Figure 3 6 Model for size dependence of fan-shaped structures on the particle

diameter of enzyme immobilized particles Larger the particle more surface area is

available for precipitation of TMB which eventually leads to bigger fan-shaped

structures

Larger particle Smaller particle

40

TMB gradient in the substrate solution might be responsible for such structures If there

is higher concentration of H2O2 on one side of the particle and assuming an even

concentration distribution of TMB there will be selective precipitation in the direction of

H2O2 When several particles come in the vicinity of higher concentration of H2O2 the

precipitation lines will converge to give fan-shaped structures (Figure 3 7)

Figure 3 7 Hypothesis for formation of fan-shaped structures

In the system consisting of GOx immobilized particles HRP immobilized particles

glucose and TMB it was observed that the fan-shaped structures formed at the liquid-air

interface and eventually settled down to the bottom of the glass slide This makes sense

as oxygen is necessary for the catalytic reaction of conversion of glucose to gluconic acid

and hydrogen peroxide which takes place on the surface of GOx immobilized particle As

a result H2O2 is primarily formed at the liquid-air interface and is then used by HRP for

precipitation of TMB

Following the observations that precipitation of TMB occurs at the surface GOx and

HRP immobilized particles are also expected to interact at the surface in the absence of

TMB Due the cascade reaction it was expected that HRP immobilized particles will be

41

attracted to GOx immobilized particles in appropriate substrate solution Experiments

were carried out to visualize inter-particle interactions at the liquid-air interface

However liquid-air interface not being a stable system to image the precise interaction

of GOx and HRP immobilized particles could not be determined Significant external

disturbances were involved at the interface To enable the cascade reaction in the bulk of

the liquid oxygen was purged into the reaction system before starting the experiment

However this did not lead to significant reaction in the bulk of the liquid and the reaction

prominently took place at the liquid-air interface where the oxygen concentration was

higher

In order to observe the interaction of GOx and HRP immobilized particles better imaging

techniques need to be used to observe diffusion of particles in absence of external

disturbances at the liquid-air interface Large quantity of oxygen needs to be purged into

the system and retained to make the reaction more pronounced in the bulk of the substrate

solution Higher concentration of oxygen in water may also be achieved by using oxygen

enriched water which is commercially available55

Alternatively a different enzyme cascade system may be used which does not require a

gaseous substrate56

For example glycogen synthase synthesizes glycogen from glucose-

1-phosphate and glycogen phosphorylase breaks down glycogen to glucose-1-

phosphate57

This system does not require a gaseous substrate and might be able to

overcome the deficiencies of GOx and HRP enzyme cascade system

42

Chapter 4

Silver-titania nanomotors

41 Introduction

Bimetallic nanowires made of platinum and gold have been developed by WPaxton

etal1 The rods move autonomously in a solution of hydrogen peroxide In the present

study silver-titania (Ag-TiO2) particles are being synthesized and investigated for motion

in presence of hydrogen peroxide and UV light With proper control of conditions and

UV light trigger it can be possible to control the speed and direction of motion of these

nanoparticles

Hydrogen peroxide is produced in the human body by phagocytes as an immune response

to foreign particles and pathogens5859

Also silver nanoparticles have been shown to be

biocompatible and capable of interacting with cells in human body without disrupting

cellular function60

Similarly it has been demonstrated that titania is compatible with

different cells DNA and proteins in human body and does not cause toxic effects Hence

silver-titania nanomotors which swim in hydrogen peroxide may open interesting

avenues in applications like targeted drug delivery in those parts of the body where

hydrogen peroxide is present

43

42 Experimental

421 Materials and methods

Silver nitrate (999995) was purchased from Alfa Aesar Gum arabic was purchased

from MP Biomedicals Hydrogen peroxide solution 50 wt in water and ascorbic acid

were purchased from Sigma-Aldrich Nanopure deionized water was used for all

experiments and preparation of all solutions Secure-Seal imaging spacers (9 mm

diameter) were obtained from Sigma Aldrich All experiments were carried out at room

temperature of 25ordmC unless otherwise mentioned Ag-TiO2 particles were synthesized by

evaporation of titania in Semicore electron beam evaporator Particles were characterized

using field-emission scanning electron microscopy Optical microscopy experiments

were performed using Axiovert 200 MAT microscope by Carl Zeiss Particles were

tracked using Physvis particle tracking software (Figure 4 1) in which the position

information of a particle on screen is stored on a frame by frame basis The data is stored

in a dat file as X and Y co-ordinates of particles tracked on the screen This information

can be converted to diffusion coefficients of the particles by further manipulation using

MathWorks-MATLABreg software

44

Figure 4 1 Physvis particle tracking software

422 Synthesis of silver colloidal particles

Silver particles were synthesized by a nucleationgrowth type reaction developed by

Goya and Matijevic61

and modified by Velikov62

Silver nitrate was used as precursor

metal salt for synthesis of silver particles Solution A was 75 ml of 50 mM AgNO3 and

035 by weight gum arabic Solution B was 75 mL of 100 mM 1-ascorbic acid and

035 by weight gum arabic Solution B was rapidly added to solution A in a 250 mL

reaction flask The reaction flask was covered and particles were stirred for 48 hrs After

48 hours particles were allowed to settle for 2-3 hours in refrigerator and supernatant

was separated from the particles Particles were then rinsed with DI water and centrifuged

thrice to obtain pure silver particles Zeta potential of these particles was reported to be

45

around -50 to -60 mV and the size was around 1μm The size of the particles can be

varied by varying the amounts of ascorbic acid and silver nitrate Using molar ratio of 31

of ascorbic acid silver nitrate can give particles of 200 nm diameter whereas ratio of

151 can give particles of upto 5 μm diameter

423 Preparation of silver-titania (Ag-TiO2) particles

A monolayer of silver particles was prepared according to the procedure by Goldenberg

et al63

Glass slides were placed on the bottom of a petridish Water was put into the

petridish so that the entire slide was immersed in water A thin layer of hexane was then

added on top of water in the petridish A suspension of silver particles in ethanol (5

ww) was slowly introduced to the water-hexane interface in the dish After the

monolayer was formed the glass slide was lifted up through the monolayer with tweezers

The silver particles-covered glass slides were left at room temperature to dry overnight

Following drying a thin film of titania was evaporated on the glass slides in Semicore

evaporator (Figure 2 7) The spheres were partially coated with a 10 nm film of titania

Ag-TiO2 particles were removed from the glass slide by gentle sonication FE-SEM was

used to characterize the particles (Figure 4 2) The thin titania layer was not easily

distinguishable due to roughness of the particles

46

Figure 4 2 FESEM image of Ag-TiO2 particles

424 Optical microscopy experiments

Imaging spacers were placed on a glass slide Suspension of Ag-TiO2 particles was

placed on the glass slide and the particles were allowed to settle Movement of particles

was observed in DI water and videos were recorded at 50x magnification of optical

microscope for 30 sec 05 H2O2 was added to the particle suspension on the slide

Videos were recorded at 50x ranging from 30 sec to 10 min to study the movement of

silver titania particles in hydrogen peroxide solution Particles in both DI water and

hydrogen peroxide were subjected to UV light and videos were recorded to study particle

dynamics in presence of UV radiation Particle tracking was performed using Physvis

particle tracking software Atleast 30 particles in every system were tracked for 10

seconds each Particle tracking data was obtained in terms of X and Y coordinates of

particles on screen This data was manipulated using MathWorks-Matlab software and

graphs of mean square displacement of particles versus time were plotted Diffusion

coefficients of particles were calculated from these graphs

47

43 Results and Discussion

Ag-TiO2 particles demonstrated autonomous motion in the presence of H2O2 No UV

light was required for this motion Motion of the particles was accelerated by addition of

fresh H2O2 to the system When subjected to UV radiation the particles displayed

momentary acceleration for 4-5 seconds

Figure 4 3 Mechanism of motion of Ag-TiO2 nanomotors

In the presence of H2O2 silver part of the Ag-TiO2 particle converts H2O2 to H2O and O2

This may lead to propulsion of the Ag-TiO2 particle due to bubble propulsion andor self-

diffusiophoresis In the presence of UV light additional reaction takes place on the

titania part of Ag-TiO2 particle which increases the velocity of the Ag-TiO2 particles

(Figure 4 4) This UV triggered photoactivity of titania is due to hole-electron separation

caused by photons having energy equal to or greater than the band gap of titania646566

Most of the holes-electrons recombine but a small fraction move to the surface and react

with the available redox species like water and oxygen to form protons (H+) superoxide

(O2-) and hydroxyl (OH) radicals

6566 This may lead to propulsion by diffusiophoresis

as well as electrophoresis

48

Figure 4 4 Silver-titania motors move faster when subjected to UV radiation

In addition to linear motion rotational motion is also observed in several particles and

particle aggregates Aggregation of particles can lead to a net circular propulsive force

which can rotate the aggregate as shown in Figure 4 5

Figure 4 5 Rotation due to particle aggregation of Ag-TiO2 particles

Particle tracking by Physvis software revealed a significant increase in diffusion

coefficient of Ag-TiO2 particles in hydrogen peroxide solution as compared to DI water

49

The Ag-TiO2 particles have a diffusion coefficient of 1491 μm2s in hydrogen peroxide

versus only 180 μm2s in DI water Thus the Ag-TiO2 particles move around more than

10 times their body length per second in hydrogen peroxide (Figure 4 6) When

subjected to UV light the particles experience a significant acceleration in hydrogen

peroxide which lasts only for a short duration of time (4-5 sec) after which the particles

return to their original velocity The diffusion coefficient of the particles during this UV

light trigger is found to be around 5093 μm2s UV light trigger does not have any visible

effect on the Ag-TiO2 particles in DI water

Table 4 1 Translational diffusion coefficients of Ag-TiO2 particles in hydrogen

peroxide and water

Mean 05 H2O2 solution DI water

Diffusion coefficient of Ag-TiO2 (μm2s)

without UV light

1491 180

Diffusion coefficient of Ag-TiO2 (μm2s)

in the presence of UV light

5093 113

50

Figure 4 6 Mean square displacement curves of Ag-TiO2 Janus particles in hydrogen

peroxide and DI water

When higher concentration (~30 vv) of Ag-TiO2 particles in hydrogen peroxide is

subjected to UV radiation the particles exhibit pattern formation (Figure 4 7)

Aggregation of Ag-TiO2 Janus particles is observed when UV light is turned on and the

particles disaggregated when UV light is switched off This can be due to predator-prey

kind of interaction between silver and titania half of the Janus particles in the presence of

UV light Oxygen and water that are produced at the silver end by decomposition of

hydrogen peroxide are used up by titania when subjected to UV radiation This can lead

to attraction between titania and silver parts of neighboring Janus particles causing

aggregation

y = 20373x

y = 59994x

y = 71848x

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10 12

Mean

sq

uare

dis

pla

cem

en

t (μ

m2s

)

Time (sec)

Mean square displacement vs Time for Ag-TiO2

particles

05 Hydrogen peroxide and UV05 Hydrogen peroxide

DI Water

51

A UV OFF B UV ON

C UV OFF

Figure 4 7 Higher concentrations of Ag-TiO2 particles showing aggregation when UV

is turned on

52

Chapter 5

Summary and Conclusion

In summary no significant enhancement in diffusion was observed in case of enzyme

immobilized Janus particles in substrate solution Presence of a linker between enzyme

and particle did not increase the diffusion coefficient of particles Alternative enzyme

immobilization methods need to be tested like thiolation of enzyme and subsequent

binding to gold surface of a Janus particle35

or expressing the enzyme with polyhistidine

or avidin tag67

This can result in higher enzyme activity upon immobilization and may

lead to enhanced diffusion of Janus particles

Predator-prey system of GOx and HRP immobilized particles showed some promising

results Aggregation of particles and formation of fan-shaped structures by TMB

oxidation were observed under optical microscope when GOx and HRP immobilized

particles glucose and TMB were present in the system The size of the fan-shaped

structures was dependent on the size of enzyme immobilized particles This can be

indicative of some kind of interaction between the GOx and HRP immobilized particles

No conclusive aggregation of GOx and HRP immobilized particles was observed in

absence of TMB Better imaging techniques are required to study the interaction of GOx

and HRP immobilized particles at the unstable liquid-air interface where majority of the

reaction takes place Other enzyme cascade systems not requiring gaseous reactants like

glycogen synthase and glycogen phosphorylase should be tested for similar activity

53

Ag-TiO2 particles were found to exhibit autonomous motion in the presence of hydrogen

peroxide solution The motion was momentarily accelerated upon exposure to UV

radiation The particles showed a diffusion coefficient of 1491 μm2s in hydrogen

peroxide versus only 180 μm2s in DI water Upon subjecting particles to UV radiation in

presence of H2O2 diffusion coefficient increased to 5093 μm2s for a period of 4-5

seconds In addition to linear motion rotating motion could also be observed in some

particle aggregates Further development of these aggregate structures can lead to

formation of rotors at nanoscale Higher concentration of silver-titania particles in

hydrogen peroxide displayed aggregation in presence of UV light and disaggregated

when UV light was switched off This may be attributed to the predator-prey kind of

interaction between silver and titania where oxygen and water that are produced by

silver by decomposition of hydrogen peroxide are used up by titania when subjected to

UV radiation This promises to be an interesting system and needs to be further

investigated to explore its properties

54

References

1 Paxton WF Sundararajan S Mallouk TE Sen A Angew Chem Int Ed Engl

2006 45 5420-5429

2 Golestanian R Liverpool TB Adjari A Phys Rev Lett 2005 94 220801

3 httpwwwsilvermedicineorgh2o2html

4 Rami Pedahzur Ovadia Lev Badri Fattal Hillel Shuval Water Science and Technology

1995 31(5-6) 123-129

5 W Went F American Scientist 1968 56

6 Adriano Cavalcanti Tad Hogg Bijan Shirinzadeh IEEE MHS 2006 International

Symposium on Micro-NanoMechatronics and Human Science 2006 226-232

7 EM Purcell American Journal of Physics 1977 45 3-11

8 Golestanian R Phys Rev Lett 2010 105 018103(1)-018103(4)

9 S S Dukhin Z R Ulberg G L Dvornichenko B V Deryagin Russian Chemical

Bulletin 1983 31(8) 1535-1544

10 Dukhin AS Goetz PJ Ultrasound for characterizing colloids Particle sizing zeta

potential rheology Elsevier 2002 59

11 HJKeh HJTu Trends in Colloid and Interface Science XIV Progress in Colloid and

Polymer Science 2000 115 174-180

12 D Zinemanas A Nir International Journal of Multiphase Flow 1995 21(5) 787-800

55

13 Stephen Ebbens Jonathan Howse Soft matter 2010 6 726-739

14 J Vicario R Eelkema W R Browne A Meetsma R M La Crois B L Feringa

Chem Commun 2005 31 3936

15 Davide Pantarotto Wesley R Browne Ben L Feringa Chem Commun 2008 1533 -

1535

16 Lisa A Cameron Matthew J Footer Alexander van Oudenaarden Julie A Theriot

PNAS 1999 96(9) 4908-4913

17 Ali Najafi Ramin Golestanian Phys Rev E 2004 69 062901

18 Jinseok Kim Jungyul Park Sungwook Yang Jeongeun Baek Byungkyu Kim Sang

Ho Lee Eui-Sung Yoon Kukjin Chun Sukho Park Lab Chip 2007 7 1504 - 1508

19 Kurt M Dubowski Clinical Chemistry 2008 5411 1919ndash1920

20 Spector DA Yang Q Wade JB Am J Physiol Renal Physiol 2007 292(1) 467-474

21 Rhee SG Chang TS Bae YS Lee SR Kang SW J Am Soc Nephrol 2003 14(8-3)

211-215

22 Sakaue T Kapral R Mikhailov A S Eur Phys J B 2010 75 381ndash387

23 Muddana H S Sengupta S Mallouk T E Sen A Butler P J J Am Chem Soc

2010 132 (7) 2110ndash2111

24 Baker CJ Deahl K Domek J Orlandi EW Arch Biochem Biophys 2000 382(2) 232-

237

25 V Vojinovic RH Carvalho F Lemos JMS Cabral LP Fonseca BS Ferreira

Biochemical Engineering Journal 2007 35 126-135

56

26

OV Lebedeva NN Ugarova Russian Chemical Bulletin 1996 45(1) 18-25

27 Nigel C Veitch Phytochemistry 2004 65(3) 249-259

28 Kenneth H Petersen Journal of Immunological Methods 2009 340(1) 86-89

29 httpwwwbrenda-enzymesorgphpresult_flatphp4ecno=11117

30 Goldenberg L M Wagner J Stumpe J Paulke B-R Goumlrnitz E Langmuir 2002 18

5627-5629

31 httpwwwgoldbiocompdf3761-Protocol201pdf

32 Av Rovisco Pais Lisboa Portugal T amp D Technology West Yorkshire UK Journal

of Molecular Catalysis B Enzymatic 2004 28(2-3) 129-135

33 httpwwwphysicsnyuedugrierlabmethodsnode11html

34 Yoon-Mee Lee1 O-Yul Kwon Yeo-Joon Yoon Keungarp Ryu Biotechnology Letters

2006 28 39ndash43

35 JC Pyun SD Kim JW Chung Analytical Biochemistry 2005 347(2) 227-233

36 George H Pimbley Jr Mathematical Biosciences 1974 20(1-2) 27-51

37 Andy Fenton Sarah E Perkins Parasitology 2010 137 1027-1038

38 Mead James G Brownell Robert L Mammal Species of the World A Taxonomic and

Geographic Reference (3rd ed) Johns Hopkins University Press 2005 2 2142

57

39

Krause KH Schweiz Med Wochenschr 2000 130(4) 97-100

40 Irina Kareva Faina Berezovskaya Carlos Castillo-Chavez Journal of Biological

Dynamics 2010 4(4)315 - 327

41 Delaittre G Reynhout I Cornelissen J Nolte R Chemistry ndash A European Journal

2009 15 12600ndash12603

42 C Buller DH Johnson RC Tschumper Investigative Ophthalmology amp Visual Science

1990 31 2156-2163

43 Robert M Mazo Brownian Motion Fluctuations Dynamics and Applications Oxford

Science Publications 2009 57

44 Vivek K Mutalik KV Venkatesh Theoretical Biology and Medical Modelling 2005 2

19

45 Satoshi Nakamura Kunimasa Koga Biochemical and Biophysical Research

Communications 1977 78(2) 806-810

46 Krishnananda Chattopadhyay Shyamalava Mazumdar Biochemistry 2000 39 (1)

263ndash270

47 Satoshi Nakamura Yasuyuki Ogura J Biochem 1968 63 (3) 308-316

48 van Dongen SF Nallani M Cornelissen JJ Nolte RJ van Hest JC Chemistry 2009

15(5) 1107-1114

49 Ronald Bentley Methods in Enzymology 1955 1 340-345

50 Vartiainen J Raumlttouml M Paulussen S Packaging Technology and Science 2005

18 243ndash251

58

51 GA Sherbeny AA Shindia YMMM Sheriff International Journal of Agriculture

and Biology 2005 7

52 D Rando G-W Kohring F Giffhorn Appl Microbiol Biotechnol 1997 48

53 Worthington enzyme manual Worthington Biochemical Corporation

httpworthington-biochemcomgopdefaulthtml

54 httpwwwbrenda-enzymesorgphpresult_flatphp4ecno=11117

55 httpwwwoxygenphwatercom

56 P Boon Chock Sue Goo Rhee and Earl R Stadtman Annu Rev Biochem 1980 49

813-841

57 Sybil Golden P A Wals Joseph Katz Analytical Biochemistry 1977 77(2) 436-445

58 R L Baehner S K Murrmann J Davis R B Johnston Jr J Clin Invest 1975 56(3)

571ndash576

59 I Bejarano M P Terroacuten S D Paredes C Barriga A B Rodriacuteguez J A Pariente

Molecular and Cellular Biochemistry 2007 296(1-2) 77-84

60 Michael C Moulton Laura K Braydich-Stolle Mallikarjuna N Nadagouda Samantha

Kunzelman Saber M Hussain Rajender S Varma Nanoscale 2010 2 763-770

61 Goya DV Matijevic E Colloids and Surfaces A 1999 146 139

62 Velikov KP Zegers GE van Blaanderen A Langmuir 2003 19 1384

63 Goldenberg L M Wagner J Stumpe J Paulke B-R Goumlrnitz E Langmuir 2002 18

5627-5629

59

64

Banerjee S Gopal J Muraleedharan P Tyagi A K Raj B Curr Sci 2006

901378ndash1383

65 Bogomolov V N Kudinov E K Firsov Y A Sov Phys Solid State 1968 35 555

66 Kudo A Miseki Y Chem Soc Rev 2009 38 253ndash278

67 Gaj T Meyer SC Ghosh I Protein expression and Purification 2007 56 54-61

20

Nanosight LM 10 (Figure 2 8) generates videos of population of moving nanoparticles in

a liquid when illuminated by laser light The particles which pass through the laser beam

path are seen by the instrument as small points of light undergoing Brownian motion

(Figure 2 9a)

The videos are further analyzed by NTA (Nanoparticle tracking analysis) software

(Figure 2 9b) which identifies and tracks each particle independently on a frame-by-

frame basis The software also calculates and subtracts any bulk flow in the system The

mean square displacement is calculated for each particle as long as it is visible If the

particle blinks that is if it disappears momentarily and then reappears on the screen the

software will consider the same particle as two separate ones All analysis data is

exported to the excel files in terms of diffusion coefficients of the tracked particles

B A

Figure 2 9 NTA video analysis (a) Particles as they are seen on the screen during

experimentation in Nanosight LM10 (b) NTA screen as the particles are being analyzed for

their diffusion coefficients

21

23 Results

231 Enzyme assay

The activity of free HRP enzyme reported by Sigma Aldrich is 150 unitsmg However

the activity of HRP upon analysis was found to be 52158 unitsmg which is much lower

than the reported value The activity of HRP was found to be further lowered to 2133

unitsmg when it was immobilized on the PS-SiO2 Janus particles in the presence of a

linker The following formula is used to calculate enzyme activity

where ΔAmin is the change in absorbance per minute

22

Figure 2 10 Graph for the assay of solution of free enzyme containing 05mgL of HRP

ΔAmin = 01716 Unitsmg of free enzyme = 52158

During EDCNHS coupling 04 mg enzyme was added to 1 ml of 025 vv suspension

of Janus particles Thus initial concentration of enzyme in the mixture was 04 mgml

Assuming retained activity of the enzyme in supernatant solution the amount of enzyme

in supernatant was calculated to be 03868 mgml from the assay Amount of enzyme

functionalized on the Janus particles was calculated as the difference between enzyme

previously added for immobilization (04 mgml) and the enzyme present in supernatant

(03868 mgml) and was found to be 00132 mgml Enzyme assay was performed on the

enzyme immobilized Janus particles to estimate the activity of immobilized enzyme

(Figure 2 11) and was found to be 2133 unitsmg

y = 0002x + 00516

0

01

02

03

04

05

06

07

08

0 50 100 150 200 250 300 350 400

Ab

sorb

an

ce

51

0 n

m

Time (sec)

Assay of free enzyme HRP

23

Figure 2 11 Graph for the assay of of immobilized enzyme on 05 μm Janus particles

(~025 vv) ΔAmin = 01853 Unitsmg of free enzyme = 2133

232 Diffusion coefficient measurements

No significant increase was observed in diffusion coefficient of HRP-immobilized Janus

particles in the presence of H2O2 as the substrate Similar results were obtained for

diffusion coefficient measurement with and without linker moieties bridging the enzymes

and particles(Table 2 1 Table 2 2) Thus the linker did not seem to assist in increasing

the particle diffusion by improving or retaining the activity of the enzyme

y = 00015x + 00953

0

01

02

03

04

05

06

07

0 50 100 150 200 250 300 350 400

Ab

sorb

an

ce

510 n

m

Time (sec)

Assay of HRP immobilized 05 μm Janus particles

24

Table 2 1 Diffusion coefficient measurements of HRP immobilized Janus particles

without linker

HRP immobilized

Janus particles in

003 H2O2

Control - Janus

particles in

003 H2O2

Control - HRP

immobilized Janus

particles in DI water

Diffusion coefficient

(10-10

cm2s)

1359416 1051777 1308675

Standard deviation 4534801 4122075 4918597

Table 2 2 Diffusion coefficient measurements of HRP immobilized Janus particles in

the presence of linker

HRP immobilized

Janus particles in

003 H2O2

Control - Janus

particles in

003 H2O2

Control - HRP

immobilized Janus

particles in DI water

Diffusion coefficient

(10-10

cm2s)

13838 1339829 1407891

Standard deviation 5644692 5649364 5797218

25

Figure 2 12 Diffusion coefficient measurements of PS-SiO2 Janus particles without and

with 6-aminohexanoic acid linker

24 Discussion

To compare the diffusion coefficients observed during the experiments theoretical

diffusion coefficient was calculated using following equation33

r

kTD altraslation

6 Equation 21

where r is the hydrodynamic radius of particle Dtranslational is translational diffusion

coefficient of the particle T is temperature of the system η is the viscosity of fluid

through which the particles are moving and k is the Boltzmann constant From FE-SEM

images the size of the particles used was determined to be 450 nm For 450 nm diameter

0

20

40

60

80

100

120

140

160

Without linker With linker

Dif

fusio

n c

oeff

icie

nt

(10

-10 c

m2s

)

HRP immobilized 05 μm Janus particles - Diffusion coefficient measurements

HRP immobilized Janus particles in 003 H2O2

Control - Janus particles in 003 H2O2

Control - HRP immobilized Janus particles in DI water

26

particle Dtranslational is equal to 109 μm2s which is comparable to the diffusion

coefficient measured for the enzyme immobilized particles

HRP was found to be active on the Janus particles however the activity of the enzyme

was reduced upon immobilization Lower enzyme activity can be due to the coupling

method used which in this case was EDCNHS covalent binding However EDCNHS

coupling method has been shown to significantly retain the enzyme activity when the

enzyme is immobilized on carboxylated multi-walled carbon nanotubes under appropriate

conditions34

Hence EDCNHS binding method by itself is not believed to be

responsible for reduction of enzyme activity upon immobilization

Direct binding of enzyme to the Janus particle may lead to reduction in the enzyme

activity because of space constraints for the enzyme and difficulty for the passage of

substrate to the active site of the enzyme due to stearic hindrance Hence a linker was

inserted between the enzyme and Janus particle during enzyme immobilization However

inserting a linker between the enzyme and the particle did not lead to enhanced diffusion

of Janus particles or increase in enzyme activity of immobilized enzyme

Lack of enhanced diffusion can be firstly attributed to the reduction in enzyme activity

upon immobilization Enzyme activity may be retained by modifying the conditions

during EDCNHS coupling or changing the binding method altogether The degree of

immobilization of HRP on carboxylated Janus particles can possibly be affected by

several factors such as temperature the concentration of the coupling reagent EDC

27

concentration HRP concentration in solution and incubation time34

Different

combinations of these factors can be tested to find optimum conditions for

immobilization of HRP Alternate binding method can involve thiolation of HRP by the

covalent conjugation of mercaptopropionic acid and then the thiolated HRP can then be

attached on the gold surface of a polystyrene-gold Janus particle35

Thus further

modifications can be made to the system in order to retain enzyme activity on the

immobilized particles and thereby power nanomotors using enzymes

28

Chapter 3

Predator-prey system of enzyme immobilized particles

31 Introduction

Predator-prey systems are widely found in nature and play a major role in biological

processes3637

Predator-prey systems include all the interactions in which one organism

pursues andor consumes another organism One familiar predator-prey system at the

macroscale is that of a tiger chasing and consuming a deer Another not so familiar one

includes Baleen whales which eat millions of microscopic planktons at once38

At the

microscale such kind of interactions can be observed in the case of phagocytes in the

body which are capable of engulfing foreign microorganisms and killing them with a

combination of microbicidal systems39

In the case of cancer the tumor is the prey and

the mature myeloid cells in the body are the predator However the tumor here has a

defense mechanism against the predator in which the tumor produces cytokines which

inhibits the maturation of the myeloid cells and thereby prevents predation40

One similarity that can be observed in all these systems is that the prey possesses some

attribute which is desired by the predator which makes the predator chase the prey A

similar relationship is seen in case of enzymes which are involved in cascade reactions41

In cascade reactions product synthesized by one enzyme is the reactant required by

another enzyme to carry out its own catalytic reaction As such there is a predator-prey

29

kind of dependence between the two enzymes which may cause one enzyme to chase the

other enzyme when placed in suitable substrate solutions However the mechanism

hypothesized for such a system is totally different than the mechanism involved in

biological systems In biological systems such as bacterial chemotaxis or phagocytes

chasing foreign particles in human body complex biochemical reactions are responsible

for the predator-prey behavior42

In the proposed enzyme cascade system there are no

complex biochemical reactions involved The principle hypothesized for predator-prey

behavior of enzyme cascade reactions can be explained as follows Motion of synthetic

motors at nanoscale involves two components translational diffusion and rotational

Brownian diffusion At very short time scales of less than 1Dr where Dr (s-1

) is

rotational Brownian diffusion coefficient the motion of particles is unidirectional For

longer time scales of greater than 1Dr the direction of motion is random giving

poweredactive diffusion43

If the chemical homogeneity is broken that is when there is

higher concentration of a substrate present in one direction of the enzyme the enzyme

might acquire a subtle bias which may lead to a spatial aggregation of the enzymes based

on the substrate concentration gradient The bias can be a result of the relationship

between the speed of enzyme diffusion and substrate concentration where higher

substrate concentration lead to higher enzyme diffusion As a result in an enzyme

cascade system one enzyme will migrate towards higher concentration of substrate

which is present near the other enzyme thus giving rise to a predator-prey behavior

Such enzyme cascades are widely found in signaling networks and metabolic pathways in

living cells and can be used to produce predator-prey like nanomotors systems44

The

30

enzymes being used in the present study are horseradish peroxidase and glucose oxidase

Both enzymes are extremely stable4546

Glucose is the substrate for glucose oxidase

which is widely available and highly stable47

Horseradish peroxidase acts on hydrogen

peroxide It is involved in a cascade reaction with glucose oxidase which produces

hydrogen peroxide by converting glucose into gluconic acid (Figure 3 1)48

Figure 3 1 Concept of particle aggregation due to cascade reactions of immobilized

enzymes

In this experiment glucose oxidase (GOx) and horseradish peroxidase (HRP) were

immobilized on polystyrene carboxylate beads of different sizes using EDCNHS

coupling method Glucose and 3355-Tetramethylbenzidine (TMB) were dissolved

together in water and this solution was used as the substrate solution All the components

were present in a single system in DI water Aggregation of particles and precipitation of

oxidized TMB from the solution was expected to be observed when all these components

were present in such a system

Properties of horseradish peroxidase have been discussed in the previous chapter (Section

211 Horseradish peroxidase)

GOxGOx HRPHRP

Glucose + O2Gluconic acid + H2O2

TMB TMB oxidizedBlue precipitate

H2O

Aggregation

31

311 Glucose oxidase

Figure 3 2 Glucose oxidase

Glucose oxidase (GOx) belongs to the group of oxidoreductases and is also called

glucose aerodehydrogenase49

It is obtained from fungi and has been identified as an

antimicrobial enzyme50

GOx binds to β-D-glucopyranose and catalyzes the oxidation

reaction to from gluconolactone while simultaneously reducing oxygen to hydrogen

peroxide51

GOx has high substrate affinity and specificity high stability and high

turnover number which makes it a suitable biocatalyst for industrial applications52

Figure 3 3 Redox reaction of glucose catalyzed by glucose oxidase53

32

GOx is a glycoprotein with molecular weight of 155 kDa and consists of two identical

polypeptide subunits linked together by a disulfide bond The optimum pH for GOx is

55 though it is stable in a broad pH range of 4 to 7 and can be stored for a long period of

time51

The functional parameters for glucose oxidase include Km of 151 mM and kcat of

337 s-1

at temperature of 20 ordmC and pH of 754

Some of the inhibitors of GOx are 2-

Deoxy-D-glucose adenine nucleotides and NaNO3

32 Experimental

321 Materials and methods

Uniform carboxyl modified polystyrene microspheres 05 microm in diameter were obtained

from Polysciences Inc The size of the purchased microspheres was verified by FESEM

Peroxidase Type I from horseradish glucose oxidase Type II-S from Aspergillus niger

N-(3-dimethyl-aminopropyl)-Nrsquo-ethylcarbodiimide (EDC) and Hydrogen peroxide

solution 50 wt in water were purchased from Sigma-Aldrich N-hydroxysuccinimide

(NHS) was purchased from Fluka D-Glucose (99) was purchased from VWR

International Secure-Seal imaging spacers (9mm diameter) for glass slides were obtained

from Sigma Aldrich All chemicals were of reagent grade and used without further

purification Nanopure deionized water was used for all experiments and preparation of

solutions All measurements were made at room temperature (25 oC) unless mentioned

otherwise The UV-Visible measurements were performed using Agilent 8453A diode-

array UV-Visible Spectrophotometer with ChemStation UV-Vis Software Optical

33

microscopy experiments were performed using Axiovert 200 MAT microscope by Carl

Zeiss Diffusion coefficient measurements were obtained using Nanosight LM10

322 Optical microscopy experiments

The enzyme substrate system was observed under optical microscope to check for

aggregation and precipitation due to cascade reactions Several such systems were

studied Each system in general consisted of GOx and HRP immobilized on different

polystyrene particles The substrate solutions consisted of 01 M glucose and different

concentrations of TMB solution The systems were studied in the presence of all the

constituents and in the absence of glucose GOxHRP and TMB Systems consisting of

different sized HRP immobilized particles in substrate solution of 003 hydrogen

peroxide and TMB were also studied Videos were recorded over a period of 24 hours to

observe changes in the system Each experiment was performed atleast thrice to assess

reproducibility of results

323 Diffusion coefficient measurements using Nanosight LM10

Diffusion coefficient of GOx and HRP immobilized particles were measured using

Nanosight LM10 nanoparticle tracking instrument with Nanoparticle Tracking Analysis

(NTA) 20 software Diffusion coefficients were measured for different systems namely

GOx particles in 01 M glucose and oxygen both GOx and HRP functionalized particles

in 01 M glucose and oxygen along with controls for the individual systems Videos were

34

recorded for 30 seconds at the rate of 30 frames per second Each experiment was carried

out in triplicate to assess reproducibility

33 Results

331 Optical microscopy

According to the enzyme cascade model precipitation of TMB was possible only upon

the interaction of GOx and HRP immobilized particles in presence of appropriate

substrate solution Hence in the first set of experiments precipitation of TMB was used

as an indication of the type of interaction between GOx and HRP immobilized particles

Fan-shaped structures were observed when both HRP and GOx immobilized particles

were present in a substrate solution consisting of glucose and TMB These structures

started forming as a single strand and then branched out and intertwined to form the fan-

shaped structures (Figure 3 4)

Table 3 1 Observations for formation of fan-shaped structures in enzyme cascade

system in presence of different components

GOx HRP Glucose TMB Observation

Yes Yes Yes Yes

Fan-shaped structures observed particles associated with

the fan-shaped structures

No Yes Yes Yes No fan-shaped structures

Yes No Yes Yes No fan-shaped structures

Yes Yes No Yes No fan-shaped structures

Yes Yes Yes No No fan-shaped structures

35

Fan-shaped structures can also be observed in the system consisting of only HRP

immobilized particles in substrate solution of hydrogen peroxide and TMB The size of

the fan-shaped structures depends on the size of the enzyme immobilized particles Free

enzymes show extremely tiny structures which are difficult to view under the optical

microscope

Figure 3 4 Formation of fan-shaped structures when both HRP and GOx

immobilized particles were present in a substrate solution consisting of glucose and

TMB A In the initial stages single strands form on the surface of liquid (5Ox) B

Single strands branch out and intertwine to form fan-shaped structures (50x) C Many

such structures form and settle down after 20 minutes (20x)

A

C

B

36

Experiments were performed in the absence of TMB that is the system consisted of GOx

and HRP immobilized particles in glucose substrate solution In the absence of TMB the

particles were expected to move closer to each other without any precipitation due to the

cascade reaction However there was no conclusive aggregation of particles observed in

the system

Free Enzyme 005 μm HRP - immobilized particle

10 μm HRP - immobilized particle 05 μm HRP - immobilized particle

Figure 3 5 Size dependence of fan-shaped structures on the size of HRP -

immobilized particles (20x)

37

Table 3 2 Observations for particle aggregation in enzyme cascade system in presence

and absence of TMB

Glucose GOx HRP TMB Observation

Present Present Present Present Aggregation and precipitate

Present Present Present Absent No conclusive aggregation

Present Absent Present Present No aggregation or ppt

Absent Present Present Present No aggregation or ppt

Present Present Absent Present No aggregation or ppt

332 Diffusion coefficient measurements

Diffusion coefficients were measured for GOx immobilized particles of 05 μm diameter

in substrate solution of 01 M glucose and oxygen Diffusion coefficients of particles

were also measured for system consisting of GOx and HRP functionalized particles in

glucose and oxygen Each experiment was done in triplicate to assess reproducibility

Nanosight visualization chamber being a closed chamber oxygen was purged into all the

solutions before starting the experiments to ensure sufficient supply of oxygen for

catalytic reaction of GOx

38

Table 3 3 Diffusion coefficients for 05 μm GOx immobilized particles in 01 M

glucose purged with O2

Mean

GOx

particles in

01 M

glucose

Control - GOx

particles in DI

water

Control - GOx

particles in 01 M

glucose without

oxygen purging

Control -

particles in 01

M Glucose

Diffusion

coefficient

(x10-10

cm2s)

13316 12888 13827 13702

Standard

deviation 6307 4926 4424 7468

Table 3 4 Diffusion coefficients for GOx and HRP immobilized particles in 01 M

glucose and purged with O2

Mean

GOx- HRP-

functionalized

particles in 01 M

glucose

Control ndash Gox-

HRP- functionalized

particles in DI water

Control - particles

in 01 M glucose

Diffusion coefficient

(x10-10

cm2s)

13005 14434 13702

Standard deviation 5263 6622 7468

No significant increase was observed in diffusion coefficient of enzyme immobilized

particles in any of the systems

39

34 Discussion

Formation of fan-shaped structures due to patterned deposition of oxidized TMB

indicates some kind of interaction between GOx and HRP immobilized particles Particle

diameter dependence of the size of structures can be explained with a simple model in

Figure 3 6 A larger particle will have a higher surface area as compared to a smaller

particle Hence more enzyme is immobilized on the larger particle As a result higher

quantity of TMB is oxidized and precipitated by the larger particle as compared to the

smaller particle This leads to thicker strands of fan-shaped structures eventually scaling

up the whole system to give bigger fan-shaped structures

The exact reason for the formation of such fan-shaped structures which are dependent on

particle size could not be determined It is hypothesized that hydrogen peroxide andor

Figure 3 6 Model for size dependence of fan-shaped structures on the particle

diameter of enzyme immobilized particles Larger the particle more surface area is

available for precipitation of TMB which eventually leads to bigger fan-shaped

structures

Larger particle Smaller particle

40

TMB gradient in the substrate solution might be responsible for such structures If there

is higher concentration of H2O2 on one side of the particle and assuming an even

concentration distribution of TMB there will be selective precipitation in the direction of

H2O2 When several particles come in the vicinity of higher concentration of H2O2 the

precipitation lines will converge to give fan-shaped structures (Figure 3 7)

Figure 3 7 Hypothesis for formation of fan-shaped structures

In the system consisting of GOx immobilized particles HRP immobilized particles

glucose and TMB it was observed that the fan-shaped structures formed at the liquid-air

interface and eventually settled down to the bottom of the glass slide This makes sense

as oxygen is necessary for the catalytic reaction of conversion of glucose to gluconic acid

and hydrogen peroxide which takes place on the surface of GOx immobilized particle As

a result H2O2 is primarily formed at the liquid-air interface and is then used by HRP for

precipitation of TMB

Following the observations that precipitation of TMB occurs at the surface GOx and

HRP immobilized particles are also expected to interact at the surface in the absence of

TMB Due the cascade reaction it was expected that HRP immobilized particles will be

41

attracted to GOx immobilized particles in appropriate substrate solution Experiments

were carried out to visualize inter-particle interactions at the liquid-air interface

However liquid-air interface not being a stable system to image the precise interaction

of GOx and HRP immobilized particles could not be determined Significant external

disturbances were involved at the interface To enable the cascade reaction in the bulk of

the liquid oxygen was purged into the reaction system before starting the experiment

However this did not lead to significant reaction in the bulk of the liquid and the reaction

prominently took place at the liquid-air interface where the oxygen concentration was

higher

In order to observe the interaction of GOx and HRP immobilized particles better imaging

techniques need to be used to observe diffusion of particles in absence of external

disturbances at the liquid-air interface Large quantity of oxygen needs to be purged into

the system and retained to make the reaction more pronounced in the bulk of the substrate

solution Higher concentration of oxygen in water may also be achieved by using oxygen

enriched water which is commercially available55

Alternatively a different enzyme cascade system may be used which does not require a

gaseous substrate56

For example glycogen synthase synthesizes glycogen from glucose-

1-phosphate and glycogen phosphorylase breaks down glycogen to glucose-1-

phosphate57

This system does not require a gaseous substrate and might be able to

overcome the deficiencies of GOx and HRP enzyme cascade system

42

Chapter 4

Silver-titania nanomotors

41 Introduction

Bimetallic nanowires made of platinum and gold have been developed by WPaxton

etal1 The rods move autonomously in a solution of hydrogen peroxide In the present

study silver-titania (Ag-TiO2) particles are being synthesized and investigated for motion

in presence of hydrogen peroxide and UV light With proper control of conditions and

UV light trigger it can be possible to control the speed and direction of motion of these

nanoparticles

Hydrogen peroxide is produced in the human body by phagocytes as an immune response

to foreign particles and pathogens5859

Also silver nanoparticles have been shown to be

biocompatible and capable of interacting with cells in human body without disrupting

cellular function60

Similarly it has been demonstrated that titania is compatible with

different cells DNA and proteins in human body and does not cause toxic effects Hence

silver-titania nanomotors which swim in hydrogen peroxide may open interesting

avenues in applications like targeted drug delivery in those parts of the body where

hydrogen peroxide is present

43

42 Experimental

421 Materials and methods

Silver nitrate (999995) was purchased from Alfa Aesar Gum arabic was purchased

from MP Biomedicals Hydrogen peroxide solution 50 wt in water and ascorbic acid

were purchased from Sigma-Aldrich Nanopure deionized water was used for all

experiments and preparation of all solutions Secure-Seal imaging spacers (9 mm

diameter) were obtained from Sigma Aldrich All experiments were carried out at room

temperature of 25ordmC unless otherwise mentioned Ag-TiO2 particles were synthesized by

evaporation of titania in Semicore electron beam evaporator Particles were characterized

using field-emission scanning electron microscopy Optical microscopy experiments

were performed using Axiovert 200 MAT microscope by Carl Zeiss Particles were

tracked using Physvis particle tracking software (Figure 4 1) in which the position

information of a particle on screen is stored on a frame by frame basis The data is stored

in a dat file as X and Y co-ordinates of particles tracked on the screen This information

can be converted to diffusion coefficients of the particles by further manipulation using

MathWorks-MATLABreg software

44

Figure 4 1 Physvis particle tracking software

422 Synthesis of silver colloidal particles

Silver particles were synthesized by a nucleationgrowth type reaction developed by

Goya and Matijevic61

and modified by Velikov62

Silver nitrate was used as precursor

metal salt for synthesis of silver particles Solution A was 75 ml of 50 mM AgNO3 and

035 by weight gum arabic Solution B was 75 mL of 100 mM 1-ascorbic acid and

035 by weight gum arabic Solution B was rapidly added to solution A in a 250 mL

reaction flask The reaction flask was covered and particles were stirred for 48 hrs After

48 hours particles were allowed to settle for 2-3 hours in refrigerator and supernatant

was separated from the particles Particles were then rinsed with DI water and centrifuged

thrice to obtain pure silver particles Zeta potential of these particles was reported to be

45

around -50 to -60 mV and the size was around 1μm The size of the particles can be

varied by varying the amounts of ascorbic acid and silver nitrate Using molar ratio of 31

of ascorbic acid silver nitrate can give particles of 200 nm diameter whereas ratio of

151 can give particles of upto 5 μm diameter

423 Preparation of silver-titania (Ag-TiO2) particles

A monolayer of silver particles was prepared according to the procedure by Goldenberg

et al63

Glass slides were placed on the bottom of a petridish Water was put into the

petridish so that the entire slide was immersed in water A thin layer of hexane was then

added on top of water in the petridish A suspension of silver particles in ethanol (5

ww) was slowly introduced to the water-hexane interface in the dish After the

monolayer was formed the glass slide was lifted up through the monolayer with tweezers

The silver particles-covered glass slides were left at room temperature to dry overnight

Following drying a thin film of titania was evaporated on the glass slides in Semicore

evaporator (Figure 2 7) The spheres were partially coated with a 10 nm film of titania

Ag-TiO2 particles were removed from the glass slide by gentle sonication FE-SEM was

used to characterize the particles (Figure 4 2) The thin titania layer was not easily

distinguishable due to roughness of the particles

46

Figure 4 2 FESEM image of Ag-TiO2 particles

424 Optical microscopy experiments

Imaging spacers were placed on a glass slide Suspension of Ag-TiO2 particles was

placed on the glass slide and the particles were allowed to settle Movement of particles

was observed in DI water and videos were recorded at 50x magnification of optical

microscope for 30 sec 05 H2O2 was added to the particle suspension on the slide

Videos were recorded at 50x ranging from 30 sec to 10 min to study the movement of

silver titania particles in hydrogen peroxide solution Particles in both DI water and

hydrogen peroxide were subjected to UV light and videos were recorded to study particle

dynamics in presence of UV radiation Particle tracking was performed using Physvis

particle tracking software Atleast 30 particles in every system were tracked for 10

seconds each Particle tracking data was obtained in terms of X and Y coordinates of

particles on screen This data was manipulated using MathWorks-Matlab software and

graphs of mean square displacement of particles versus time were plotted Diffusion

coefficients of particles were calculated from these graphs

47

43 Results and Discussion

Ag-TiO2 particles demonstrated autonomous motion in the presence of H2O2 No UV

light was required for this motion Motion of the particles was accelerated by addition of

fresh H2O2 to the system When subjected to UV radiation the particles displayed

momentary acceleration for 4-5 seconds

Figure 4 3 Mechanism of motion of Ag-TiO2 nanomotors

In the presence of H2O2 silver part of the Ag-TiO2 particle converts H2O2 to H2O and O2

This may lead to propulsion of the Ag-TiO2 particle due to bubble propulsion andor self-

diffusiophoresis In the presence of UV light additional reaction takes place on the

titania part of Ag-TiO2 particle which increases the velocity of the Ag-TiO2 particles

(Figure 4 4) This UV triggered photoactivity of titania is due to hole-electron separation

caused by photons having energy equal to or greater than the band gap of titania646566

Most of the holes-electrons recombine but a small fraction move to the surface and react

with the available redox species like water and oxygen to form protons (H+) superoxide

(O2-) and hydroxyl (OH) radicals

6566 This may lead to propulsion by diffusiophoresis

as well as electrophoresis

48

Figure 4 4 Silver-titania motors move faster when subjected to UV radiation

In addition to linear motion rotational motion is also observed in several particles and

particle aggregates Aggregation of particles can lead to a net circular propulsive force

which can rotate the aggregate as shown in Figure 4 5

Figure 4 5 Rotation due to particle aggregation of Ag-TiO2 particles

Particle tracking by Physvis software revealed a significant increase in diffusion

coefficient of Ag-TiO2 particles in hydrogen peroxide solution as compared to DI water

49

The Ag-TiO2 particles have a diffusion coefficient of 1491 μm2s in hydrogen peroxide

versus only 180 μm2s in DI water Thus the Ag-TiO2 particles move around more than

10 times their body length per second in hydrogen peroxide (Figure 4 6) When

subjected to UV light the particles experience a significant acceleration in hydrogen

peroxide which lasts only for a short duration of time (4-5 sec) after which the particles

return to their original velocity The diffusion coefficient of the particles during this UV

light trigger is found to be around 5093 μm2s UV light trigger does not have any visible

effect on the Ag-TiO2 particles in DI water

Table 4 1 Translational diffusion coefficients of Ag-TiO2 particles in hydrogen

peroxide and water

Mean 05 H2O2 solution DI water

Diffusion coefficient of Ag-TiO2 (μm2s)

without UV light

1491 180

Diffusion coefficient of Ag-TiO2 (μm2s)

in the presence of UV light

5093 113

50

Figure 4 6 Mean square displacement curves of Ag-TiO2 Janus particles in hydrogen

peroxide and DI water

When higher concentration (~30 vv) of Ag-TiO2 particles in hydrogen peroxide is

subjected to UV radiation the particles exhibit pattern formation (Figure 4 7)

Aggregation of Ag-TiO2 Janus particles is observed when UV light is turned on and the

particles disaggregated when UV light is switched off This can be due to predator-prey

kind of interaction between silver and titania half of the Janus particles in the presence of

UV light Oxygen and water that are produced at the silver end by decomposition of

hydrogen peroxide are used up by titania when subjected to UV radiation This can lead

to attraction between titania and silver parts of neighboring Janus particles causing

aggregation

y = 20373x

y = 59994x

y = 71848x

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10 12

Mean

sq

uare

dis

pla

cem

en

t (μ

m2s

)

Time (sec)

Mean square displacement vs Time for Ag-TiO2

particles

05 Hydrogen peroxide and UV05 Hydrogen peroxide

DI Water

51

A UV OFF B UV ON

C UV OFF

Figure 4 7 Higher concentrations of Ag-TiO2 particles showing aggregation when UV

is turned on

52

Chapter 5

Summary and Conclusion

In summary no significant enhancement in diffusion was observed in case of enzyme

immobilized Janus particles in substrate solution Presence of a linker between enzyme

and particle did not increase the diffusion coefficient of particles Alternative enzyme

immobilization methods need to be tested like thiolation of enzyme and subsequent

binding to gold surface of a Janus particle35

or expressing the enzyme with polyhistidine

or avidin tag67

This can result in higher enzyme activity upon immobilization and may

lead to enhanced diffusion of Janus particles

Predator-prey system of GOx and HRP immobilized particles showed some promising

results Aggregation of particles and formation of fan-shaped structures by TMB

oxidation were observed under optical microscope when GOx and HRP immobilized

particles glucose and TMB were present in the system The size of the fan-shaped

structures was dependent on the size of enzyme immobilized particles This can be

indicative of some kind of interaction between the GOx and HRP immobilized particles

No conclusive aggregation of GOx and HRP immobilized particles was observed in

absence of TMB Better imaging techniques are required to study the interaction of GOx

and HRP immobilized particles at the unstable liquid-air interface where majority of the

reaction takes place Other enzyme cascade systems not requiring gaseous reactants like

glycogen synthase and glycogen phosphorylase should be tested for similar activity

53

Ag-TiO2 particles were found to exhibit autonomous motion in the presence of hydrogen

peroxide solution The motion was momentarily accelerated upon exposure to UV

radiation The particles showed a diffusion coefficient of 1491 μm2s in hydrogen

peroxide versus only 180 μm2s in DI water Upon subjecting particles to UV radiation in

presence of H2O2 diffusion coefficient increased to 5093 μm2s for a period of 4-5

seconds In addition to linear motion rotating motion could also be observed in some

particle aggregates Further development of these aggregate structures can lead to

formation of rotors at nanoscale Higher concentration of silver-titania particles in

hydrogen peroxide displayed aggregation in presence of UV light and disaggregated

when UV light was switched off This may be attributed to the predator-prey kind of

interaction between silver and titania where oxygen and water that are produced by

silver by decomposition of hydrogen peroxide are used up by titania when subjected to

UV radiation This promises to be an interesting system and needs to be further

investigated to explore its properties

54

References

1 Paxton WF Sundararajan S Mallouk TE Sen A Angew Chem Int Ed Engl

2006 45 5420-5429

2 Golestanian R Liverpool TB Adjari A Phys Rev Lett 2005 94 220801

3 httpwwwsilvermedicineorgh2o2html

4 Rami Pedahzur Ovadia Lev Badri Fattal Hillel Shuval Water Science and Technology

1995 31(5-6) 123-129

5 W Went F American Scientist 1968 56

6 Adriano Cavalcanti Tad Hogg Bijan Shirinzadeh IEEE MHS 2006 International

Symposium on Micro-NanoMechatronics and Human Science 2006 226-232

7 EM Purcell American Journal of Physics 1977 45 3-11

8 Golestanian R Phys Rev Lett 2010 105 018103(1)-018103(4)

9 S S Dukhin Z R Ulberg G L Dvornichenko B V Deryagin Russian Chemical

Bulletin 1983 31(8) 1535-1544

10 Dukhin AS Goetz PJ Ultrasound for characterizing colloids Particle sizing zeta

potential rheology Elsevier 2002 59

11 HJKeh HJTu Trends in Colloid and Interface Science XIV Progress in Colloid and

Polymer Science 2000 115 174-180

12 D Zinemanas A Nir International Journal of Multiphase Flow 1995 21(5) 787-800

55

13 Stephen Ebbens Jonathan Howse Soft matter 2010 6 726-739

14 J Vicario R Eelkema W R Browne A Meetsma R M La Crois B L Feringa

Chem Commun 2005 31 3936

15 Davide Pantarotto Wesley R Browne Ben L Feringa Chem Commun 2008 1533 -

1535

16 Lisa A Cameron Matthew J Footer Alexander van Oudenaarden Julie A Theriot

PNAS 1999 96(9) 4908-4913

17 Ali Najafi Ramin Golestanian Phys Rev E 2004 69 062901

18 Jinseok Kim Jungyul Park Sungwook Yang Jeongeun Baek Byungkyu Kim Sang

Ho Lee Eui-Sung Yoon Kukjin Chun Sukho Park Lab Chip 2007 7 1504 - 1508

19 Kurt M Dubowski Clinical Chemistry 2008 5411 1919ndash1920

20 Spector DA Yang Q Wade JB Am J Physiol Renal Physiol 2007 292(1) 467-474

21 Rhee SG Chang TS Bae YS Lee SR Kang SW J Am Soc Nephrol 2003 14(8-3)

211-215

22 Sakaue T Kapral R Mikhailov A S Eur Phys J B 2010 75 381ndash387

23 Muddana H S Sengupta S Mallouk T E Sen A Butler P J J Am Chem Soc

2010 132 (7) 2110ndash2111

24 Baker CJ Deahl K Domek J Orlandi EW Arch Biochem Biophys 2000 382(2) 232-

237

25 V Vojinovic RH Carvalho F Lemos JMS Cabral LP Fonseca BS Ferreira

Biochemical Engineering Journal 2007 35 126-135

56

26

OV Lebedeva NN Ugarova Russian Chemical Bulletin 1996 45(1) 18-25

27 Nigel C Veitch Phytochemistry 2004 65(3) 249-259

28 Kenneth H Petersen Journal of Immunological Methods 2009 340(1) 86-89

29 httpwwwbrenda-enzymesorgphpresult_flatphp4ecno=11117

30 Goldenberg L M Wagner J Stumpe J Paulke B-R Goumlrnitz E Langmuir 2002 18

5627-5629

31 httpwwwgoldbiocompdf3761-Protocol201pdf

32 Av Rovisco Pais Lisboa Portugal T amp D Technology West Yorkshire UK Journal

of Molecular Catalysis B Enzymatic 2004 28(2-3) 129-135

33 httpwwwphysicsnyuedugrierlabmethodsnode11html

34 Yoon-Mee Lee1 O-Yul Kwon Yeo-Joon Yoon Keungarp Ryu Biotechnology Letters

2006 28 39ndash43

35 JC Pyun SD Kim JW Chung Analytical Biochemistry 2005 347(2) 227-233

36 George H Pimbley Jr Mathematical Biosciences 1974 20(1-2) 27-51

37 Andy Fenton Sarah E Perkins Parasitology 2010 137 1027-1038

38 Mead James G Brownell Robert L Mammal Species of the World A Taxonomic and

Geographic Reference (3rd ed) Johns Hopkins University Press 2005 2 2142

57

39

Krause KH Schweiz Med Wochenschr 2000 130(4) 97-100

40 Irina Kareva Faina Berezovskaya Carlos Castillo-Chavez Journal of Biological

Dynamics 2010 4(4)315 - 327

41 Delaittre G Reynhout I Cornelissen J Nolte R Chemistry ndash A European Journal

2009 15 12600ndash12603

42 C Buller DH Johnson RC Tschumper Investigative Ophthalmology amp Visual Science

1990 31 2156-2163

43 Robert M Mazo Brownian Motion Fluctuations Dynamics and Applications Oxford

Science Publications 2009 57

44 Vivek K Mutalik KV Venkatesh Theoretical Biology and Medical Modelling 2005 2

19

45 Satoshi Nakamura Kunimasa Koga Biochemical and Biophysical Research

Communications 1977 78(2) 806-810

46 Krishnananda Chattopadhyay Shyamalava Mazumdar Biochemistry 2000 39 (1)

263ndash270

47 Satoshi Nakamura Yasuyuki Ogura J Biochem 1968 63 (3) 308-316

48 van Dongen SF Nallani M Cornelissen JJ Nolte RJ van Hest JC Chemistry 2009

15(5) 1107-1114

49 Ronald Bentley Methods in Enzymology 1955 1 340-345

50 Vartiainen J Raumlttouml M Paulussen S Packaging Technology and Science 2005

18 243ndash251

58

51 GA Sherbeny AA Shindia YMMM Sheriff International Journal of Agriculture

and Biology 2005 7

52 D Rando G-W Kohring F Giffhorn Appl Microbiol Biotechnol 1997 48

53 Worthington enzyme manual Worthington Biochemical Corporation

httpworthington-biochemcomgopdefaulthtml

54 httpwwwbrenda-enzymesorgphpresult_flatphp4ecno=11117

55 httpwwwoxygenphwatercom

56 P Boon Chock Sue Goo Rhee and Earl R Stadtman Annu Rev Biochem 1980 49

813-841

57 Sybil Golden P A Wals Joseph Katz Analytical Biochemistry 1977 77(2) 436-445

58 R L Baehner S K Murrmann J Davis R B Johnston Jr J Clin Invest 1975 56(3)

571ndash576

59 I Bejarano M P Terroacuten S D Paredes C Barriga A B Rodriacuteguez J A Pariente

Molecular and Cellular Biochemistry 2007 296(1-2) 77-84

60 Michael C Moulton Laura K Braydich-Stolle Mallikarjuna N Nadagouda Samantha

Kunzelman Saber M Hussain Rajender S Varma Nanoscale 2010 2 763-770

61 Goya DV Matijevic E Colloids and Surfaces A 1999 146 139

62 Velikov KP Zegers GE van Blaanderen A Langmuir 2003 19 1384

63 Goldenberg L M Wagner J Stumpe J Paulke B-R Goumlrnitz E Langmuir 2002 18

5627-5629

59

64

Banerjee S Gopal J Muraleedharan P Tyagi A K Raj B Curr Sci 2006

901378ndash1383

65 Bogomolov V N Kudinov E K Firsov Y A Sov Phys Solid State 1968 35 555

66 Kudo A Miseki Y Chem Soc Rev 2009 38 253ndash278

67 Gaj T Meyer SC Ghosh I Protein expression and Purification 2007 56 54-61

21

23 Results

231 Enzyme assay

The activity of free HRP enzyme reported by Sigma Aldrich is 150 unitsmg However

the activity of HRP upon analysis was found to be 52158 unitsmg which is much lower

than the reported value The activity of HRP was found to be further lowered to 2133

unitsmg when it was immobilized on the PS-SiO2 Janus particles in the presence of a

linker The following formula is used to calculate enzyme activity

where ΔAmin is the change in absorbance per minute

22

Figure 2 10 Graph for the assay of solution of free enzyme containing 05mgL of HRP

ΔAmin = 01716 Unitsmg of free enzyme = 52158

During EDCNHS coupling 04 mg enzyme was added to 1 ml of 025 vv suspension

of Janus particles Thus initial concentration of enzyme in the mixture was 04 mgml

Assuming retained activity of the enzyme in supernatant solution the amount of enzyme

in supernatant was calculated to be 03868 mgml from the assay Amount of enzyme

functionalized on the Janus particles was calculated as the difference between enzyme

previously added for immobilization (04 mgml) and the enzyme present in supernatant

(03868 mgml) and was found to be 00132 mgml Enzyme assay was performed on the

enzyme immobilized Janus particles to estimate the activity of immobilized enzyme

(Figure 2 11) and was found to be 2133 unitsmg

y = 0002x + 00516

0

01

02

03

04

05

06

07

08

0 50 100 150 200 250 300 350 400

Ab

sorb

an

ce

51

0 n

m

Time (sec)

Assay of free enzyme HRP

23

Figure 2 11 Graph for the assay of of immobilized enzyme on 05 μm Janus particles

(~025 vv) ΔAmin = 01853 Unitsmg of free enzyme = 2133

232 Diffusion coefficient measurements

No significant increase was observed in diffusion coefficient of HRP-immobilized Janus

particles in the presence of H2O2 as the substrate Similar results were obtained for

diffusion coefficient measurement with and without linker moieties bridging the enzymes

and particles(Table 2 1 Table 2 2) Thus the linker did not seem to assist in increasing

the particle diffusion by improving or retaining the activity of the enzyme

y = 00015x + 00953

0

01

02

03

04

05

06

07

0 50 100 150 200 250 300 350 400

Ab

sorb

an

ce

510 n

m

Time (sec)

Assay of HRP immobilized 05 μm Janus particles

24

Table 2 1 Diffusion coefficient measurements of HRP immobilized Janus particles

without linker

HRP immobilized

Janus particles in

003 H2O2

Control - Janus

particles in

003 H2O2

Control - HRP

immobilized Janus

particles in DI water

Diffusion coefficient

(10-10

cm2s)

1359416 1051777 1308675

Standard deviation 4534801 4122075 4918597

Table 2 2 Diffusion coefficient measurements of HRP immobilized Janus particles in

the presence of linker

HRP immobilized

Janus particles in

003 H2O2

Control - Janus

particles in

003 H2O2

Control - HRP

immobilized Janus

particles in DI water

Diffusion coefficient

(10-10

cm2s)

13838 1339829 1407891

Standard deviation 5644692 5649364 5797218

25

Figure 2 12 Diffusion coefficient measurements of PS-SiO2 Janus particles without and

with 6-aminohexanoic acid linker

24 Discussion

To compare the diffusion coefficients observed during the experiments theoretical

diffusion coefficient was calculated using following equation33

r

kTD altraslation

6 Equation 21

where r is the hydrodynamic radius of particle Dtranslational is translational diffusion

coefficient of the particle T is temperature of the system η is the viscosity of fluid

through which the particles are moving and k is the Boltzmann constant From FE-SEM

images the size of the particles used was determined to be 450 nm For 450 nm diameter

0

20

40

60

80

100

120

140

160

Without linker With linker

Dif

fusio

n c

oeff

icie

nt

(10

-10 c

m2s

)

HRP immobilized 05 μm Janus particles - Diffusion coefficient measurements

HRP immobilized Janus particles in 003 H2O2

Control - Janus particles in 003 H2O2

Control - HRP immobilized Janus particles in DI water

26

particle Dtranslational is equal to 109 μm2s which is comparable to the diffusion

coefficient measured for the enzyme immobilized particles

HRP was found to be active on the Janus particles however the activity of the enzyme

was reduced upon immobilization Lower enzyme activity can be due to the coupling

method used which in this case was EDCNHS covalent binding However EDCNHS

coupling method has been shown to significantly retain the enzyme activity when the

enzyme is immobilized on carboxylated multi-walled carbon nanotubes under appropriate

conditions34

Hence EDCNHS binding method by itself is not believed to be

responsible for reduction of enzyme activity upon immobilization

Direct binding of enzyme to the Janus particle may lead to reduction in the enzyme

activity because of space constraints for the enzyme and difficulty for the passage of

substrate to the active site of the enzyme due to stearic hindrance Hence a linker was

inserted between the enzyme and Janus particle during enzyme immobilization However

inserting a linker between the enzyme and the particle did not lead to enhanced diffusion

of Janus particles or increase in enzyme activity of immobilized enzyme

Lack of enhanced diffusion can be firstly attributed to the reduction in enzyme activity

upon immobilization Enzyme activity may be retained by modifying the conditions

during EDCNHS coupling or changing the binding method altogether The degree of

immobilization of HRP on carboxylated Janus particles can possibly be affected by

several factors such as temperature the concentration of the coupling reagent EDC

27

concentration HRP concentration in solution and incubation time34

Different

combinations of these factors can be tested to find optimum conditions for

immobilization of HRP Alternate binding method can involve thiolation of HRP by the

covalent conjugation of mercaptopropionic acid and then the thiolated HRP can then be

attached on the gold surface of a polystyrene-gold Janus particle35

Thus further

modifications can be made to the system in order to retain enzyme activity on the

immobilized particles and thereby power nanomotors using enzymes

28

Chapter 3

Predator-prey system of enzyme immobilized particles

31 Introduction

Predator-prey systems are widely found in nature and play a major role in biological

processes3637

Predator-prey systems include all the interactions in which one organism

pursues andor consumes another organism One familiar predator-prey system at the

macroscale is that of a tiger chasing and consuming a deer Another not so familiar one

includes Baleen whales which eat millions of microscopic planktons at once38

At the

microscale such kind of interactions can be observed in the case of phagocytes in the

body which are capable of engulfing foreign microorganisms and killing them with a

combination of microbicidal systems39

In the case of cancer the tumor is the prey and

the mature myeloid cells in the body are the predator However the tumor here has a

defense mechanism against the predator in which the tumor produces cytokines which

inhibits the maturation of the myeloid cells and thereby prevents predation40

One similarity that can be observed in all these systems is that the prey possesses some

attribute which is desired by the predator which makes the predator chase the prey A

similar relationship is seen in case of enzymes which are involved in cascade reactions41

In cascade reactions product synthesized by one enzyme is the reactant required by

another enzyme to carry out its own catalytic reaction As such there is a predator-prey

29

kind of dependence between the two enzymes which may cause one enzyme to chase the

other enzyme when placed in suitable substrate solutions However the mechanism

hypothesized for such a system is totally different than the mechanism involved in

biological systems In biological systems such as bacterial chemotaxis or phagocytes

chasing foreign particles in human body complex biochemical reactions are responsible

for the predator-prey behavior42

In the proposed enzyme cascade system there are no

complex biochemical reactions involved The principle hypothesized for predator-prey

behavior of enzyme cascade reactions can be explained as follows Motion of synthetic

motors at nanoscale involves two components translational diffusion and rotational

Brownian diffusion At very short time scales of less than 1Dr where Dr (s-1

) is

rotational Brownian diffusion coefficient the motion of particles is unidirectional For

longer time scales of greater than 1Dr the direction of motion is random giving

poweredactive diffusion43

If the chemical homogeneity is broken that is when there is

higher concentration of a substrate present in one direction of the enzyme the enzyme

might acquire a subtle bias which may lead to a spatial aggregation of the enzymes based

on the substrate concentration gradient The bias can be a result of the relationship

between the speed of enzyme diffusion and substrate concentration where higher

substrate concentration lead to higher enzyme diffusion As a result in an enzyme

cascade system one enzyme will migrate towards higher concentration of substrate

which is present near the other enzyme thus giving rise to a predator-prey behavior

Such enzyme cascades are widely found in signaling networks and metabolic pathways in

living cells and can be used to produce predator-prey like nanomotors systems44

The

30

enzymes being used in the present study are horseradish peroxidase and glucose oxidase

Both enzymes are extremely stable4546

Glucose is the substrate for glucose oxidase

which is widely available and highly stable47

Horseradish peroxidase acts on hydrogen

peroxide It is involved in a cascade reaction with glucose oxidase which produces

hydrogen peroxide by converting glucose into gluconic acid (Figure 3 1)48

Figure 3 1 Concept of particle aggregation due to cascade reactions of immobilized

enzymes

In this experiment glucose oxidase (GOx) and horseradish peroxidase (HRP) were

immobilized on polystyrene carboxylate beads of different sizes using EDCNHS

coupling method Glucose and 3355-Tetramethylbenzidine (TMB) were dissolved

together in water and this solution was used as the substrate solution All the components

were present in a single system in DI water Aggregation of particles and precipitation of

oxidized TMB from the solution was expected to be observed when all these components

were present in such a system

Properties of horseradish peroxidase have been discussed in the previous chapter (Section

211 Horseradish peroxidase)

GOxGOx HRPHRP

Glucose + O2Gluconic acid + H2O2

TMB TMB oxidizedBlue precipitate

H2O

Aggregation

31

311 Glucose oxidase

Figure 3 2 Glucose oxidase

Glucose oxidase (GOx) belongs to the group of oxidoreductases and is also called

glucose aerodehydrogenase49

It is obtained from fungi and has been identified as an

antimicrobial enzyme50

GOx binds to β-D-glucopyranose and catalyzes the oxidation

reaction to from gluconolactone while simultaneously reducing oxygen to hydrogen

peroxide51

GOx has high substrate affinity and specificity high stability and high

turnover number which makes it a suitable biocatalyst for industrial applications52

Figure 3 3 Redox reaction of glucose catalyzed by glucose oxidase53

32

GOx is a glycoprotein with molecular weight of 155 kDa and consists of two identical

polypeptide subunits linked together by a disulfide bond The optimum pH for GOx is

55 though it is stable in a broad pH range of 4 to 7 and can be stored for a long period of

time51

The functional parameters for glucose oxidase include Km of 151 mM and kcat of

337 s-1

at temperature of 20 ordmC and pH of 754

Some of the inhibitors of GOx are 2-

Deoxy-D-glucose adenine nucleotides and NaNO3

32 Experimental

321 Materials and methods

Uniform carboxyl modified polystyrene microspheres 05 microm in diameter were obtained

from Polysciences Inc The size of the purchased microspheres was verified by FESEM

Peroxidase Type I from horseradish glucose oxidase Type II-S from Aspergillus niger

N-(3-dimethyl-aminopropyl)-Nrsquo-ethylcarbodiimide (EDC) and Hydrogen peroxide

solution 50 wt in water were purchased from Sigma-Aldrich N-hydroxysuccinimide

(NHS) was purchased from Fluka D-Glucose (99) was purchased from VWR

International Secure-Seal imaging spacers (9mm diameter) for glass slides were obtained

from Sigma Aldrich All chemicals were of reagent grade and used without further

purification Nanopure deionized water was used for all experiments and preparation of

solutions All measurements were made at room temperature (25 oC) unless mentioned

otherwise The UV-Visible measurements were performed using Agilent 8453A diode-

array UV-Visible Spectrophotometer with ChemStation UV-Vis Software Optical

33

microscopy experiments were performed using Axiovert 200 MAT microscope by Carl

Zeiss Diffusion coefficient measurements were obtained using Nanosight LM10

322 Optical microscopy experiments

The enzyme substrate system was observed under optical microscope to check for

aggregation and precipitation due to cascade reactions Several such systems were

studied Each system in general consisted of GOx and HRP immobilized on different

polystyrene particles The substrate solutions consisted of 01 M glucose and different

concentrations of TMB solution The systems were studied in the presence of all the

constituents and in the absence of glucose GOxHRP and TMB Systems consisting of

different sized HRP immobilized particles in substrate solution of 003 hydrogen

peroxide and TMB were also studied Videos were recorded over a period of 24 hours to

observe changes in the system Each experiment was performed atleast thrice to assess

reproducibility of results

323 Diffusion coefficient measurements using Nanosight LM10

Diffusion coefficient of GOx and HRP immobilized particles were measured using

Nanosight LM10 nanoparticle tracking instrument with Nanoparticle Tracking Analysis

(NTA) 20 software Diffusion coefficients were measured for different systems namely

GOx particles in 01 M glucose and oxygen both GOx and HRP functionalized particles

in 01 M glucose and oxygen along with controls for the individual systems Videos were

34

recorded for 30 seconds at the rate of 30 frames per second Each experiment was carried

out in triplicate to assess reproducibility

33 Results

331 Optical microscopy

According to the enzyme cascade model precipitation of TMB was possible only upon

the interaction of GOx and HRP immobilized particles in presence of appropriate

substrate solution Hence in the first set of experiments precipitation of TMB was used

as an indication of the type of interaction between GOx and HRP immobilized particles

Fan-shaped structures were observed when both HRP and GOx immobilized particles

were present in a substrate solution consisting of glucose and TMB These structures

started forming as a single strand and then branched out and intertwined to form the fan-

shaped structures (Figure 3 4)

Table 3 1 Observations for formation of fan-shaped structures in enzyme cascade

system in presence of different components

GOx HRP Glucose TMB Observation

Yes Yes Yes Yes

Fan-shaped structures observed particles associated with

the fan-shaped structures

No Yes Yes Yes No fan-shaped structures

Yes No Yes Yes No fan-shaped structures

Yes Yes No Yes No fan-shaped structures

Yes Yes Yes No No fan-shaped structures

35

Fan-shaped structures can also be observed in the system consisting of only HRP

immobilized particles in substrate solution of hydrogen peroxide and TMB The size of

the fan-shaped structures depends on the size of the enzyme immobilized particles Free

enzymes show extremely tiny structures which are difficult to view under the optical

microscope

Figure 3 4 Formation of fan-shaped structures when both HRP and GOx

immobilized particles were present in a substrate solution consisting of glucose and

TMB A In the initial stages single strands form on the surface of liquid (5Ox) B

Single strands branch out and intertwine to form fan-shaped structures (50x) C Many

such structures form and settle down after 20 minutes (20x)

A

C

B

36

Experiments were performed in the absence of TMB that is the system consisted of GOx

and HRP immobilized particles in glucose substrate solution In the absence of TMB the

particles were expected to move closer to each other without any precipitation due to the

cascade reaction However there was no conclusive aggregation of particles observed in

the system

Free Enzyme 005 μm HRP - immobilized particle

10 μm HRP - immobilized particle 05 μm HRP - immobilized particle

Figure 3 5 Size dependence of fan-shaped structures on the size of HRP -

immobilized particles (20x)

37

Table 3 2 Observations for particle aggregation in enzyme cascade system in presence

and absence of TMB

Glucose GOx HRP TMB Observation

Present Present Present Present Aggregation and precipitate

Present Present Present Absent No conclusive aggregation

Present Absent Present Present No aggregation or ppt

Absent Present Present Present No aggregation or ppt

Present Present Absent Present No aggregation or ppt

332 Diffusion coefficient measurements

Diffusion coefficients were measured for GOx immobilized particles of 05 μm diameter

in substrate solution of 01 M glucose and oxygen Diffusion coefficients of particles

were also measured for system consisting of GOx and HRP functionalized particles in

glucose and oxygen Each experiment was done in triplicate to assess reproducibility

Nanosight visualization chamber being a closed chamber oxygen was purged into all the

solutions before starting the experiments to ensure sufficient supply of oxygen for

catalytic reaction of GOx

38

Table 3 3 Diffusion coefficients for 05 μm GOx immobilized particles in 01 M

glucose purged with O2

Mean

GOx

particles in

01 M

glucose

Control - GOx

particles in DI

water

Control - GOx

particles in 01 M

glucose without

oxygen purging

Control -

particles in 01

M Glucose

Diffusion

coefficient

(x10-10

cm2s)

13316 12888 13827 13702

Standard

deviation 6307 4926 4424 7468

Table 3 4 Diffusion coefficients for GOx and HRP immobilized particles in 01 M

glucose and purged with O2

Mean

GOx- HRP-

functionalized

particles in 01 M

glucose

Control ndash Gox-

HRP- functionalized

particles in DI water

Control - particles

in 01 M glucose

Diffusion coefficient

(x10-10

cm2s)

13005 14434 13702

Standard deviation 5263 6622 7468

No significant increase was observed in diffusion coefficient of enzyme immobilized

particles in any of the systems

39

34 Discussion

Formation of fan-shaped structures due to patterned deposition of oxidized TMB

indicates some kind of interaction between GOx and HRP immobilized particles Particle

diameter dependence of the size of structures can be explained with a simple model in

Figure 3 6 A larger particle will have a higher surface area as compared to a smaller

particle Hence more enzyme is immobilized on the larger particle As a result higher

quantity of TMB is oxidized and precipitated by the larger particle as compared to the

smaller particle This leads to thicker strands of fan-shaped structures eventually scaling

up the whole system to give bigger fan-shaped structures

The exact reason for the formation of such fan-shaped structures which are dependent on

particle size could not be determined It is hypothesized that hydrogen peroxide andor

Figure 3 6 Model for size dependence of fan-shaped structures on the particle

diameter of enzyme immobilized particles Larger the particle more surface area is

available for precipitation of TMB which eventually leads to bigger fan-shaped

structures

Larger particle Smaller particle

40

TMB gradient in the substrate solution might be responsible for such structures If there

is higher concentration of H2O2 on one side of the particle and assuming an even

concentration distribution of TMB there will be selective precipitation in the direction of

H2O2 When several particles come in the vicinity of higher concentration of H2O2 the

precipitation lines will converge to give fan-shaped structures (Figure 3 7)

Figure 3 7 Hypothesis for formation of fan-shaped structures

In the system consisting of GOx immobilized particles HRP immobilized particles

glucose and TMB it was observed that the fan-shaped structures formed at the liquid-air

interface and eventually settled down to the bottom of the glass slide This makes sense

as oxygen is necessary for the catalytic reaction of conversion of glucose to gluconic acid

and hydrogen peroxide which takes place on the surface of GOx immobilized particle As

a result H2O2 is primarily formed at the liquid-air interface and is then used by HRP for

precipitation of TMB

Following the observations that precipitation of TMB occurs at the surface GOx and

HRP immobilized particles are also expected to interact at the surface in the absence of

TMB Due the cascade reaction it was expected that HRP immobilized particles will be

41

attracted to GOx immobilized particles in appropriate substrate solution Experiments

were carried out to visualize inter-particle interactions at the liquid-air interface

However liquid-air interface not being a stable system to image the precise interaction

of GOx and HRP immobilized particles could not be determined Significant external

disturbances were involved at the interface To enable the cascade reaction in the bulk of

the liquid oxygen was purged into the reaction system before starting the experiment

However this did not lead to significant reaction in the bulk of the liquid and the reaction

prominently took place at the liquid-air interface where the oxygen concentration was

higher

In order to observe the interaction of GOx and HRP immobilized particles better imaging

techniques need to be used to observe diffusion of particles in absence of external

disturbances at the liquid-air interface Large quantity of oxygen needs to be purged into

the system and retained to make the reaction more pronounced in the bulk of the substrate

solution Higher concentration of oxygen in water may also be achieved by using oxygen

enriched water which is commercially available55

Alternatively a different enzyme cascade system may be used which does not require a

gaseous substrate56

For example glycogen synthase synthesizes glycogen from glucose-

1-phosphate and glycogen phosphorylase breaks down glycogen to glucose-1-

phosphate57

This system does not require a gaseous substrate and might be able to

overcome the deficiencies of GOx and HRP enzyme cascade system

42

Chapter 4

Silver-titania nanomotors

41 Introduction

Bimetallic nanowires made of platinum and gold have been developed by WPaxton

etal1 The rods move autonomously in a solution of hydrogen peroxide In the present

study silver-titania (Ag-TiO2) particles are being synthesized and investigated for motion

in presence of hydrogen peroxide and UV light With proper control of conditions and

UV light trigger it can be possible to control the speed and direction of motion of these

nanoparticles

Hydrogen peroxide is produced in the human body by phagocytes as an immune response

to foreign particles and pathogens5859

Also silver nanoparticles have been shown to be

biocompatible and capable of interacting with cells in human body without disrupting

cellular function60

Similarly it has been demonstrated that titania is compatible with

different cells DNA and proteins in human body and does not cause toxic effects Hence

silver-titania nanomotors which swim in hydrogen peroxide may open interesting

avenues in applications like targeted drug delivery in those parts of the body where

hydrogen peroxide is present

43

42 Experimental

421 Materials and methods

Silver nitrate (999995) was purchased from Alfa Aesar Gum arabic was purchased

from MP Biomedicals Hydrogen peroxide solution 50 wt in water and ascorbic acid

were purchased from Sigma-Aldrich Nanopure deionized water was used for all

experiments and preparation of all solutions Secure-Seal imaging spacers (9 mm

diameter) were obtained from Sigma Aldrich All experiments were carried out at room

temperature of 25ordmC unless otherwise mentioned Ag-TiO2 particles were synthesized by

evaporation of titania in Semicore electron beam evaporator Particles were characterized

using field-emission scanning electron microscopy Optical microscopy experiments

were performed using Axiovert 200 MAT microscope by Carl Zeiss Particles were

tracked using Physvis particle tracking software (Figure 4 1) in which the position

information of a particle on screen is stored on a frame by frame basis The data is stored

in a dat file as X and Y co-ordinates of particles tracked on the screen This information

can be converted to diffusion coefficients of the particles by further manipulation using

MathWorks-MATLABreg software

44

Figure 4 1 Physvis particle tracking software

422 Synthesis of silver colloidal particles

Silver particles were synthesized by a nucleationgrowth type reaction developed by

Goya and Matijevic61

and modified by Velikov62

Silver nitrate was used as precursor

metal salt for synthesis of silver particles Solution A was 75 ml of 50 mM AgNO3 and

035 by weight gum arabic Solution B was 75 mL of 100 mM 1-ascorbic acid and

035 by weight gum arabic Solution B was rapidly added to solution A in a 250 mL

reaction flask The reaction flask was covered and particles were stirred for 48 hrs After

48 hours particles were allowed to settle for 2-3 hours in refrigerator and supernatant

was separated from the particles Particles were then rinsed with DI water and centrifuged

thrice to obtain pure silver particles Zeta potential of these particles was reported to be

45

around -50 to -60 mV and the size was around 1μm The size of the particles can be

varied by varying the amounts of ascorbic acid and silver nitrate Using molar ratio of 31

of ascorbic acid silver nitrate can give particles of 200 nm diameter whereas ratio of

151 can give particles of upto 5 μm diameter

423 Preparation of silver-titania (Ag-TiO2) particles

A monolayer of silver particles was prepared according to the procedure by Goldenberg

et al63

Glass slides were placed on the bottom of a petridish Water was put into the

petridish so that the entire slide was immersed in water A thin layer of hexane was then

added on top of water in the petridish A suspension of silver particles in ethanol (5

ww) was slowly introduced to the water-hexane interface in the dish After the

monolayer was formed the glass slide was lifted up through the monolayer with tweezers

The silver particles-covered glass slides were left at room temperature to dry overnight

Following drying a thin film of titania was evaporated on the glass slides in Semicore

evaporator (Figure 2 7) The spheres were partially coated with a 10 nm film of titania

Ag-TiO2 particles were removed from the glass slide by gentle sonication FE-SEM was

used to characterize the particles (Figure 4 2) The thin titania layer was not easily

distinguishable due to roughness of the particles

46

Figure 4 2 FESEM image of Ag-TiO2 particles

424 Optical microscopy experiments

Imaging spacers were placed on a glass slide Suspension of Ag-TiO2 particles was

placed on the glass slide and the particles were allowed to settle Movement of particles

was observed in DI water and videos were recorded at 50x magnification of optical

microscope for 30 sec 05 H2O2 was added to the particle suspension on the slide

Videos were recorded at 50x ranging from 30 sec to 10 min to study the movement of

silver titania particles in hydrogen peroxide solution Particles in both DI water and

hydrogen peroxide were subjected to UV light and videos were recorded to study particle

dynamics in presence of UV radiation Particle tracking was performed using Physvis

particle tracking software Atleast 30 particles in every system were tracked for 10

seconds each Particle tracking data was obtained in terms of X and Y coordinates of

particles on screen This data was manipulated using MathWorks-Matlab software and

graphs of mean square displacement of particles versus time were plotted Diffusion

coefficients of particles were calculated from these graphs

47

43 Results and Discussion

Ag-TiO2 particles demonstrated autonomous motion in the presence of H2O2 No UV

light was required for this motion Motion of the particles was accelerated by addition of

fresh H2O2 to the system When subjected to UV radiation the particles displayed

momentary acceleration for 4-5 seconds

Figure 4 3 Mechanism of motion of Ag-TiO2 nanomotors

In the presence of H2O2 silver part of the Ag-TiO2 particle converts H2O2 to H2O and O2

This may lead to propulsion of the Ag-TiO2 particle due to bubble propulsion andor self-

diffusiophoresis In the presence of UV light additional reaction takes place on the

titania part of Ag-TiO2 particle which increases the velocity of the Ag-TiO2 particles

(Figure 4 4) This UV triggered photoactivity of titania is due to hole-electron separation

caused by photons having energy equal to or greater than the band gap of titania646566

Most of the holes-electrons recombine but a small fraction move to the surface and react

with the available redox species like water and oxygen to form protons (H+) superoxide

(O2-) and hydroxyl (OH) radicals

6566 This may lead to propulsion by diffusiophoresis

as well as electrophoresis

48

Figure 4 4 Silver-titania motors move faster when subjected to UV radiation

In addition to linear motion rotational motion is also observed in several particles and

particle aggregates Aggregation of particles can lead to a net circular propulsive force

which can rotate the aggregate as shown in Figure 4 5

Figure 4 5 Rotation due to particle aggregation of Ag-TiO2 particles

Particle tracking by Physvis software revealed a significant increase in diffusion

coefficient of Ag-TiO2 particles in hydrogen peroxide solution as compared to DI water

49

The Ag-TiO2 particles have a diffusion coefficient of 1491 μm2s in hydrogen peroxide

versus only 180 μm2s in DI water Thus the Ag-TiO2 particles move around more than

10 times their body length per second in hydrogen peroxide (Figure 4 6) When

subjected to UV light the particles experience a significant acceleration in hydrogen

peroxide which lasts only for a short duration of time (4-5 sec) after which the particles

return to their original velocity The diffusion coefficient of the particles during this UV

light trigger is found to be around 5093 μm2s UV light trigger does not have any visible

effect on the Ag-TiO2 particles in DI water

Table 4 1 Translational diffusion coefficients of Ag-TiO2 particles in hydrogen

peroxide and water

Mean 05 H2O2 solution DI water

Diffusion coefficient of Ag-TiO2 (μm2s)

without UV light

1491 180

Diffusion coefficient of Ag-TiO2 (μm2s)

in the presence of UV light

5093 113

50

Figure 4 6 Mean square displacement curves of Ag-TiO2 Janus particles in hydrogen

peroxide and DI water

When higher concentration (~30 vv) of Ag-TiO2 particles in hydrogen peroxide is

subjected to UV radiation the particles exhibit pattern formation (Figure 4 7)

Aggregation of Ag-TiO2 Janus particles is observed when UV light is turned on and the

particles disaggregated when UV light is switched off This can be due to predator-prey

kind of interaction between silver and titania half of the Janus particles in the presence of

UV light Oxygen and water that are produced at the silver end by decomposition of

hydrogen peroxide are used up by titania when subjected to UV radiation This can lead

to attraction between titania and silver parts of neighboring Janus particles causing

aggregation

y = 20373x

y = 59994x

y = 71848x

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10 12

Mean

sq

uare

dis

pla

cem

en

t (μ

m2s

)

Time (sec)

Mean square displacement vs Time for Ag-TiO2

particles

05 Hydrogen peroxide and UV05 Hydrogen peroxide

DI Water

51

A UV OFF B UV ON

C UV OFF

Figure 4 7 Higher concentrations of Ag-TiO2 particles showing aggregation when UV

is turned on

52

Chapter 5

Summary and Conclusion

In summary no significant enhancement in diffusion was observed in case of enzyme

immobilized Janus particles in substrate solution Presence of a linker between enzyme

and particle did not increase the diffusion coefficient of particles Alternative enzyme

immobilization methods need to be tested like thiolation of enzyme and subsequent

binding to gold surface of a Janus particle35

or expressing the enzyme with polyhistidine

or avidin tag67

This can result in higher enzyme activity upon immobilization and may

lead to enhanced diffusion of Janus particles

Predator-prey system of GOx and HRP immobilized particles showed some promising

results Aggregation of particles and formation of fan-shaped structures by TMB

oxidation were observed under optical microscope when GOx and HRP immobilized

particles glucose and TMB were present in the system The size of the fan-shaped

structures was dependent on the size of enzyme immobilized particles This can be

indicative of some kind of interaction between the GOx and HRP immobilized particles

No conclusive aggregation of GOx and HRP immobilized particles was observed in

absence of TMB Better imaging techniques are required to study the interaction of GOx

and HRP immobilized particles at the unstable liquid-air interface where majority of the

reaction takes place Other enzyme cascade systems not requiring gaseous reactants like

glycogen synthase and glycogen phosphorylase should be tested for similar activity

53

Ag-TiO2 particles were found to exhibit autonomous motion in the presence of hydrogen

peroxide solution The motion was momentarily accelerated upon exposure to UV

radiation The particles showed a diffusion coefficient of 1491 μm2s in hydrogen

peroxide versus only 180 μm2s in DI water Upon subjecting particles to UV radiation in

presence of H2O2 diffusion coefficient increased to 5093 μm2s for a period of 4-5

seconds In addition to linear motion rotating motion could also be observed in some

particle aggregates Further development of these aggregate structures can lead to

formation of rotors at nanoscale Higher concentration of silver-titania particles in

hydrogen peroxide displayed aggregation in presence of UV light and disaggregated

when UV light was switched off This may be attributed to the predator-prey kind of

interaction between silver and titania where oxygen and water that are produced by

silver by decomposition of hydrogen peroxide are used up by titania when subjected to

UV radiation This promises to be an interesting system and needs to be further

investigated to explore its properties

54

References

1 Paxton WF Sundararajan S Mallouk TE Sen A Angew Chem Int Ed Engl

2006 45 5420-5429

2 Golestanian R Liverpool TB Adjari A Phys Rev Lett 2005 94 220801

3 httpwwwsilvermedicineorgh2o2html

4 Rami Pedahzur Ovadia Lev Badri Fattal Hillel Shuval Water Science and Technology

1995 31(5-6) 123-129

5 W Went F American Scientist 1968 56

6 Adriano Cavalcanti Tad Hogg Bijan Shirinzadeh IEEE MHS 2006 International

Symposium on Micro-NanoMechatronics and Human Science 2006 226-232

7 EM Purcell American Journal of Physics 1977 45 3-11

8 Golestanian R Phys Rev Lett 2010 105 018103(1)-018103(4)

9 S S Dukhin Z R Ulberg G L Dvornichenko B V Deryagin Russian Chemical

Bulletin 1983 31(8) 1535-1544

10 Dukhin AS Goetz PJ Ultrasound for characterizing colloids Particle sizing zeta

potential rheology Elsevier 2002 59

11 HJKeh HJTu Trends in Colloid and Interface Science XIV Progress in Colloid and

Polymer Science 2000 115 174-180

12 D Zinemanas A Nir International Journal of Multiphase Flow 1995 21(5) 787-800

55

13 Stephen Ebbens Jonathan Howse Soft matter 2010 6 726-739

14 J Vicario R Eelkema W R Browne A Meetsma R M La Crois B L Feringa

Chem Commun 2005 31 3936

15 Davide Pantarotto Wesley R Browne Ben L Feringa Chem Commun 2008 1533 -

1535

16 Lisa A Cameron Matthew J Footer Alexander van Oudenaarden Julie A Theriot

PNAS 1999 96(9) 4908-4913

17 Ali Najafi Ramin Golestanian Phys Rev E 2004 69 062901

18 Jinseok Kim Jungyul Park Sungwook Yang Jeongeun Baek Byungkyu Kim Sang

Ho Lee Eui-Sung Yoon Kukjin Chun Sukho Park Lab Chip 2007 7 1504 - 1508

19 Kurt M Dubowski Clinical Chemistry 2008 5411 1919ndash1920

20 Spector DA Yang Q Wade JB Am J Physiol Renal Physiol 2007 292(1) 467-474

21 Rhee SG Chang TS Bae YS Lee SR Kang SW J Am Soc Nephrol 2003 14(8-3)

211-215

22 Sakaue T Kapral R Mikhailov A S Eur Phys J B 2010 75 381ndash387

23 Muddana H S Sengupta S Mallouk T E Sen A Butler P J J Am Chem Soc

2010 132 (7) 2110ndash2111

24 Baker CJ Deahl K Domek J Orlandi EW Arch Biochem Biophys 2000 382(2) 232-

237

25 V Vojinovic RH Carvalho F Lemos JMS Cabral LP Fonseca BS Ferreira

Biochemical Engineering Journal 2007 35 126-135

56

26

OV Lebedeva NN Ugarova Russian Chemical Bulletin 1996 45(1) 18-25

27 Nigel C Veitch Phytochemistry 2004 65(3) 249-259

28 Kenneth H Petersen Journal of Immunological Methods 2009 340(1) 86-89

29 httpwwwbrenda-enzymesorgphpresult_flatphp4ecno=11117

30 Goldenberg L M Wagner J Stumpe J Paulke B-R Goumlrnitz E Langmuir 2002 18

5627-5629

31 httpwwwgoldbiocompdf3761-Protocol201pdf

32 Av Rovisco Pais Lisboa Portugal T amp D Technology West Yorkshire UK Journal

of Molecular Catalysis B Enzymatic 2004 28(2-3) 129-135

33 httpwwwphysicsnyuedugrierlabmethodsnode11html

34 Yoon-Mee Lee1 O-Yul Kwon Yeo-Joon Yoon Keungarp Ryu Biotechnology Letters

2006 28 39ndash43

35 JC Pyun SD Kim JW Chung Analytical Biochemistry 2005 347(2) 227-233

36 George H Pimbley Jr Mathematical Biosciences 1974 20(1-2) 27-51

37 Andy Fenton Sarah E Perkins Parasitology 2010 137 1027-1038

38 Mead James G Brownell Robert L Mammal Species of the World A Taxonomic and

Geographic Reference (3rd ed) Johns Hopkins University Press 2005 2 2142

57

39

Krause KH Schweiz Med Wochenschr 2000 130(4) 97-100

40 Irina Kareva Faina Berezovskaya Carlos Castillo-Chavez Journal of Biological

Dynamics 2010 4(4)315 - 327

41 Delaittre G Reynhout I Cornelissen J Nolte R Chemistry ndash A European Journal

2009 15 12600ndash12603

42 C Buller DH Johnson RC Tschumper Investigative Ophthalmology amp Visual Science

1990 31 2156-2163

43 Robert M Mazo Brownian Motion Fluctuations Dynamics and Applications Oxford

Science Publications 2009 57

44 Vivek K Mutalik KV Venkatesh Theoretical Biology and Medical Modelling 2005 2

19

45 Satoshi Nakamura Kunimasa Koga Biochemical and Biophysical Research

Communications 1977 78(2) 806-810

46 Krishnananda Chattopadhyay Shyamalava Mazumdar Biochemistry 2000 39 (1)

263ndash270

47 Satoshi Nakamura Yasuyuki Ogura J Biochem 1968 63 (3) 308-316

48 van Dongen SF Nallani M Cornelissen JJ Nolte RJ van Hest JC Chemistry 2009

15(5) 1107-1114

49 Ronald Bentley Methods in Enzymology 1955 1 340-345

50 Vartiainen J Raumlttouml M Paulussen S Packaging Technology and Science 2005

18 243ndash251

58

51 GA Sherbeny AA Shindia YMMM Sheriff International Journal of Agriculture

and Biology 2005 7

52 D Rando G-W Kohring F Giffhorn Appl Microbiol Biotechnol 1997 48

53 Worthington enzyme manual Worthington Biochemical Corporation

httpworthington-biochemcomgopdefaulthtml

54 httpwwwbrenda-enzymesorgphpresult_flatphp4ecno=11117

55 httpwwwoxygenphwatercom

56 P Boon Chock Sue Goo Rhee and Earl R Stadtman Annu Rev Biochem 1980 49

813-841

57 Sybil Golden P A Wals Joseph Katz Analytical Biochemistry 1977 77(2) 436-445

58 R L Baehner S K Murrmann J Davis R B Johnston Jr J Clin Invest 1975 56(3)

571ndash576

59 I Bejarano M P Terroacuten S D Paredes C Barriga A B Rodriacuteguez J A Pariente

Molecular and Cellular Biochemistry 2007 296(1-2) 77-84

60 Michael C Moulton Laura K Braydich-Stolle Mallikarjuna N Nadagouda Samantha

Kunzelman Saber M Hussain Rajender S Varma Nanoscale 2010 2 763-770

61 Goya DV Matijevic E Colloids and Surfaces A 1999 146 139

62 Velikov KP Zegers GE van Blaanderen A Langmuir 2003 19 1384

63 Goldenberg L M Wagner J Stumpe J Paulke B-R Goumlrnitz E Langmuir 2002 18

5627-5629

59

64

Banerjee S Gopal J Muraleedharan P Tyagi A K Raj B Curr Sci 2006

901378ndash1383

65 Bogomolov V N Kudinov E K Firsov Y A Sov Phys Solid State 1968 35 555

66 Kudo A Miseki Y Chem Soc Rev 2009 38 253ndash278

67 Gaj T Meyer SC Ghosh I Protein expression and Purification 2007 56 54-61