Conducting polymers in

bioanalytical systems

Prof. dr. habil. Arūnas Ramanavičius (1)Assoc. prof. dr. Almira Ramanavičienė (2)

1. Department of Physical Chemistry Faculty of Chemistry, Vilnius University,

2. Center of NanoTechnology and Materials science, Faculty of Chemistry, Vilnius University, Naugarduko 22, 2006 Vilnius, Lithuania;

Center of NanoTechnology and Materials science - NanoTechnas

Faculty of Chemistry, Vilnius University

Center of NanoTechnology and Material science -

NanoTechnas

Center of NanoTechnology and Material science –NanoTechnas established within 2005-2008

NanoTechnasestablished within 2005-2008

Outline

• Introduction;

• Structure of conducting polymers (CP);

• Conducting polymers in biosensor design; in catalytic

biosensors, immunosensors, DNA sensors, sensors, molecularly biosensors, immunosensors, DNA sensors, sensors, molecularly

imprinted polymer based sensors, biofuel cells;

• Some properties of conducting polymers;

• New methods for synthesis of CP based nanoparticles and

encapsulation of nanoparticles within CP;

• Biocompatibility of coducting polymers;

• Concluding remarks.

Some our research directions related to conducting polymers

Sensors and Biosensors

Fuel cells and Biofuel cells

Catalytic DNA Immuno- Moleculary Catalytic

Enzymatic

Sensors

DNA

Sensors

Immuno-

Sensors

Moleculary imprinted polymer based

Sensors

Conducting polymers

Biomedicine: Implants; Drug delivery systems

Nn

Sn

n

Chemical structures of common conducting polymers / Selection principles

Can be electrochemically synthesized on the conducting surfaces.

Can be doped by biological objects e.g.: proteins.

n n

Polypyrrole Polythiophene

n

Polyacetylene PolyfuraneH

Sn

Polythiophene Polyaniline

Nn

On

Polyfurane

H

conducting surfaces.

Can by synthesized at neutral pH. (It is especially important during immobilization of biological objects)

.

The layer is stabile and elastic

Some advantages of conducting polymers

�From the analytical point of view interactions on the solid-liquid surface are very important for successful construction of electrochemical sensors.�Conducting polymers might be applied, since they are: (i) biocompatible and, hence, causes minimal and reversible disturbance to the working environment; (ii) capable to transduce the energy arising from interaction (ii) capable to transduce the energy arising from interaction between immune reagents into electrical signals that are easily monitored; (iii) protects electrodes from fouling and interfering materials such as electroactive anions; (iv) can be modified in a controlled fashion.

Conducting (π-π conjugated) polymers can be successfully applied to solve immobilization problem of bio-molecules

Charge transfer via conjugated polymeric backbone

Part of biological recognition (catalyst)

Signal transducer

AnalyteProducts

Biosensors

Registration

device

Fundamental task –stabile and effective attachment of biological objects on the surface of signal transducer.

In electrochemical biosensors electron transfer is requested

Glc

Gluconolactone 2H O 2 2

GODM

M

ox.

red.

2e-2e

-2e

-

Ele

ctr

ode

O 2

2H O2

+

M =PM S; K3[Fe(CN)6]; ferocene derivates …

Glc

Gluconolactone

GDHM

M

ox.

red.

2e-2e

-2e

-

Ele

ctr

ode

PQQ-

M =PM S; DCPIP; BQ

Enzymes in design of catalytic biosensors

ferocene derivates …

Glc

Gluconolactone

GDHM

M

ox.

red.

2e-2e

-2e

-

Ele

ctr

ode

2NADH

2NAD+

NAD-

Alcohol

Aldehyde

HQ-ADH

e-

2e-

Ele

ctr

ode

PQQ

e-

e-

e-

e-

hem

e chem

e c

hem

e c

hem

e c

Major methods for functionalization of polymers by biomolecules

�Adsorption;

� Covalent attachment;

� Entrapment within polymers;

�Molecular imprinting of polymers;

�Application of SAM’s followed by covalent �Application of SAM’s followed by covalent attachment.

Conducting polymers might be very useful: (i) for modification, (ii) and for protection of biosensor surfaces.

Ramanavicius A. Malinauskas A. Ramanaviciene A. (2004) Catalytic biosensors based on conducting polymers. In: D.W. Thomas (ed.) Advanced

Biomaterials for Medical Applications. Kluwer Academic Publishers, Netherlands, pp. 93-109.

The Electrochemical synthesis of conducting polymers (e.g. polypyrrole)

NH

NH+.

NH

NH

e-

2 e-

NH

N

NH

NH

NH

NH n

(2 n -1 )e-

H

NH

NH

NHH n

N H

R

R

O 2m e-

R C O H

C O O H

C H 2 O H

BM

BM

BM

Immobilization of bio-molecules within electrochemically formed polypyrrole

(Entrapment of bio-molecules within Ppy)

P

Pt

elec

trod

e

p yBM

P

BM

p yNH

NH+.

NH

e-

2e-

.

BM

BMBM

BM

Pt

elec

trod

eBM

E, mV

t

950

0

NH

NH

H

NH

NH

NH

NH n

(2n-1)e-

H

NH

NH

NHH n

NHR

R

O2me-

R COH

COOH

CH2OH

-600

-400

-200

0

200

400

600

800

I, nA

8 5 0

Pulse chrono-amperogram, registered during synthesis of polypyrrole by potential-pulse mode.

Control of Ppy film growth

-800

-600

0 20 40 60t, s

0 5 1 0 1 5 2 0 2 5 3 04 0 0

4 5 0

5 0 0

5 5 0

6 0 0

6 5 0

7 0 0

7 5 0

8 0 0

I, n

A

I m p u l s u s k a i c i u s ( n )

y=(Z-K)/(1+(L*x) D)+K;Where:Z=441.0±8.1;K=825.0±2.45;L=0.3751±0.0112;D=2. (1,95 ±0,03)

Dependence of anodic peaks on the pulse number during the polymerization procedure.

Pulse number

Immobilization ofbio-molecules onthe surface of

Pt e

lect

rode

PPy

Pt e

lect

rode

+PirolasElektrocheminėpolimerizacijaElectrochemical

polymerisation

+Cu(NO )3 2

PPy

Pt e

lect

rode

–NO2

–NO2

–NO2

–NO2

–NO2

-

Attachment ofnitrogroups

+

Electrochemicalreduction

-

-H O2

PPy–NH 2

+H +e

PPy+EDC–NH 2H

OO

C– the surface of

electrochemicallysynthesizedpolypyrrole

PPy

Pt e

lect

rode

–NH 2

–NH 2

–NH 2

–NH 2

–NH 2

Electrochemicalreduction

2

PPy

Pt e

lect

rode

+EDC–NH–CO–

–NH–CO–

–NH

–NH 2

QH-ADH

QH-ADH-H O2

HO

OC

–

QH-ADH

Activation of HOOC-by EDC

Ramanavicius A., Habermüller K., Razumiene J., Meskys R., Marcinkeviciene L., Bachmatova I., Csöregi E., Laurinavicius V., and Schuhmann W. (2000) An

oxygen-independent ethanol sensor based on quinohemoprotein alcohol dehydrogenase covalently bound to a functionalized polypyrrole film,

Progress in Colloid and Polymer Science, 116, 143-148.

“Grafted” conducting polymers

Y. Oztekin, A. Ramanaviciene, N., Ryskevic, Z., Yazicigil, Z., Ustundag, A.O. Solak, A. Ramanavicius, 1,10-Phenanthroline modified glassy carbon electrode for voltammetric determination of cadmium(II) ions Sensors and Actuators B Chemical 2011, 157, 146– 153.

Part of biological recognition

Signal transducer

AnalyteProducts

Biosensors

Registration

device

Ramanavicius A. Malinauskas A. Ramanaviciene A. (2004) Catalytic biosensors based on conducting polymers. In: D.W. Thomas (ed.) Advanced Biomaterials for Medical Applications. Kluwer Academic Publishers, Netherlands, pp. 93-109.

GDH

GDH

Glucose

Glucono-lactone

Glucose

PQQ

PQQ

PMSoks.

PMSred.

COO -ICOO -I

COO -I

COO -I

COO -I

COO -I COO -

ICOO -I

COO -I COO -

ICOO -I

COO -ICOO -

ICOO -ICOO -

ICOO -ICOO -

ICOO -I

COO -I

COO -I COO -

ICOO -I

COO -ICOO -

ICOO -ICOO -

ICOO -ICOO -

ICOO -I

COO -I

Glucose

Ascorbic a. Urate- -PMS mediated biosensor.

Based on PQQ-GDH entrapped within

conducting polymer

Protection from interfering chemicals, by over-oxidized Ppy layer

Catalytic conversion of GDH

e-Glucono-lactone

PMSred.PMSoks.

Pt

Catalytic conversion of analyte

Transfer of electrons from enzyme to conducting surface by diffusing “electron shuttles”

Ramanavicius A. (2000) Electrochemical study of permeability and charge-transfer in polypyrrole films, Biologija, 2, 64-66.

Laurinavicius V., Razumiene J., Ramanavicius A., Ryabov A.D., (2004) Wiring of PQQ–dehydrogenases, Biosensors & Bioelectronics 20 (6), 1217-1222.

COO-I COO-

ICOO-I

COO-ICOO-

ICOO-ICOO-

ICOO-ICOO-

ICOO-I

COO-I

Glucose

M

Os

PQQ

GDH

Glukoz ė

Gliukono-laktonas

Gliukoz ė

PQQ

M

GDH

e-

COO-ICOO-I

COO-I COO-

I

COO-I

COO-I

COO-I

COO-I

COO-I

COO-I

COO-I

COO-I

COO-I COO-

ICOO-I

COO-ICOO-

ICOO-ICOO-

ICOO-ICOO-

ICOO-I

COO-I

Glucose

Ascorbic a. Urate.

Gluconolactone

Glucose

Glucose

Insoluble mediator mediated biosensor.Based on PQQ-GDH

entrapped within conducting polymer

Protection from interfering chemicals, by over-oxidized Ppy layer

Catalytic conversion of

M

M

M

M

M

GDH

Gliukono-laktonas

PQQ

OsMMM

M M MM M

M

M

M M

M

e-

e-

MM M

MMMMMM

M M M

M

Electrode

Gluconolactone

Catalytic conversion of analyte

Transfer of electrons from enzyme to conducting surface by mean of “electron hopping”

Application ofmultilayeredconductingpolymers basedstructures

OsGDHGlucose

PQQe-

Os

Os

Os OsOs

Os

Os

COO -ICOO -I

COO -I COO -

I

COO -I

COO-I

COO -I

COO-I

COO-I

COO-I

COO -I

COO-I

COO-I COO-

ICOO-I

COO-ICOO-

ICOO-ICOO-

ICOO-ICOO-

ICOO-I

COO-I

Glucose

Os

OsOs

Os

Os

GDHGlucose

Glucono-

PQQe-

e-

Os

Os

Os OsOs

Os

Os

COO-ICOO-I

COO-I COO-

I

COO-I

COO-I

COO-I

COO-I

COO-I

COO-I

COO-I

COO-I

COO-I COO-

ICOO-I

COO-ICOO-

ICOO-ICOO-

ICOO-ICOO-

ICOO-I

COO-I

Glucose

Ascorbate Urate- -

Protection layer based on over-oxidized Ppy layer.Catalytic conversion of

Os

Os

Os

Os

Os

Os

Os

Os

Os

Os

Os

Os

Os

Os

Os

Os

Os

Os

Os

Os

Os

Os

Os

GDH

Glucono-lactone

Glucono-lactone

Glucose

PQQ

e-

e-

e-

e-

e-

e-

e-

e-

e-

e-

e-

e-

e-

e-

e-

Os

Os

Os

Os

Os

Os

Os

Os

Os

Os

Os

Os

Os

Os

Os

Os

Os

Os

Os

Os

Os

GDH

Glucono-lactone

Glucono-lactone

Glucose

PQQ

e-

e-

e-

e-

e-

e-

e-

e-

e-

e-

e-

e-

e-

e-

Pt

analyte

Transfer of electrons from enzyme to conducting surface by mean of “electron hopping”

Habermuller, K., Ramanavicius A., Laurinavicius V., and Schuhmann W.

(2000) An oxygen-insensitive reagentless glucose biosensor based on

osmium-complex modified polypyrrole, Electroanalysis, 12, 1383-1389.

Polypyrrole

Hemas-c Hemas-c

Hemas-cI IIeeee

e

eHeme-c Heme-c

Heme-c

PQQ Hemas-c

I II

IIIActo r. Aldehidas

eee

e

Ethanol Aldehyde of Acetic Acid

Heme-c

Ramanavičius A., Habermüller K. Csöregi, E., Laurinavičius V., and Schuhmann. W. (1999) Polypyrrole entrappedquinohemoprotein alcohol dehydrogenase. evidence for dir ect electron transfer via conducting polymer chains , AnalyticalChemistry, 71, 3581-3586.

Res

pons

e, %

Met

hano

l

QH-ADH within PPy.Native QH-ADH QH-ADH covalently attached on the surface of PPy.

Eth

anol

1-pr

opan

ol

1-bu

tanl

ol

Isob

utan

ol

Res

pons

e, %

heme-c

PQQ

acetic aldehyde

ee

e

e

ee

eheme-c

heme-c

heme-c

polypyrrole chains

I II

III

Fig.1. Electron transfer scheme in biosensor coate d with QH-ADH entrapped within chains of conducting polymer

Specificity of QH-ADH andQH-ADH/PPy basedbiosensors.

+Cu(NO )3 2

PPy

Pt e

lect

rode

PPy

Pt e

lect

rode

–NO2

–NO2

–NO2

–NO2

–NO2

+

-

El. reduction

-

-H O2

2

Pt e

lect

rode

+Pyrrole

Electrochemicalpolymerization

PPy

Pt e

lekt

roda

s

PPy

Pt e

lekt

roda

s

–NH2

–NH2

–NH2

–NH2

–NH2

+EDC

–NH2

–NH–CO–

–NH–CO–

–NH

–NH2

Enzyme

Enzyme-H O2

HO

OC

–

Enzyme

+H +e

El. reduction aktivation of HOOC-

Possible application of CP’s in Enzymatic Biofuel cells

Ramanavicius A. Kausaite A., Ramanaviciene A, (2005) Biofuel cell based on direct bioelectrocatalysis, Biosensors and Bioelectronics,. 20: 1962-1967.

Ramanavicius A., Kausaite A., Ramanaviciene A., (2006) Potentiometric Study of Quinohemoprotein Alcohol Dehydrogenase Immobilized on the Carbon Rod Electrode, Sensors and Actuators B: Chemical, 113, 435-444.

Potentials of (1) MP-8 functionalized cathode as

function of hydrogen peroxide concentration; (2) MP-

8/GOx functionalized carbon rod electrode as a function

of hydrogen peroxide concentration; (3) MP-8/GOx

functionalized carbon rod electrode as a function of

glucose concentration; (4) QH-ADH functionalized

anode as a function of ethanol concentration.

Investigations performed in 50 mM Na acetate solution,

pH 6,

Schematic representation of glucose oxidase coating bypolypyrrole initiated by catalytic action of this enzyme.

Polypyrrole Coated Glucose Oxidase Nanoparticles

Schematic representation of application of polypyrrole coated glucose oxidase nanoparticles in PMS mediated biosensor design.Ramanavicius A., Kausaite A., Ramanaviciene A. (2005) Polypyrrole Coated Glucose Oxidase Nanoparticles for Biosensor Design, Sensors and Actuators

B-Chemical 111-112, 532-539. /// Ramanavicius A., Kausaite A., Ramanaviciene A., Acaite J., Malinauskas A., (2006) Redox enzyme – glucose oxidase

– initiated synthesis of polypyrrole Synthetic Metals, 156, 409-413

Atomic force microscopy (AFM)

Ramanaviciene A, Schuhmann W, Ramanavicius A. (2006) AFM Study of Conducting Polymer Polypyrrole Nanoparticles Formed by

Redox Enzyme – Glucose Oxidase – Initiated Polymerisation Colloids and Surfaces B: Biointerfaces 48, 159-166.

S. W. Kim, H. G. Cho, C. R. Park, Fabrication of Unagglomerated Polypyrrole Nanospheres with Controlled Sizes From a Surfactant-Free Emulsion System Langmuir 2009, 25, 9030–9036.

S. W. Kim, H. G. Cho, C. R. Park, Fabrication of Unagglomerated Polypyrrole Nanospheres with Controlled Sizes From a Surfactant-Free Emulsion System Langmuir 2009, 25, 9030–9036.

S. W. Kim, H. G. Cho, C. R. Park, Fabrication of Unagglomerated Polypyrrole Nanospheres with Controlled Sizes From a Surfactant-Free Emulsion System Langmuir 2009, 25, 9030–9036.

Polypyrrole beads vs. latex beads

NH

+ 7FeCl3* 6H2O3

NH

NH

HN

n

+

Cl-

+ 7FeCl2 + 6HCl

0.4 µm latex beads on a glass fibre pad

0.4 µm latex beads on a glass fibre pad

PPy

Signal transducer

Part of biological recognition

AnalitėAnalitėAnalyteAnalyte Analyte

Registration

deviceFundamental task – stabile and effective attachment of biological objects on the surface of signal transducer and efficient signal transduction

Ramanaviciene A., Ramanavicius A. (2004) Affinity sensors based

on nano-structured p-p conjugated polymer polypyrrole. In: D.W.

Thomas (ed.) Advanced Biomaterials for Medical Applications. Kluwer Academic Publishers, Netherlands, pp. 111-125.

+Cu(NO )3 2

PPy

Pt e

lect

rode

PPy

Pt e

lect

rode

–NO2

–NO2

–NO2

–NO2

–NO2

+

El. reduction

-

-H O2

Pt e

lect

rode

+Pyrrole

Electrochemicalpolymerization

–NH –NH

+H +e

Covalent attachment

2

PPy

Pt e

lekt

roda

s

PPy

Pt e

lekt

roda

s

–NH 2

–NH 2

–NH 2

–NH 2

–NH 2

+EDC

–NH 2

–NH–CO–

–NH–CO–

–NH

–NH 2

gp51

gp51-H O2

HO

OC

–

gp51

El. reduction aktivation ofHOOC-

gp51 – Glicoprotein from bovine leukemia virus (antigen)

2

PPy

Pt e

lect

rode

–NH 2

–NH–CO

–NH

gp51

2

PPy

Pt e

lect

rode

–NH2

–NH–CO

–NH

gp51

Y

Y

Y

++

++

++

++

++

++

+

++

++

++

++

++

++

+

- -

- -

- -

- -

Application of covalently attached proteins in design of immunosensors

Pt e

lect

rode

–NH–CO

–NH2

gp51

Pt e

lect

rode

–NH–CO

–NH2

gp51

++

++

++

++

++

++

+

++

++

++

++

++

++

+

- -

- -

- -

- -Incubation

gp51 – Glicoprotein from bovine leukemia virus (antigen) Y – Specific antibodies

Polypyrrolefilm

Pt e

lect

rode gp51

Polypyrrolefilm

Pt e

lect

rode

Y

Y

Y

++

++

++

++

++

++

+

++

++

++

++

++

++

+

- -

- -

- -

- -

gp51

Application of conducting polymers doped by proteins in design of immunosensors

Pt e

lect

rode

gp51

Pt e

lect

rode

++

++

++

++

++

++

+

++

++

++

++

++

++

+

- -

- -

- -

- -

gp51Incubation

gp51 – Glicoprotein from bovine leukemia virus (antigen) Y – Specific antibodies

Polypyrrolefilm

Pt e

lect

rode

Polypyrrolefilm

Pt e

lect

rode

Y

++

++

++

++

++

++

+

++

++

++

++

++

++

+

- -

- -

- -

- -Y Y Y

Electrochemical Immunosensors for Detection of Antigens (proteins etc.)

Monoklonaii

Pt e

lect

rode

Pt e

lect

rode

Y

++

++

++

++

++

++

+

++

++

++

++

++

++

+

- -

- -

- -

- -

Incubation

– Protein of interset Y– Monoclonar

antibodies specific toprotein of interest

Y Y

PPy

Pt e

lect

rode

BM

PPy

Pt e

lect

rode

BM

BM

BM

Pt e

lect

rode

PPy

BM

BM

Incubation

solventextraction

Electrochemical Affinity Sensors Based on Molecularly Imprinted Polypyrrole

BM

- biologically activecompound (analyte of

interest)

Ramanaviciene A, Ramanavicius A. (2004) Molecularly imprinted polypyrrole-based synthetic receptor for direct detection of bovine leukemia virus

glycoproteins , Biosensors & Bioelectronics 2004, 20 (6), 1076-1082.

Ramanaviciene A., Finkelsteinas A., Ramanavicius A., (2006) Basic Electrochemistry Meets Nanotechnology: Electrochemical Preparation of Artificial

Receptors Based on a Nanostructured Conducting Polymer, Polypyrrole, Journal of chemical Education 83, 1212-1214.

Signal transducer

Target DNA

DNA-sensor

Registering device

Detection of interaction between ssDNA and target DNA

Ramanavičienė A. Ramanavičius A. (2004) Pulsed amperometric detection of DNA with an ssDNA/ polypyrrole modified

electrode Journal of Analytical and Bioanalytical Chemistry, 2004; 379 (2): 287-293.

Imag

inar

y Im

peda

nce

(Z")

, KO

hm

100

120

-15

-10

-5

0

5

10

15

20

25

0 20 40 60 80 100 120

Detection by potential-pulse amperometry200 mV/s

-400

-300

-200

-100

0

100

200

300

0 100 200 300 400 500 600 700 800

p rie š in k u b a v imą p o in k u b a v im o

After incubationBefore incubation

Detection by cyclic voltammetry

Real Impedance (Z'), KOhm

0 20 40 60 80 100 120

Imag

inar

y Im

peda

nce

(Z")

, KO

hm

0

20

40

60

80

100

i ii-25

-20

-15

Before incubation

After incubation

Ramanaviciene A. Ramanavicius A. (2004) Pulsed amperometric detection of DNA with an ssDNA/polypyrrole modified electrode Journal of Analytical and Bioanalytical Chemistry, 2004; 379 (2): 287-293.

Impedance spectroscopy

Before incubation

Quantum Dots

• • 1 – 10 nm size, 100 – 1000 atoms• • Materials: conductors/semi-conductors• (CdS, CdTe, CdSe, ZnSe, etc. Other materials,

Au, Si, Ge, etc.)• • Electro-positive holes // electron capture• – Color shift• – Color shift• – Conductivity change

Stabilization of Q-dots (CdSe )ZnS by polypyrrole

Ramanavicius A., Karabanovas V., Ramanaviciene A., Rotomskis R. (2008) Stabilization of (CdSe)ZnS Quantum Dots with Polypyrrole Formed by UV/VIS

Irradiation Initiated Polymerization Journal of Nanoscience and Nanotechnology 8, 1-7.

Ramanavicius A., Kurilcik

N., Jursenas S.,

Finkelsteinas A.,

RamanavicieneA. (2007)

Conducting Polymer

Based Fluorescence

Quenching as a new

Approach to Increase the

Selectivity of Selectivity of

Immunosensors

Biosensors and

Bioelectronics, 23, 499-

505.

Ramanaviciene A.,

Ramanavicius A. (2004)

Towards the hybrid

biosensors based on

biocompatible conducting

polymers. In: Shur M.S.,

Zukauskas A. (eds.) UV

Solid-State Light Emitters

and Detectors. Kluwer

Academic Publishers,

Netherlands, pp. 287-296.

Biocompatibility of polypyrrole in vivo

Ramanaviciene A., Kausaite A., Tautkus S., Ramanavicius A. , (2007) Biocompatibility of polypyrrole particles: an in vivo study inmice Journal of Pharmacy and Pharmacology, 59, 311-315.

Prof. habil. dr. Arūnas RamanavičiusAssoc. prof. dr. Almira Ramanavičienė

Dėkoju už dėmesį!

Thank you for attention!

Main activities: Development of bio-, immuno- and DNA-sensors;Development of bio-analytical methods for the food, environmental analysisand biomedical application; Synthesis and application of conducting polymers.

Center of NanoTechnology and Materials science -NanoTechnas,Faculty of Chemistry, Vilnius University,Naugarduko 22, 2006 Vilnius, Lithuania.e-mail: Arunas.Ramanavicius@chf.vu.lt.