acoustic wave biosensor for the detection of the breast ... · ii acoustic wave biosensor for the...

i

Acoustic Wave Biosensor for the Detection of the Breast Cancer Metastasis Biomarker Protein

PTHrP

by

Victor Serban Crivianu-Gaita

A thesis submitted in conformity with the requirements

for the degree of Master of Science in the

Department of Chemistry

University of Toronto

Copyright by Victor Serban Crivianu-Gaita 2016

ii

Acoustic Wave Biosensor for the Detection of the Breast

Cancer Metastasis Biomarker Protein PTHrP


Master of Science

Department of Chemistry

University of Toronto

2016

Abstract

This manuscript illustrates the three stage development of a biosensor capable of

detecting a breast cancer metastasis biomarker protein. In the first stage of development, a

new reducing agent was discovered for the formation of antibody fragment antigen-

binding (Fab) units. This reducing agent, dithiobutylamine (DTBA), was found to be 213

times more efficient than the previously accepted leading reducing agent, dithiothreitol

(DTT), at cleaving antibody F(ab)2 fragments. The second stage of development studied

the anti-fouling properties of Fab fragments on various homogeneous and mixed

alkylsilane monolayers. Immobilized Fab fragments were determined to impart anti-

fouling properties onto these surfaces, making them ideal for use in biosensors. The last

stage of development compared whole antibody and Fab fragment-based biosensors for

the detection of the cancer metastasis protein in buffer, with the whole antibody-based

biosensor yielding the lowest limit of detection of 61 ng/mL.

iii

Acknowledgments

First, I would like to express my utmost gratitude to my supervisor, Professor

Michael Thompson, for his guidance, advice, and friendship of these past few years. I

feel extremely thankful for the opportunity I have had through working under Professor

Thompson.

I would also like to thank Dr. Alexander Romaschin for his insightful discussions

and for the time he has given to analyze my thesis. I would like to thank Dr. Jack Sheng

for his help with the EMPAS system throughout the years.

I would like to thank all of the Thompson research group members for their

support and encouragement throughout my research period. I would like to especially

mention Mohamed Aamer who was intimately involved in the biosensor research. I

would like to thank Brian De La Franier, Ruben Machado, Rohan Ravindranath,

Jenise Chen, Dr. Christophe Blaszykowski, and all other current/previous groups

members for their help through this period.

Finally, I would like to thank my parents Iosif Crivianu-Gaita and Daniela

Crivianu-Gaita for always supporting my choices and encouraging my dreams. I would

also like to thank my significant other, Tasha Stoltz, for giving me strength and

endurance when times were difficult. Thus, I dedicate my thesis to them.


June 2016

iv

Table of Contents

Abstract .......................................................................................................................................... ii

Acknowledgments ........................................................................................................................ iii

Table of Contents ......................................................................................................................... iv

List of Abbreviations ................................................................................................................. viii

List of Tables ................................................................................................................................ xi

List of Figures .............................................................................................................................. xii

1. Introduction ................................................................................................................................1

1.1. Cancer Background ..............................................................................................................1

1.2. PTHrP as a Marker for Metastatic Breast Cancer ................................................................3

1.3. Introduction to Biosensors ...................................................................................................4

1.4. Fouling and Non-Specific Adsorption .................................................................................6

1.4.1 Theory .........................................................................................................................6

1.4.2. Amino Acid and Peptide-based Anti-Fouling Agents ...............................................7

1.4.3. Ethylene Glycol-based Anti-Fouling Agents .............................................................9

1.5. Self-Assembling Monolayers (SAMs) ...............................................................................12

1.5.1. Theory ......................................................................................................................12

1.5.2. Trichlorosilyl-derived SAMs ...................................................................................13

1.5.3. Mixed Trichlorosilyl-derived SAMs........................................................................15

1.6. Surface Characterization Techniques .................................................................................16

1.6.1. Contact Angle (CA) Goniometry .............................................................................16

1.6.2. X-ray Photoelectron Spectroscopy (XPS) ...............................................................17

1.6.3. Atomic Force Microscopy (AFM) ...........................................................................19

1.7. Biosensing Elements ..........................................................................................................21

1.7.1. Introduction ..............................................................................................................21

v

1.7.2. Comparison of Whole Antibodies, Fab, scFv, and Aptamers ................................24

1.7.3. Immobilization of Whole Antibodies ......................................................................27

1.7.4. Immobilization of Fab fragments ...........................................................................29

1.8. Whole Antibody Cleavage for the Production of Fab Fragments ....................................31

1.9. Detecting the Target Analyte .............................................................................................34

1.9.1. Acoustic Wave Devices ...........................................................................................34

1.9.2. Bulk Acoustic Wave (BAW) Devices .....................................................................35

1.9.3. ElectroMagnetic Piezoelectric Acoustic Sensor (EMPAS) .....................................37

1.10. Thesis Project ...................................................................................................................39

1.10.1. Fab Cleavage and Optimization ...........................................................................39

1.10.2. Anti-Fouling Behaviour of Fab Fragments ..........................................................40

1.10.3 Biosensor Development and Testing ......................................................................41

2. Experimental ............................................................................................................................42

2.1. General Remarks ................................................................................................................42

2.1.1. Chemistry .................................................................................................................42

2.1.2 Biochemistry .............................................................................................................42

2.2. Fab Cleavage and Optimization ........................................................................................43

2.2.1. Optimization of Whole Antibody to F(ab)2 Cleavage .............................................43

2.2.2. SDS-PAGE Analyses ...............................................................................................44

2.2.3. Kinetic Analyses of F(ab)2 Reduction .....................................................................44

2.2.4. Ellmans Test for Fab Nucleophilic Sulfides .........................................................45

2.2.5. Kinetic Comparisons of DTT, MEA, and DTBA ....................................................45

2.3. Anti-Fouling Behaviour of Fab Fragments .......................................................................46

2.3.1. Quartz Disk Preparation ...........................................................................................46

2.3.2. Isolation and Characterization of Fab Fragments ...................................................46

2.3.3. Silanization of Simple Adlayers ..............................................................................46

vi

2.3.4. Silanization of Mixed Adlayers ...............................................................................47

2.3.5. Immobilization of Fab Fragments onto Silanized Surfaces....................................47

2.3.6. EMPAS Measurements ............................................................................................47

2.3.7. Contact Angle Measurements ..................................................................................48

2.3.8. X-ray Photoelectron Spectroscopy (XPS) Analyses ................................................48

2.3.9. Atomic Force Microscopy (AFM) Studies ..............................................................48

2.4. Biosensor Development and Testing .................................................................................48

2.4.1. Quartz Disk Preparation ...........................................................................................48

2.4.2. Isolation and Characterization of Fab Fragments ...................................................49

2.4.3. Silanization of the Quartz Disks ..............................................................................49

2.4.4. Surfaces Containing Whole Antibodies ...................................................................49

2.4.5. Surfaces Containing Fab Fragments .......................................................................50

2.4.6. EMPAS Measurements ............................................................................................50

2.4.7. Calculation of Limit of Detection ............................................................................51

3. Results and Discussion .............................................................................................................52

3.1. Fab Cleavage and Optimization ........................................................................................52

3.1.1. Optimization of the Pepsin Cleavage Protocols.......................................................52

3.1.2. Kinetic Comparisons of DTT, MEA, and DTBA ....................................................53

3.1.3. Conclusions ..............................................................................................................61

3.2. Anti-Fouling Behaviour of Fab Fragments .......................................................................62

3.2.1. Surface Characterization of Immobilized Fab Fragments ......................................62

3.2.2. Analysis of Simple Adlayers ...................................................................................64

3.2.3. Analysis of Mixed Adlayers ....................................................................................68

3.2.4. Analysis of Fab Immobilized Adlayers ..................................................................70

3.2.5. AFM Analysis of Fab Fragment Distribution.........................................................73

3.2.6. Detection of PTHrP from Mouse Serum .................................................................74

vii

3.2.7. Conclusions ..............................................................................................................76

3.3. Biosensor Development and Testing .................................................................................77

3.3.1. TUBTS-based Surfaces and Fab Fragment Binding ..............................................77

3.3.2. PFP-based Surfaces ..................................................................................................80

3.3.3. Analyte and Fouling Signal Optimization of Surfaces ............................................81

3.3.4. Calibration Curves for Whole Antibodies and Fab Fragment Biosensors .............85

3.3.5. Biosensor Regenerability .........................................................................................88

3.3.6. Conclusions ..............................................................................................................90

4. Final Conclusions .....................................................................................................................92

5. Future Work .............................................................................................................................94

References .....................................................................................................................................95

Appendix XPS Spectra ...........................................................................................................104

Copyright Acknowledgements ..................................................................................................116

viii

List of Abbreviations

2TP: 2,2-dithiopyridine

AFM: Atomic Force Microscopy

BAW: Bulk Acoustic Wave

-ME: -mercaptoethanol

BSA: Bovine Serum Albumin

CA: Contact Angle

CTCs: Circulating Tumor Cells

CYS: Cysteine

DNA: deoxyribonucleic acid

DPPE: N-(-maleimidocaproyl)pipalmitoylphosphatidylethanolamine

dsFv: disulfide-stabilized Fv

DTT: dithiothreitol

EDC: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide

EDTA: Ethylenediaminetetraacetic Acid

EMPAS: ElectroMagnetic Piezoelectric Acoustic Sensor

ETH: Ethanolamine

GMA: glycidyl methacrylate

HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

HPLC: High Pressure Liquid Chromatography

ix

HTS: Hexyltrichlorosilane

IDC: Invasive Duct Carcinoma

IGF-1: Insulin-Like Growth Factor

IgG: Immunoglobulin G

ILC: Invasive Lobular Carcinoma

L-DOPA: L-3,4-dihydroxyphenylalanine

LOD: Limit of Detection

MARS: Magnetic Acoustic Resonator Sensor

MBC: Metastatic Breast Cancer

MEA: Mercaptoethylamine

MEG-OMe: Ethylene glycol 3-trichlorosilylpropyl methyl ether

MEG-TFA: 2-(3-trichlorosilylpropoxy)-ethyl trifluoroacetate

NBS: Nucleotide Binding Sites

NHS: N-hydroxysuccinimide

OEG: Oligoethylene Glycol

OPG: Osteoprotegerin

OTS-TFA: 6-trichlorosilyl-hexanyl trifluoroacetate

PBS: Phosphate-Buffered Saline

PBS: Phosphate-Buffered Saline

PEG: Polyethylene Glycol

x

pFv: permutated Fv

PMPC: 2-metacrylolyloxyethyl phosphorylcholine

PTH: Parathyroid Hormone

PTHrP: Parathyroid Hormone-Related Peptide

QCM: Quartz Crystal Microbalance

RANKL: Receptor Activator of Nuclear Factor Kappa-B Ligand

RNA: 2-deoxyribonucleic acid

SAM: Self-Assembling Monolayer

SAW: Surface Acoustic Wave

scFv: single-chain Fv

SDS: Sodium Dodecyl Sulfate

SDS-PAGE: Sodium Dodecyl Sulfate PolyAcrylamide Gel Electrophoresis

SELEX: Systematic Evolution of Ligands by Exponential Enrichment

SPR: Surface Plasmon Resonance

SSMCC: sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate

TCEP: tris(2-carboxyethyl)phoshpine

TGF-: Tumor Growth Factor Beta

TSM: Thickness Shear Mode

TUBTS: S-(11-trichlorosilyl-undecanyl)-benzothiosulfonate

XPS: X-ray Photoelectron Spectroscopy

xi

List of Tables

Table 1. Rate constants (s-1

) for the two experiments at varying temperatures with 2.0 mM

reducing agent to 1 mg/mL polyclonal rabbit anti-goat IgG F(ab)2 concentrations.......53


) for the experiment varying the concentration of DTBA at

room temperature (22C) for the reduction of polyclonal rabbit anti-goat IgG F(ab)2....57


) for the experiment at room temperature with 2.0 mM

reducing agent to 1 mg/mL monoclonal mouse anti-human IgG1 F(ab)2 concentration....58

Table 4. Low resolution XPS analyses of the different surfaces in this study for the

determination of relative atomic percentages..67

Table 5. XPS data obtained with a 20 take-off angle relative to the normal. The data was

normalized to the C1s 285 eV peak using the Avantage software...78

Table 6. Assessment of all of the surfaces through the ratio of analyte signal/fouling

signal. *Bovine serum albumin (BSA) (1 mg/mL in pH 7.4 EMPAS buffer) was injected

prior to the sample injection as an anti-fouling agent.....83

xii

List of Figures

Figure 1. Secondary bone metastases can be developed through the metastatic cycle

involving PTHrP2

Figure 2. Elevated levels of PTHrP have been detected in various types of cancers. The

presence of this biomarker in breast and prostate cancer patients is indicative of

metastasis. PTHrP is directly involved in the metastatic pathway of these two cancers,

resulting in bone metastases......4

Figure 3. General representation of a biosensor. A wide variety of biosensors can be

developed from the many combinations of biosensing elements and transducers5

Figure 4. (A) L-cysteine. (B) Glutathione peptide. (C) Anti-fouling (left to right): aspartic

acid, asparagines, serine. Fouling (left to right): tyrosine, leucine, alanine. (D) Serine

pentapeptide. (E) Ser3-Asp2 pentapeptide. (F) (Leu-His-Asp)2 hexapeptide....8

Figure 5. (A) Tetraglyme. (B)(C) Oligoethylene glycol-based alkyl thiol anti-fouling

agents. (D) OEG dendritic adsorbates with substrate-anching L-DOPA and dopamine

catechol residues..10

Figure 6. MEG-TFA: 2-(3-trichlorosilylpropyloxy)-ethyl trifluoroacetate). MEG-OMe:

ethylene glycol 3-trichlorosilylpropyl methyl ether. MEG-OH: hydrolyzed MEG-TFA.

OTS-OH: hydrolyzed 6-trichlorosilyl-hexanyl trifluoroacetate (OTS-TFA)..11

Figure 7. The general arrangement of a self-assembled monolayer composed of an

endgroup (R1), a backbone, and a headgroup (R2). Biosensing elements react with the

endgroups and are immobilized to the surfaces of transducers. The endgroups also dictate

the surface properties of the assembled monolayer.12

Figure 8. (A) Schematic representation of the silanization process after forming of the

trisilanol group, hydrogen bonding occurs, following by condensation for the formation of

surface-bound silanols. (B) After binding to the surfaces, neighbouring linkers undergo

hydrogen bonding through their silanols followed by condensation to form interlinker

siloxanes..14

Figure 9. Contact angle picture of a water droplet on a quartz surface showing the four

components of Youngs equation....17

xiii

Figure 10. Schematic representation of an X-ray photoelectron spectroscopy instrument.

The take-off angle () can be varied in angle-resolved XPS in order to probe the surface at

different depths....18

Figure 11. General setup of an atomic force microscope...20

Figure 12. An illustration of whole antibodies (Ab), F(ab)2, Fab, Fab, Fv, and aptamers.

*Compared to antibodies and their fragments, aptamer shape and size varies from one

aptamer to another. The blue helix has been chosen to designate a general aptamer in this

thesis, however, it should be noted that not all aptamers form helices...22

Figure 13. Systematic evolution of ligands by exponential enrichment SELEX. The

process begins with a large DNA/RNA library and allows binding of the analyte.

Aptamers that bound the analyte are then amplified and taken to the next round for further

selection. In the end, the ideal aptamer will only bind its target with high selectivity and

specificity....26

Figure 14. General setup for the oriented immobilization of whole antibodies using an

immobilizing protein, such as protein G....28

Figure 15. Examples of the chemical structures of the Fab fragment immobilization

linkers. The highlighted (red) portion of the linker indicates where the Fab C-terminal

thiols react...31

Figure 16. Chemical structures of various thiol reducing agents that are used in protein

biochemistry....33

Figure 17. Representation of the formation of a thickness shear mode acoustic wave (1st

harmonic) and the associated mechanical displacement along the x-axis (x) when an

electrode-plated AT-cut quartz crystal is subjected to a perpendicular electrical field

(E)....36

Figure 18. (A) A picture overview of the entire EMPAS setup. (B) A schematic overview

of the EMPAS. (C) The quartz crystal cell holder illustrating the proximal AC powered

electromagnetic coil. (D) A picture of the parallel RLC circuit..38

Figure 19. Cleavage scheme for a general IgG antibody using either papain or pepsin

followed by a reducing agent..40

Figure 20. Structures of the Fab linker and spacers used in this study to explore the anti-

fouling properties of Fab fragments. TUBTS S-(11-trichlorosilylundecanyl)-

xiv

benzothiosulfonate. HTS hexyltrichlorosilane. MEG-TFA 2-(3-trichlorosilyl-

propyloxy)-ethyl trifluoroacetate. MEG-OH monoethylene glycolated-OH..40

Figure 21. A graph illustrating the change in F(ab)2 concentration (anti-goat IgG

antibodies) versus time comparing the three reducing agents at room temperature

(22C)..53

Figure 22. 12% SDS-PAGE gel for the 2.0 mM DTBA cleavage at room temperature

(22C) of polyclonal rabbit anti-goat IgG F(ab)2....54

Figure 23. 12% SDS-PAGE gel for the 2.0 mM DTT cleavage at room temperature


Figure 24. 12% SDS-PAGE gel for the 2.0 mM MEA cleavage at room temperature



antibodies) versus time comparing the three reducing agents at physiological temperature

(37C)..56


antibodies) versus time comparing three different concentrations of DTBA at room

temperature (22C)..57

Figure 27. A graph illustrating the change in F(ab)2 concentration (anti-human IgG1


(22C)..58

Figure 28. 12% SDS-PAGE gel for the 2.0 mM DTBA cleavage at room temperature

(22C) of monoclonal mouse anti-human IgG F(ab)2.59

Figure 29. 12% SDS-PAGE gel for the 2.0 mM DTT cleavage at room temperature

(22C) of monoclonal mouse anti-human IgG F(ab)2.....59

Figure 30. 12% SDS-PAGE gel for the 2.0 mM MEA cleavage at room temperature

(22C) of monoclonal mouse anti-human IgG F(ab)2.60

Figure 31. A graph of the concentration of forming Fab fragments (anti-human IgG


(22C)..61

xv

Figure 32. XPS narrow scans of the S2p signal for quartz, TUBTS, and TUBTS/Fab

surfaces. The decrease of the sulfone peak at 169 eV from the TUBTS to TUBTS/Fab

surfaces is indicative of the binding of the Fab fragments in an oriented fashion through

their C-terminal nucleophilic sulfides. Contact angles for the quartz, TUBTS, and

TUBTS/Fab surfaces were 16, 69, and 44, respectively. The relative atomic percentage

of the C1s, O1s, Si2p, S2p, and N1s XPS signals is also shown for the three different

surfaces63

Figure 33. EMPAS frequency shifts of the different simple adlayers when tested against

BSA (red, 45 mg/mL) and goat IgG (blue, 0.1 mg/mL).65

Figure 34. Contact angle measurements of A bare quartz (16), B TUBTS (69), C

HTS (89), D MEG-TFA (71), E MEG-OH (35), F TUBTS/HTS (1:1) (73), G

TUBTS/MEG-TFA (1:1) (76), H TUBTS/MEG-OH (1:1) (54), I TUBTS/HTS

(1:10) (69), J TUBTS/MEG-TFA (1:10) (72), K TUBTS/MEG-OH (1:10) (62), L

TUBTS/HTS (1:1)/Fab (33), M TUBTS/MEG-OH (1:1)/Fab (44), N TUBTS/HTS

(1:10)/Fab (43), O TUBTS/MEG-OH (1:10)/Fab (31), P TUBTS/Fab (44)...66

Figure 35. EMPAS frequency shifts of the different mixed adlayers when tested against

BSA (red, 45 mg/mL) and goat IgG (blue, 0.1 mg/mL).....69

Figure 36. EMPAS frequency shifts of the different Fab immobilized surfaces when

tested against BSA (red, 45 mg/mL) and goat IgG (blue, 0.1 mg/mL). In this case, the goat

IgG is the analyte and the frequency shift is proportional to the amount of analyte

detected....71

Figure 37. Atomic force microscopy topography images of A bare, B TUBTS, C

TUBTS/HTS (1:10), D TUBTS/MEG-OH (1:10), E TUBTS/Fab, F TUBTS/HTS

(1:10)/Fab, and G TUBTS/MEG-OH (1:10)/Fab surfaces.

Figure 38. EMPAS frequency shifts for various surfaces exposed to the fouling agent

mouse serum....74

Figure 39. Calibration curve for the specific detection of PTHrP. Unless specified, the

PTHrP concentrations indicate PTHrP in mouse serum. The frequency shifts of 100

g/mL PTHrP in EMPAS buffer and 0 ng/mL PTHrP mouse serum sum to a value that is

close to that observed by the 100 g/mL PTHrP in mouse serum, indicative of specific

binding.76

Figure 40. (A) Illustration of the TUBTS/Fab surfaces and the orientation of the Fab

fragments. (B) Illustration of the PFP/Oriented Ab surfaces where recombinant protein G

is used to orient whole antibodies. (C) The XPS relative atomic percentages of the

xvi

TUBTS/Fab surfaces and the (D) PFP/Oriented Ab surfaces. (E) S2p XPS signals

illustrating the decrease of the sulfone (169 ev)/sulfide (164 eV) peak ratios upon Fab

fragment binding.79

Figure 41. EMPAS frequency shifts for simple monolayers tested against (red) mouse

serum (fouling signal) and (blue) goat IgG (0.1 mg/mL in pH 7.4 EMPAS buffer, analyte

signal)..........82

Figure 42. EMPAS frequency shifts for whole antibody-based surfaces tested against

(red) mouse serum (fouling signal) and (blue) goat IgG (0.1 mg/mL in pH 7.4 EMPAS

buffer, analyte signal)..84

Figure 43. EMPAS frequency shifts for Fab fragment-based surfaces tested against (red)

mouse serum (fouling signal) and (blue) goat IgG (0.1 mg/mL in pH 7.4 EMPAS buffer,

analyte signal)..84

Figure 44. Calibration curves of the non-mass amplified and mass-amplified

PFP/Oriented Ab/ETH/BSA* surfaces. *BSA was injected prior to the sample injection

asnd was used as an anti-fouling agent...86

Figure 45. Calibration curves for the non-mass amplified and mass-amplified

TUBTS/Fab/BSA* surfaces. *BSA was injected prior to the sample injection and was

used as an anti-fouling agent.......87

Figure 46. General EMPAS profile illustrating the frequency shifts for BSA (f1), PTHrP

(f2), and secondary antibody (f3)....89

Figure 47. Regenerability curves for the whole antibody-based and Fab fragment-based

biosensors. The first mass-amplified PTHrP frequency shift is taken as a relative point for

which the second to the fifth injections are compared to....90

Fig. A.1. XPS spectra of the C1s (285 eV) peak of A bare quartz, B TUBTS, C HTS,

D MEG-TFA, E MEG-OH, F TUBTS/HTS (1:1), G TUBTS/MEG-TFA (1:1), H

TUBTS/MEG-OH (1:1), I TUBTS/HTS (1:10), J TUBTS/MEG-TFA (1:10), K

TUBTS/MEG-OH (1:10), L TUBTS/HTS (1:10)/Fab, M TUBTS/MEG-OH

(1:10)/Fab, N TUBTS/HTS (1:10)/Fab, O TUBTS/MEG-OH (1:10)/Fab, P

TUBTS/Fab..104

Fig. A.2. XPS spectra of the O1s (532 eV) peak of A bare quartz, B TUBTS, C HTS,




xvii


TUBTS/Fab..105

Fig. A.3. XPS spectra of the Si2p (103 eV) peak of A bare quartz, B TUBTS, C HTS,





TUBTS/Fab..106

Fig. A.4. XPS spectra of the S2p (164 eV) peak of A bare quartz, B TUBTS, C HTS,





TUBTS/Fab..107

Fig. A.5. XPS spectra of the N1s (400 eV) peak of A bare quartz, B TUBTS, C HTS,





TUBTS/Fab..108

Fig. A.6. XPS spectra of the F1s (688 eV) peak of A bare quartz, B TUBTS, C HTS,





TUBTS/Fab..109

Fig. A.7. XPS spectra of the C1s (285 eV) peak of A bare quartz, B PFP, C TUBTS,

D PFP/Non-oriented Ab, E PFP/Non-oriented Ab/ETH, F PFP/Oriented Ab, G

PFP/Oriented Ab/ETH, H PFP/Non-oriented Fab, I PFP/Non-oriented Fab/ETH, J

TUBTS/ Oriented Fab, K TUBTS/ Oriented Fab/CYS...110

Fig. A.8. XPS spectra of the O1s (532 eV) peak of A bare quartz, B PFP, C TUBTS,




xviii

Fig. A.9. XPS spectra of the Si2p (103 eV) peak of A bare quartz, B PFP, C TUBTS,




Fig. A.10. XPS spectra of the S2p (164 eV) peak of A bare quartz, B PFP, C TUBTS,




Fig. A.11. XPS spectra of the N1s (400 eV) peak of A bare quartz, B PFP, C

TUBTS, D PFP/Non-oriented Ab, E PFP/Non-oriented Ab/ETH, F PFP/Oriented

Ab, G PFP/Oriented Ab/ETH, H PFP/Non-oriented Fab, I PFP/Non-oriented

Fab/ETH, J TUBTS/ Oriented Fab, K TUBTS/ Oriented Fab/CYS...114

Fig. A.12. XPS spectra of the F1s (688 eV) peak of A bare quartz, B PFP, C TUBTS,




1

1. Introduction

1.1. Cancer Background

Cancer is the uncontrolled division of cells known as mitosis gone wild. For most

non-cancerous cells, the goal for cell division is the accurate duplication and even

distribution of the genome into two daughter cells.1 Accumulation of genomic alterations

(i.e. mutations on specific genes, deletions of chromosome segments, etc.) may disrupt the

cell division cycle causing the cell to either die or to enter an uncontrollable, unstoppable

replication/division cycle. Once a cell has become cancerous, it has one of two possible

fates to become benign or malignant.2

The former is involves a cell that does not spread

to other regions of the body. A malignant cancerous cell is able to spread to other regions

of the body and thus it is said to have metastatic ability.

Initially, cancer begins through the formation of a primary tumor one that is

localized to only one area of the body.3 When primary tumor cancerous cells become

malignant, they enter a metastatic cascade. This metastatic cascade begins with the cells

acquiring invasive properties, going through nearby tissues, and then into the circulatory

system. These circulating tumor cells (CTCs) then extravasate into secondary tissues and

form secondary tumors called metastases.

Every year worldwide, approximately one million women are diagnosed with

breast cancer.4 Compared to other forms of cancer, carcinomas of the breast are diagnosed

relatively early on. Metastasis of the cancer into other areas of the body results in an

immediate reduction of patient quality of life.4 Although metastatic breast cancer (MBC)

is treatable, it is not curable and median survival of patients is not more than two or three

years.4 The two most common subtypes of breast carcinomas are invasive duct carcinoma

(IDC) and invasive lobular carcinoma (ILC), occurring in 75% and 15% of breast cancers,

respectively.4 Metastases caused by the former type of carcinoma are typically found in

the bone, lung, liver, and lymph nodes. It has been estimated that those with MBC have an

85% chance to develop bone metastases.4

Certain tumors, particularly those showing preferential metastasis to the bone,

produce parathyroid-hormone related peptide (PTHrP) (Figure 1). This key molecule

2

activates osteoblasts to produce the receptor activator of nuclear factor kappa-B ligand

(RANKL) and downregulates osteoprotegerin (OPG). The next step in this cycle involves

the activation of osteoclast precursors.4 Osteolysis is observed as there is an increase in

extracellular calcium (Ca2+

) levels. Other growth factors, such as tumor growth factor beta

(TGF-) and insulin-like growth factor one (IGF-1), released from the activation of

osteoclast precursors, promote tumor cell proliferation and stimulate production of

PTHrP.4

Figure 1. Secondary bone metastases can be developed through the metastatic cycle

involving PTHrP.

3

1.2. PTHrP as a Marker for Metastatic Breast Cancer

Significant amounts of resources have been put towards understanding the

structure of PTHrP, the effects it causes in regards to metastasis, and to therapies targeting

it. Although it is derived from the same ancestral gene as parathyroid hormone (PTH),

PTHrP is involved in the regulation of cartilage growth during bone development. After

early childhood, this peptide ceases to have physiological function in adulthood.5 The

presence of PTHrP outside of childhood is an indicator of one of two possible scenarios:

(1) the PTHrP is causing humoral hypercalcemia of malignancy but is not involved in the

metastasis of the cancer (2) the PTHrP is directly involved in the metastatic pathway

through the growth and differentiation of neoplastic cells.5-6

PTHrP has similar biological

activity to PTH as both bind efficiently to the PTH1R receptor. The biological activity of

the former peptide is found in residues 1-34.7 However, it has been found that apart from

residues 8-13 that are similar to those found in PTH, the overall sequence and structure of

PTHrP is different from PTH.

Thus far, very few applications involving PTHrP have been developed. For

example, anti-PTHrP antibody therapies targeting PTHrP and avoiding PTH have been

studied extensively in the pursuit of preventing the formation of bone metastases8-12

.

However, prior to this study, it has not been used as a metastatic cancer biomarker in any

field.

Currently, there are no methods to efficiently and reliably detect cancer

metastasis. The very few biosensors that have been developed focus on the sequestration

and detection of CTCs.13-15

However, CTCs are not often present in the bloodstream and

an attempt to detect them is akin to finding a needle in a haystack. Typically, cancer

metastasis can only be detected once it has occurred and the affected individuals chance

of survival has been significantly decreased.

In this study, PTHrP is used as a biomarker protein for the detection of breast

cancer using a specifically engineered biosensor. Primary tumors of many cancers produce

PTHrP, however, it is only indicative of metastasis in breast and prostate cancer (Figure

2).16

The most significant benefit of using PTHrP as a biomarker is that it is constantly

4

present in the bloodstream of patients who have metastatic breast or prostate cancer. This

allows for a more reliable and efficient detection of metastasis compared to CTCs.

Figure 2. Elevated levels of PTHrP have been detected in various types of cancers. The

presence of this biomarker in breast and prostate cancer patients is indicative of

metastasis. PTHrP is directly involved in the metastatic pathway of these two cancers,

resulting in bone metastases.

1.3. Introduction to Biosensors

Through the development of biosensor technology, biosensors have received

significant attention as tools in analytical and diagnostic applications. These analytical

devices are being developed for use in fields such as, but not limited to, environmental

analysis, food analysis, drug analysis, and clinical diagnosis.17-20

Biosensors use

immobilized biomolecules to detect analytes in complex media in a selective, specific, and

sensitive manner.21

Biosensors offer real-time, label-free detections in non-destructive

manners allowing for quick and accurate analyses to be performed of target analytes.

Generally, a biosensor is composed of three parts a biorecognition (or biosensing

element), a transducer, and a signal processing unit.21

5

A biosensing element is defined as any biologically-derived or biologically-

mimetic molecule that is capable of binding to a target analyte. The ideal biosensing

element has a high affinity and low dissociation constant towards its target analyte, is

resilient towards denaturation, does not bind non-specifically, and can be manipulated

easily during the biosensor development. The interaction between the biosensor

biorecognition element and its analyte produces a signal that is then converted into a

measurable electrical response through the transducer. Depending on the combination of

biosensing elements and transducers, various biosensors may be developed (Figure 3).

Figure 3. General representation of a biosensor. A wide variety of biosensors can be

developed from the many combinations of biosensing elements and transducers.

Biosensors offer several advantages as analytical devices rapid/reliable analyses,

minimal operating time, high reproducibility, and high sensitivity/selectivity.22

The ideal

biosensor has all of these qualities, however, in practice only some of these categories

may be achieved. For example, it a biosensor may display extremely high reproducibility

and low limits of detection at a trade-off for a longer operating time. Biosensors are

constantly being developed to improve on the existing models. With time, it will be

possible to engineer a biosensor that is close to the ideal biosensor.

With all of the benefits that biosensors offer, there are a few disadvantages related

to their development. The first is the controlled immobilization of the biorecognition

elements. This process must be designed such that it is user friendly, reproducible, and as

cheap as possible.23

The resulting biointerface must be stable over a range of temperatures,

detection media conditions, and over time. The difficulty with the immobilization of

biomolecules is that an immobilization method must be developed that will not denature

the biomolecules or render their analyte-binding capabilities. Furthermore, various

6

transducer surfaces interact with biosensing elements in different ways some of them are

more likely to denature the biosensing elements. Another disadvantage related to

biosensors is that many times they have difficulty detecting the target analyte in complex

media. For example, many biosensors are capable of detecting analytes in simple buffers,

however, complex media such as blood serum complicates the detection of the analyte

through signals caused by non-specific detection.

1.4. Fouling and Non-Specific Adsorption

1.4.1 Theory

Fouling is defined as the undesired adsorption of chemical species to surfaces

resulting in analyte signal interference. The ideal biosensor ignores all of the other

compounds in solution and only binds to the target analyte. In practice, there are many

complex interactions between biosensor surfaces and the surrounding media. For this

study, the most important non-specific interactions are between serum proteins and the

biosensor surfaces. This non-specific adsorption process begins with the spontaneous

adsorption of water molecules and ions to a surface to form a water/electrical double

layer.24-25

Through a combination of non-covalent interactions (i.e. van der Waals,

hydrophobic interactions, and hydrogen bonding), serum proteins are able to adsorb to the

water/electrical double layer.26-27

Dehydration of both the proteins and the material

surfaces is required for adsorption to occur.28-29

Proteins initially adsorb to the surfaces in

their native state and then undergo a conformational shift in order to optimize the energy

of interaction with the surfaces.30-31

Generally, the first proteins to undergo surface adsorption are those in the

sample media that have affinity towards the surface and are present in the largest

concentration. As time passes, these proteins are replaced by others having greater

affinities for the surface, such as the analyte of interest.32

It is possible that some of the

undesirable proteins adsorb to the surfaces and then aggregate, preventing their

displacement by the analyte. When this occurs, a false representation of the concentration

of detected analyte is observed as part of the signal is due to non-specific adsorption. As

mentioned earlier, biosensors operating in complex media such as serum or blood are

frequently challenged by this problem. Thus, a lot of effort and research is placed towards

7

the development of surfaces that are resistant to non-specific adsorption that are anti-

fouling.

1.4.2. Amino Acid and Peptide-based Anti-Fouling Agents

A common method of altering the fouling properties of surfaces is through the

immobilization of amino acids or peptides onto those surfaces. One of the first of these

studies immobilized L-cysteine (Figure 4A) monolayers directly onto gold surfaces via

the formation of the energetically favourable gold-sulfur (Au-S) bonds.33

Fouling of the

surfaces using 10% human plasma diluted in phosphate-buffered saline (PBS) was only

marginally lower than what was observed with unmodified gold. When the anti-fouling

agent was changed to glutathione peptide (Figure 4B), in situ ellipsometry indicated a

considerable reduction in protein fouling (

8

Figure 4. (A) L-cysteine. (B) Glutathione peptide. (C) Anti-fouling (left to right): aspartic

acid, asparagines, serine. Fouling (left to right): tyrosine, leucine, alanine. (D) Serine

pentapeptide. (E) Ser3-Asp2 pentapeptide. (F) (Leu-His-Asp)2 hexapeptide.

9

1.4.3. Ethylene Glycol-based Anti-Fouling Agents

The most commonly encountered anti-fouling agents apart from amino acids and

peptides are those derived from oligoethylene glycol (OEG) or polyethylene glycol (PEG)

units. These anti-fouling agents are either adsorbed to surfaces or covalently immobilized

depending on the substrate choice and application. For example, a popular anti-fouling

agent that is adsorbed is tetraglyme (Figure 5A).38

Protein fouling using human plasma

(10%, 50%, or 100%) of gold-covered surfaces was determined to be as low as = 4.8

ng/cm2 for 10% human plasma solutions and as high as = 24.1 ng/cm

2 for 100% plasma

solutions. Bare gold exhibited fouling levels of = 244 ng/cm2 just from 1% plasma

solutions.

This structure of tetraglyme was also incorporated into alkylthiol linkers that were

immobilized onto gold surfaces (Figure 5B). However, these linkers exhibited poor anti-

fouling properties in 10% human plasma ( = 200 ng/cm2) and excellent anti-fouling

properties in 10% human serum ( = 15 ng/cm2).

39-40 These studies show that although

some linkers may exhibit strong anti-fouling properties in complex human serum, the

clotting proteins of human plasma may cause more fouling than can be prevented. Another

example of this is using a similar linker (Figure 5C) that differed by the number of

ethylene glycol repeats.41

This research group tested this gold-immobilized linker against

undiluted fetal bovine serum and against undiluted human plasma. The fouling levels

associated with the former were found to be = 26.1 ng/cm2 and those with the latter were

found to be = 71.0 ng/cm2.

As research of ethylene glycol-based anti-fouling agents continued, larger and

larger anti-fouling agents were developd. PEG-based anti-fouling agents between the sizes

of 2-20 kDa have been found to display fouling levels of = 6-10 ng/cm2 when exposed

to 10% fetal bovine serum.42

Branched dendritic structures (Figure 5D) have also been

developed to combat anti-fouling. These anti-fouling agents are composed of L-3,4-

dihydroxyphenylalanine (L-DOPA)/dopamine multidentate oligomers and OEG dendrons,

which are to assemble on TiO2 surfaces. Ellipsometry analyses indicated that fouling was

dependent on the surface coverage of the anti-fouling dendritic coating. Exposure to

undiluted blood serum yielded fouling levels of = 2 ng/cm2, which is below the limit of

detection (LOD) for that particular technique.

10

Figure 5. (A) Tetraglyme. (B)(C) Oligoethylene glycol-based alkyl thiol anti-fouling

agents. (D) OEG dendritic adsorbates with substrate-anching L-DOPA and dopamine

catechol residues.

However, it should be noted that there are other ways to increase the anti-fouling

character of a chemical species. For example, four different anti-fouling agents (Figure 6)

11

were immobilized separately onto quartz surfaces. Using the electromagnetic piezoelectric

acoustic sensor (EMPAS), the fouling levels of the surfaces were tested with undiluted

fetal bovine serum.43

MEG-OH displayed the best anti-fouling properties as the EMPAS

frequency shift associated with it was ~2000 Hz. Compared to bare quartz that is

extremely fouling (~30,000 Hz frequency shift), there is almost a 95% reduction in surface

fouling. The precursor of MEG-OH, MEG-TFA, also displayed good anti-fouling

behavior with a frequency shift of ~5000 Hz. However, this precursor is not stable in an

aqueous solution as the trifluoroacetate group can be cleaved with ease. The superior anti-

fouling properties of MEG-OH are attributed to the presence of a single ether oxygen and

to the interaction of that oxygen atom with the distal hydroxyl group. For comparison,

OTS-OH displayed a frequency shift of ~20,000 Hz, with the only difference between it

and MEG-OH being the internal ether oxygen. When the distal hydroxyl group was

blocked in MEG-OMe, the fouling frequency shift was observed to be ~5000 Hz. Not as

large of a difference as was observed when the internal ether oxygen was removed,

however, still a significant increase in fouling. Further studies of the MEG-OH anti-

fouling agent revealed that the internal ether oxygen was responsible for forming a water

barrier that served to combat anti-fouling.44

Neutron reflectometry studies for MEG-OH

and OTS-OH indicated water layers of 40 and 20 , respectively.

Figure 6. MEG-TFA: 2-(3-trichlorosilylpropyloxy)-ethyl trifluoroacetate). MEG-OMe:

ethylene glycol 3-trichlorosilylpropyl methyl ether. MEG-OH: hydrolyzed MEG-TFA.

OTS-OH: hydrolyzed 6-trichlorosilyl-hexanyl trifluoroacetate (OTS-TFA).

12

1.5. Self-Assembling Monolayers (SAMs)

1.5.1. Theory

Long organic chemical species that spontaneously form ordered molecular layers

on surfaces (most often inorganic) are termed SAMs. These SAMs contain three parts a

headgroup binding to the surface, a backbone, and an endgroup that determines the surface

properties of the assembled monolayer (Figure 4).45

It is possible to form SAMs in the gas

and liquid phases, however, the latter is most common.46

Figure 7. The general arrangement of a self-assembled monolayer composed of an

endgroup (R1), a backbone, and a headgroup (R2). Biosensing elements react with the

endgroups and are immobilized to the surfaces of transducers. The endgroups also dictate

the surface properties of the assembled monolayer.

The SAM substrates are chosen based on the intended application (ex. gold for

electrochemical biosensors, quartz for piezoelectric biosensors, etc). Thus, SAMs must be

developed to contain a headgroup that is capable of reacting with the chosen substrate.

Similarly, the endgroup of the SAM must be tailored to be able to react with the chosen

biosensor biorecognition elements. The backbone acts as a spacer unit, providing a set

distance between the head group and the endgroup. The customizability of SAMs is

immense as all three of these parts can be altered for any application. For example, one

can vary the number of backbone atoms, choose to make the endgroup chemically inert or

reactive, or attach a specific functional group for post-assembly reaction.47-48

SAMs with

post-assembly reaction functional groups are termed linkers and can be used to

immobilize biorecognition elements.

13

It should be noted that although there are virtually infinite combinations of

headgroups, backbones, and endgroups, practically the combinations are limited. One

must take into consideration whether or not the backbone and head groups are chemically

compatible. For example, the trichlorosilyl moiety (Cl3Si-) endgroup does not tolerate

nucleophilic head groups such as alcohols, carboxylic acids, amines, etc. In order for these

functional groups to be used with trichlorosilyl SAMs, they must be protected in the

synthesis process and then deprotected post-assembly onto the surfaces.48

The self-assembly process of the SAMs is driven by the spontaneous adsorption of

the chemical chains onto the surfaces and by non-covalent intermolecular forces between

backbones.49-50

SAMs with more than 10 carbons in their backbones display enhanced

stability and order due to stronger intermolecular forces.51

Linkers with backbones

between 8 and 18 carbons in length form densely-packed crystalline-like rigid

monolayers. The size of the endgroups (i.e. small vs large/bulky) also influence the

stability and order of the monolayers.52

SAMs offer several benefits with respects to the development of a biosensor. First,

self-assembling monolayers are usually prepared with ease and are associated with low

costs.53

Second, only a small amount of surface modifier is required to coat large substrate

areas (~1014

molecules/cm2, or ~1 nmol/cm

2).

54 Third, linkers can be engineered

specifically for reaction with target biosensing elements, allowing for covalent, oriented

immobilization of those elements. Finally, these linkers can be tuned to impart anti-

fouling properties to the surfaces, increasing the selectivity and specificity of the

developed biosensor.

1.5.2. Trichlorosilyl-derived SAMs

One of the most common headgroups used for the formation of SAMs is the

trichlorosilyl group.55-56

This functional group allows for attachment onto hydroxylated

surfaces. The act of forming a SAM from trichlorosilyl linkers is termed silanization and

is generally performed in dilute organic solutions (i.e. toluene).57

Trichlorosilyl SAM

formation is a much more complex process, compared to thiolate SAM chemistry, and is

therefore more difficult to reproduce. This process first involves the hydrolysis of the

trichlorosilane functional group to yield trisilanol [(OH)3Si-] with water that is adsorbed

14

onto the surfaces or in solution.58-59

Although anhydrous solvents are required for

silanization, they should contain a few water molecules for the hydrolysis of the

trichlorosilane functional group. The second step of the process involves the hydrogen

bonding of the trisilanol group onto the hydroxylated surface (Figure 5). Once the linkers

have undergone this physisorption process, the trisilanol groups may then condense with

surface hydroxyls to form surface-bound silanols.59

In a similar process, the surface bound

linkers may then undergo hydrogen bonding with neighbouring linkers followed by

another condensation reaction to form siloxane (-Si-O-Si) linkages.60

The formation of

SAMs occurs in molecular islands that nucleate over the surface of the substrate. A few

water molecules in the anhydrous solvents are also favour island-type growth.56

SAMs

formed from these linkers are more stable than other linkers as they are bonded to both the

surfaces and to other neighbouring linkers.

Figure 8. (A) Schematic representation of the silanization process after forming of the

trisilanol group, hydrogen bonding occurs, following by condensation for the formation of

surface-bound silanols. (B) After binding to the surfaces, neighbouring linkers undergo

hydrogen bonding through their silanols followed by condensation to form interlinker

siloxanes.

The reproducibility of these monolayers is largely dependent on the stringent

protocols used for silanization. For example, choice of solvent, temperature, precise

silanization time, and substrate properties (i.e. hydroxyl density, contamination, etc.) are

all important in the trichlorosilyl-based SAM formation.61

Even the amount of water in

solution is important if there is not enough water, incomplete SAM formation occurs

15

whereas if there is too much, polymerization of partially hydrolyzed linkers occurs in

solution.

1.5.3. Mixed Trichlorosilyl-derived SAMs

Homogeneous SAMs are common throughout the literature, however much less

research has been devoted towards developing mixed, heterogenous SAM chemistry. The

individual linkers used to form self-assembling monolayers already display a great amount

of customizability. Creating a monolayer from two or more linkers further increases the

potential surface-altering properties of that mixed monolayer. Generally, to form an

evenly mixed SAM, one must react the substrate with both the linkers at the same time in

the same solution. The two linkers must be compatible with one another (i.e. no

nucleophilic functional groups to destroy the trichlorosilane groups) in order for a mixed

SAM system to work.

One commonly used mixed SAM involves the use of a bifunctional linker (used to

immobilize biosensing elements) and a shorter, monofunctional diluent. This latter

chemical is used to space out the bifunctional linkers, relieving congestion and steric

hindrance around the bifunctional linkers.48

Thus, these mixed SAMs allow for better

immobilization of biosensing elements as the bifunctional linker endgroups are more

accessible for reaction.62-63

Mixed SAMs may also be developed that incorporate anti-

fouling linkers to reduce the fouling properties of the biosensor surfaces. These mixed

monolayers may be developed through sequential deposition of the individual linkers or

through simultaneous deposition of both linkers.64-65

The former is also referred to as

back-filling since one linker is fully deposited onto the surface before the second is

allowed to fill in the remaining gaps. Regardless of the method that is used, the ratio of

one linker to another on the substrate surface does not necessarily reflect the ratio that is

found in the silanizing solution.66-67

The surface composition (homogeneity of the

monolayer and ratio of different linkers) varies from one mixed SAM to another. Linkers

with similar backbone length and backbone properties tend to form mixed SAMs that are

interspersed by both linkers. Those possessing backbones with significantly different

lengths or properties and/or possessing endgroups that are bulky tend to form segregated

islands.68-69

16

1.6. Surface Characterization Techniques

Surfaces that have been altered for biosensor development have to be analyzed and

confirmed using various techniques. These analytical techniques are used to evaluate

chemical composition, hydrophobicity/hydrophilicity, order, thickness, and roughness of

the surfaces. From the many surface characterization techniques available, there are three

that excellent for the development of biosensor surfaces. The following techniques

optimize sensitivity, effectiveness, availability, time, and cost of surface analysis: contact

angle goniometry (CA), X-ray photoelectron spectroscopy (XPS), and atomic force

microscopy (AFM).

1.6.1. Contact Angle (CA) Goniometry

Contact angle goniometry is a technique that is used to analyze the wettability of a

surface and its polarity. Initially, a liquid droplet is place over a flat surface forming a half

sphere on the substrate. The finite contact angle () and the shape the droplet makes with

the substrate is dependent of the interfacial free energies between the liquid-vapour (LV),

solid-liquid (SL), and solid-vapour (SV) interfaces (Figure 6). The relationship between

all four of these factors is given by Youngs equation (Equation 1) and is strictly followed

in the case of an ideal surface one which is perfectly flat, smooth, level, and rigid).70

SV = SL + LVcos where 0 180 (1)

This relationship is rarely valid for practical surface analyses as they are generally

not homogeneous surfaces in thermodynamic equilibrium. Many mathematical systems

have been developed in order to take into account a surfaces heterogeneity, roughness,

and defects.71

A contact angle of 0 is indicative of a completely wetted surfaces, where as

one with a contact angle greater than 90 exhibits no wetting. A contact angle between

these two values indicates a partially wetted surface. When water is used as the liquid

phase agent, a contact angles close to 0 are associated with hydrophilic surfaces. As the

contact angle increases, an increase in hydrophobicity and decrease in hydrophilicity is

observed. This technique is useful to show large changes in polarity between surfaces

for example between a bare substrate surface and one modified with a monolayer.72

This

17

technique is by no means quantitative and is used as a qualitative indicator that surface

modifications have occurred.

Figure 9. Contact angle picture of a water droplet on a quartz surface showing the four

components of Youngs equation.

1.6.2. X-ray Photoelectron Spectroscopy (XPS)

A more quantitative approach to determining the changes that occur on surfaces

from one modification to another is X-ray photoelectron spectroscopy (XPS). This

technique allows a researcher to study the relative atomic composition of a sample at

depths of 1-10 nm.71

The photoelectric effect occurs when electrons are ejected from the

surface atoms of a sample provided enough energy is applied to overcome the work

function. XPS uses a monochromatic X-ray source (energy in the range of 1000-2000 eV)

to eject core electrons from the K, L, or M shells of the atoms on the surface.73

h = Eb + EK + (2)

18

Figure 10. Schematic representation of an X-ray photoelectron spectroscopy instrument.

The take-off angle () can be varied in angle-resolved XPS in order to probe the surface at

different depths.

The energy of the irradiated light (h) is used and distributed into three

components (Equation 2). First enough energy must be applied to the core electrons to

overcome their binding energy (Eb). Once this happens, the electrons leave the surface

with a kinetic energy (EK) and are sorted by these energy levels by an electron energy

analyzer (Figure 7). There is also a work function () added by the spectrometer that

needs to be accounted for by the equation above. For every energy level, the number of

electrons going through the analyzer are counted and recorded at a detector. The electrons

that have lost significant amounts of their kinetic energy through inelastic collisions

19

within the sample will be observed as the background noise in the XPS spectrum.

However, ejected electrons that were able to escape without any energy losses will be

observed as the main signal peaks.

The binding energies of electrons vary from atom to atom and thus it is possible to

identify different elements on the surface being analyzed. Every element in a specific

oxidation state will display its own unique set of binding energies. Using the proper

software and peak fitting options, it is possible to quantitatively analyze the elemental

composition of the surface being analyzed.74

The number of photoelectrons detected each

second is directly proportional to the number of atoms, the flux of the incident X-ray

beam, and the sensitivity factor for the target element. Low resolution XPS is able to

quantitatively determine the elemental composition of the surface that is being analyzed.

However, it is possible to discover additional information about the chemical environment

of the surface through high resolution XPS.75

Using angle-resolved XPS, it is possible to

analyze the surface at different depths. This technique is important for the development of

biosensors as there may be multiple layers of different chemical species on the surfaces.

This technique is achieved through the variation of the photoelectron take-off angle

(Figure 7) during data collection. A smaller angle allows for probing of a greater depth

whereas a larger angle will probe less of the bulk substrate and more of the overlying

chemical layers.

1.6.3. Atomic Force Microscopy (AFM)

One very useful tool in the characterization of biosensor surfaces is the atomic

force microscope.76

The topography of surfaces may be characterized using this analytical

method, showing clearly any objects in the nanometer range (i.e. proteins, biosensing

elements, etc.). The probe in the AFM is used to scan the surfaces vertically using a force

feedback system that is distance-sensitive. Alternatively, the AFM can also be used to

apply a force to the surface and analyze the subsequent response for structural changes,

kinetics, etc.

One principle behind the AFM is Hookes law (F = kx), which states that the

force (F) required to compress a spring (k = spring constant) through a distance (x) is

proportional to that distance. The AFM generally uses a silicon or silicon nitride cantilever

20

with a sharp tip to probe a surface. As the tip is brought closer to the surface, the

cantilever experiences a deflection according to Hookes law due to the interactive forces

between the surface and the tip. The following are some of the force interactions that may

be observed with the AFM: mechanical contact foce, chemical bonding, electrostatic

forces, capillary forces, and magnetic forces. The deflection of the cantilever causes a

change in the location of a reflected laser spot that is detected on an array of position-

sensitive photodiodes. The piezoelectric scanner is used as a feedback mechanism to

ensure that the tip does not run into anything on the surface. Thus, if a constant height is

set for the tip, this height is relative to the top of the surface and can be adjusted as the tip

is dragged along the surface.

Figure 11. General setup of an atomic force microscope.

A common operating mode for the AFM is the tapping mode imaging.77-78

Using

piezoelectric actuation, the cantilever is made to oscillate along the surface. A unique

amplitude and phase relative to the driving oscillator is associated with the cantilever

when it is sufficiently far from the surface. The amplitude and phase change as the tip is

brought closer to the surface due to oscillation damping. This imaging mode uses

21

amplitude as feedback such that the distance of the tip base is adjusted in order to maintain

constant amplitude. These height adjustments generate topography images of the surfaces

being analyzed. The benefit of this AFM imaging mode is that it allows for the analysis of

surface bound proteins or chemical species that are in the nanometer size. Although it is

possible to confirm the presence of proteins to surfaces using XPS, the AFM is able to

show the distribution of these proteins on the surfaces. Even monolayer deposition can be

visualized using the AFM as a topographic change.

1.7. Biosensing Elements

1.7.1. Introduction

Since biosensors are developed to provide rapid and reliable analyses of target

analytes, one crucial step in the optimization of a biosensor is the choice of the biosensing

elements. Four of the most prominent biorecognition elements are whole monoclonal

antibodies (mAb), fragment-antigen binding (Fab) units, single-chain Fv fragments

(scFv), and aptamers (Figure 12).

Immunoglobulins (Ig), or antibodies, are large proteins produced by the immune

system that have extremely high affinities and specificities for their target analytes.79

Of

the many classes of immunoglobulins (i.e. IgE, IgM, IgG, etc.), the immunoglobulin G

(IgG, ~150 kDa) is the most prominently used class in the field of biosensing. The

structure of an IgG antibody consists of two heavy protein chains and two light protein

chains. The heavy chains are each composed of three constant domains (CH1, CH2, CH3)

and one variable domain (VH) whereas the light chains each contain only one constant

domain (CL) and one variable domain (VL). The two antibody halves (each half containing

one heavy and one light chain) are held together via disulfide bonds in the hinge region

(Figure 12).80

The number of disulfide bonds in the hinge region varies depending on

antibody species and antibody class.81

The paratope of the antibody the region that

recognizes and binds to the target analyte (or antigen) involves the top of the VL and VH

domains.

22

Figure 12. An illustration of whole antibodies (Ab), F(ab)2, Fab, Fab, Fv, and aptamers.

*Compared to antibodies and their fragments, aptamer shape and size varies from one

aptamer to another. The blue helix has been chosen to designate a general aptamer in this

thesis, however, it should be noted that not all aptamers form helices.

The antibody contains two Fab fragments, each one consisting of the VL, VH, CL,

and CH1 domains. These two fragments are held together by the key hinge disulfide

bridges.80

Below the disulfide bridges resides the fragment crystallizable (Fc) region. Fab

fragments may be obtained in one of two possible ways: via recombinant synthesis or

proteolytic cleavage of the parent antibody.82-83

Fragments including disulfide bridge

thiols (Figure 12) are called Fab fragments whereas those lacking the thiol functional

group are termed Fab fragments. The thiol functional group of the the Fab fragments

allows for easy immobilization onto biosensor surfaces.79

Even smaller than the Fab fragment is the antibody Fv fragment (Figure 12),

consisting of only the VH and VL domains. These fragments can only be obtained reliably

23

via recombinant synthesis and are held together by relatively weak non-covalent

interactions.84-85

As a result, several modified types of Fv fragments have been developed

including, but not limited to, single-chain Fv (scFv), disulfide-stabilized Fv (dsFv),

diabodies (divalent dimers), and permutated Fv (pFv) fragments.86-89

However, scFv

fragments are the most prominent of the Fv-derived antibody fragments used as

biosensing elements.

Compared to Fab and scFv fragments, aptamers are not derived from antibodies.

Aptamers are single stranded ribonucleic acid (RNA) or 2-deoxyribonucleic acid (DNA)

chains that have affinities and specificities for their target analytes on orders of magnitude

comparable to or better than antibodies.90

The development process begins with a massive

random library of RNA or DNA that is then subjected to bind the target analyte.91

The

nucleic acid chains binding the analyte successfully are then isolated, amplified, and sent

towards the next round of enrichment (Fig. 6). Multiple rounds of this process (~8-15

cycles) result in the exponential increase of the nucleic acids displaying the greatest

binding affinities towards the analyte. This iterative process is termed the systematic

evolution of ligands by exponential enrichment, or SELEX, and it can take several

months to accomplish.90

Generally, aptamers developed by the SELEX process are around

70-80 nucleotides long, although it is possible to create functional aptamers smaller than

40 nucleotides long.92-93

It is possible to shorten the length of aptamers through the

removal of fixed sequence primer regions as most of the time they do not participate in

aptamer function.90

Once the sequence of the aptamer is determined, chemical synthesis of

the aptamer can be accomplished relatively quickly in 2-3 days.

Traditional SELEX development required the use of a pure and soluble analyte,

devoid of impurities. However, due to recent developments it is now possible to isolate

functional aptamers in complex mixtures such as plasma and cell-surface proteins.94

It is

now also possible to increase the affinity of the aptamer further by using specific

subdomains of the target analyte during the SELEX process.95

In addition, many SELEX

processes also offer counter-SELEX the discarding of potential nucleic acid aptamers

that bind efficiently to the analyte and to closely related structural analogs.90

This ensures

that the developed aptamer has extremely high selectivity and specificity for only the

target analyte. The size of aptamers (~1-2 nm), however, is much smaller than that of

24

whole antibodies (~10-15 nm) allowing them to be immobilized in higher densities on

surfaces, resulting in higher sensitivities and lower limits of detection (LOD) in

biosensors.96

The same phenomenon is observed with Fab fragments and scFv

fragments.79,97

Thus, Fab, scFv, and aptamers have greater potential in biosensor

development.

1.7.2. Comparison of Whole Antibodies, Fab, scFv, and Aptamers

The optimal biosensor is cheap, easy to make, easy to use, reproducible, sensitive,

specific, and rapid to detect. The optimization of the biosensing elements and their

immobilization onto transducer surfaces are arguably the most important steps in the

development of a biosensor. Biosensing elements need to be judged for their cost and ease

of development, variability and ease of immobilization, affinity towards the analyte

(selectivity and specificity), and stability.

Of the four biosensing elements described, whole antibodies and Fab fragments

are the cheapest and quickest to obtain. For Fab fragments, once the whole antibodies are

received, it is only a matter of days before they are cleaved and Fab fragments are

obtained. The only downside is that there is usually some loss of immunological activity

of the antibody fragments via chemical cleavage.98

Due to the use of recombinant

antibody technology, scFv fragments are significantly more expensive and require several

weeks of development before their use. The development process is also very laborious

and requires skilled researchers that are well versed in cell culture practices. Overall,

aptamers are the most costly and require the longest optimization time. This is largely due

to the SELEX process, which lasts several months and costs tens of thousands of dollars.

The actual synthesis of the aptamers is very fast (a few days) and can be quite cheap (on

the scale of dollars per gram) when synthesized in large quantities.90

In terms of biosensing element immobilization, scFv fragments have the potential

to be immobilized onto the largest variety of surfaces due to how highly customizable they

are (ex. addition of a C-terminal thiol or a functional immobilizing C-terminal peptide,

etc.). However, it is arguable that whole antibodies, Fab fragments, and aptamers can be

immobilized onto surfaces with greater ease. The most common modification of scFv

fragments is the addition of a C-terminal fusion peptide that is used for immobilization.

25

Thus, the researcher has to worry about avoiding scFv denaturation and fusion peptide

denaturation. Also, many of these immobilizing peptides require specific substrates that

need to be on the surfaces. In comparison, Fab fragments and most aptamers only have

one small functional group (ex. thiol, amine, etc.) that can react with surface functional

groups for immobilization in a simple manner. Most of the time whole antibodies do not

require any modifications and can be immobilized easily using covalent or non-covalent

interactions to surfaces. In terms of surface density, aptamers can be immobilized with the

greatest number of binding sites per unit area due to their small size (~1-2 nm) compared

to whole antibodies (~15 nm) Fab fragments (~7 nm) and scFv fragments (~4 nm).91,99-100

The optimal biosensing element has an affinity that is highly selective and specific

for the target analyte. Aptamers dominate this category as their affinities can be improved

through more rounds of SELEX and through the use of counter-SELEX (Figure 13). This

ensures that the best aptamers have affinities in the nanomolar/picomolar range and do not

bind to close derivates of the analyte. Compared to whole antibodies, aptamers have

similar or better affinities for their analyte whereas Fab fragments and scFv fragments

have lower affinities.90

Fab fragments are in third place for affinity as they have been

shown to have smaller dissociation constants compared to scFv fragments.101

However,

the affinity of scFv fragments can be increased through dimerization (increased avidity) or

through affinity maturation in the recombinant process. For example, Torrance et al.

showed that after three affinity maturation cycles, the affinity of the scFv fragments was

increased by 100-fold.102

In terms of selectivity and specificity, scFv fragments come in

second place behind aptamers. Recombinant synthesis of the scFv fragments ensures that

they are highly specific for their target analyte, binding to only one epitope. On the other

hand, whole antibodies and Fab fragments can be obtained from monoclonal or

polyclonal antibodies. The latter involve antibodies that bind to one target analyte, but

through multiple potential epitopes. These antibodies are prone to non-specific binding to

close derivatives of the analyte.90

Overall, aptamers are the superior biosensing elements, however, all four of these

biorecognition elements have their place in the biosensing world. This study uses whole

antibodies and Fab fragments for the development of the biosensors are they are cheap,

quick, and easy to use with relatively good analyte affinity, specificity, and selectivity.

26

Future work for this biosensor involves its optimization with either scFv fragments or

aptamers for an increase in sensitivity and a decrease in limit of detection.

Figure 13. Systematic evolution of ligands by exponential enrichment SELEX. The

process begins with a large DNA/RNA library and allows binding of the analyte.

Aptamers that bound the analyte are then amplified and taken to the next round for further

selection. In the end, the ideal aptamer will only bind its target with high selectivity and

specificity.

27

1.7.3. Immobilization of Whole Antibodies

First reported in 1967, antibody immobilization has grown rapidly in the last few

decades and has proven extremely important to many fields.103

A short list of surfaces that

antibodies may be immobilized onto includes gold, quartz, silica, Sepharose, agarose,

cellulose, dextran, polystyrene, polyacrylamide, magnetite, steel, hydroxyapetite, and

niobium oxide.104

An older method of whole antibody immobilization to surfaces is adsorption via

non-covalent interactions. Hydrogen bonds, hydrophobic interactions, and van der Waals

are some of the forces involved with the adsorption of antibodies.103

This type of

immobilization technique results in randomly oriented antibodies. Furthermore, this

results in an unpredictable number of inaccessible antigen-binding sites. Diminished

binding capacities and efficiencies are associated with surfaces containing adsorbed

antibodies. For applications involving liquid flow over the surface, leaching of the

adsorbed antibody also occurs, resulting in deterioration of the engineered surface.103

Thus, it was determined that antibodies needed to be immobilized in a controlled and

oriented fashion, in order to maximize the number of available antigen-binding sites and

prevent surface degradation.

Immobilization of whole antibodies via covalent methods was the first step

towards this goal. Soluble activators, such as, carbodiimide and succinimide allow for the

binding of antibodies via free amino groups (typically from lysine residues).97,103,105

Other

soluble activators such as cyanuric chloride and phenylene diisocyanate help immobilize

antibodies via free carboxylic acid groups (typically from aspartic acid and glutamic acid

residues).103

It is also possible to attach bifunctional linkers, such as glutaraldehyde, that

will covalently bind to antibodies. Modification of the solid-phase support to produce a

surface rich in epoxy groups, N-hydroxysuccinimide (NHS) ester groups, and maleic

anhydride groups will result in binding antibodies covalently through free amine

groups.103,105

Although these methods immobilize antibodies covalently, the large number

of similar amino acid residues, and thus binding sites, still results in a significant amount

of different binding orientations. However, antibodies bound in this manner will have

more favourable orientations compared to those adsorbed on a surface and will be more

resilient towards degradation.

28

Figure 14. General setup for the oriented immobilization of whole antibodies using an

immobilizing protein, such as protein G.

Immobilization techniques that result in very specifically oriented antibodies

involve binding of the Fc fragment. For example, activation of the carboxylic acid group

of the C-terminus via 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and NHS

allows for binding to amino-rich surfaces.105

Another method of binding involves the

initial immobilization of Protein A, Protein G, or variants of the two (i.e. Protein A/G,

Protein G, etc.) through a linker (Figure 14).97,105

These proteins selectively bind the Fc

fragment, resulting in antibodies that maintain a high level of accessibility to antigen-

binding sites. An alternative approach is the oxidation of the carbohydrate species found

in the Fc region. The resulting aldehyde groups can then be reacted with hydrazide-

activated surfaces allowing for oriented immobilization of antibodies.103

Recently, another

29

method has been proposed for the immobilization of specifically oriented antibodies via

nucleotide binding sites (NBS) on the variable regions of the Fab chains.106

Upon

exposure to UV light, the aromatic amino acids of the NBS form radicals which are then

used to couple to biotin ligands. These biotin anchors link the antibodies to a streptavidin

surface, where one of the Fab fragments points in a perpendicular direction to the surface.

1.7.4. Immobilization of Fab fragments

Many types of immobilization techniques have been developed over the last 30

years for Fab fragments. These biosensing elements have been immobilized onto gold,

inorganic, plastic-based, polysaccharide-based, silicon-based, and magnetic surfaces.79

This has allowed for the development of a wide range of biosensors having very different

surface chemistries.

One of the oldest methods of Fab fragment immobilization involves the direct

adsorption of the fragments onto surfaces.103

The weak non-covalent interactions binding

the Fab fragments to surfaces are not strong enough when liquid flow occurs, resulting in

the leaching of the biosensing elements. The development of covalent immobilization

techniques centered around the functional C-terminal thiols prevent fragment leaching and

allow for oriented immobilization of the fragments onto surfaces.79

As the C-terminal thiols are nucleophilic, one popular method for immobilization

involves the reaction with maleimide-rich surfaces. For example, Prisyazhnoy et al.

activated AH-sepharose 4B with N-maleonyl--alanine to form maleimide-terminated

surfaces, which were then used to covalently immobilize Fab fragments (Fig. 15, A).107

Viitala et al. immobilized the antibody fragments onto gold surfaces containing a

polymerized lipid matrix containing N-(-

maleimidocaproyl)dilinoleoylphosphatidylethanolamine (Fig. 15, B1/B2).108

One benefit

of polymer matrices is that they avoid the phase separation and disruption of the

monolayer during deposition. Similarly, Vikholm et al. used N-(-

maleimidocaproyl)dipalmitoylphosphatidylethanolamine (DPPE) to immobilize Fab

fragments onto quartz surfaces through a mixed polymer matrix containing cholesterol.109

Surfaces without cholesterol exhibited Fab binding constants that were 10-fold less than

those containing cholesterol. Medina-Casanellas et al. used sulfosuccinimidyl 4-(N-

30

maleimidomethyl)cyclohexane-1-carboxylate (SSMCC) (Fig. 15, C) to immobilize the

antibody fragments onto amine-terminated silica particles (125 ).110

Capon used amine-

coated magnetic beads and the bifunctional linker SM(PEG)24 to immobilize Fab

fragments. SM(PEG)24 contained a surface-binding NHS functional group and a

maleimide functional group for Fab immobilization.111

Thus, it can be concluded from

these examples that the maleimide-immobilization of Fab fragments is widely applicable

to many different types of surfaces.

Another common immobilization technique involves the reaction of the Fab C-

terminal th

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