Acoustic Wave Biosensor for the Detection of the Breast ... · ii Acoustic Wave Biosensor for the...
Transcript of Acoustic Wave Biosensor for the Detection of the Breast ... · ii Acoustic Wave Biosensor for the...
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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
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Acoustic Wave Biosensor for the Detection of the Breast
Cancer Metastasis Biomarker Protein PTHrP
Victor Serban Crivianu-Gaita
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.
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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.
Victor Serban Crivianu-Gaita
June 2016
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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
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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
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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
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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
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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
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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
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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
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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
Table 2. Rate constants (s-1
) 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
Table 3. Rate constants (s-1
) 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
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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
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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)-
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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
(22C) of polyclonal rabbit anti-goat IgG F(ab)2....55
Figure 24. 12% SDS-PAGE gel for the 2.0 mM MEA cleavage at room temperature
(22C) of polyclonal rabbit anti-goat IgG F(ab)2....55
Figure 25. A graph illustrating the change in F(ab)2 concentration (anti-goat IgG
antibodies) versus time comparing the three reducing agents at physiological temperature
(37C)..56
Figure 26. A graph illustrating the change in F(ab)2 concentration (anti-goat IgG
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
antibodies) versus time comparing the three reducing agents at room temperature
(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
antibodies) versus time comparing the three reducing agents at room temperature
(22C)..61
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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
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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,
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
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(1:10)/Fab, N TUBTS/HTS (1:10)/Fab, O TUBTS/MEG-OH (1:10)/Fab, P
TUBTS/Fab..105
Fig. A.3. XPS spectra of the Si2p (103 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..106
Fig. A.4. XPS spectra of the S2p (164 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..107
Fig. A.5. XPS spectra of the N1s (400 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..108
Fig. A.6. XPS spectra of the F1s (688 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..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,
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...111
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xviii
Fig. A.9. XPS spectra of the Si2p (103 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...112
Fig. A.10. XPS spectra of the S2p (164 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...113
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,
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...115
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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
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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).
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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.
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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
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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
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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
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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
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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)
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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
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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
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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.
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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
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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
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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.
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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.
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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.
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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.
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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
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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-
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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