Development of an Electrochemical Immunosensor for the Detection of HIV Antibodies Using Surface Modification of

SU-8

by

Alyajahan Bhimji

A thesis submitted in conformity with the requirements for the degree of Master of Science

Pharmaceutical Sciences University of Toronto

© Copyright by Alyajahan Bhimji 2013

ii

Development of an Electrochemical Immunosensor for the

Detection of HIV Antibodies Using Surface Modification of SU-8

Alyajahan Bhimji

Master of Science

Pharmaceutical Sciences University of Toronto

2013

Abstract

The negative epoxy-based photoresist of SU-8 has a variety of applications within

microelectromechanical systems (MEMS) and lab-on-a-chip systems. Herein, SU-8 was

functionalized with antigenic peptides to HIV-1 gp41 or HIV-2 gp36 and the detection of

antibody against HIV-1/2 was carried out by an electrochemical immunoassay combining an

alkaline phosphatase conjugated secondary antibody and p-aminophenyl phosphate. The by-

product of the reaction (p-aminophenol) was quantitated electrochemically using differential

pulse voltammetry, and the current derived from the oxidation of the hydrolysis product

increased linearly over a wide primary antibody concentration range (0.001 – 1 µg mL-1), with a

detection limit of 1 ng mL-1 (6.7 pM) for both HIV-1 and HIV-2. This level of sensitivity is

clinically relevant, and feasibility of this approach for clinical sample testing was also evaluated

with HIV clinical patient samples.

iii

Acknowledgments

First and foremost, I would like to thank my supervisor, Dr. Shana Kelley, for providing me with

the opportunity to work in her lab and enabling my studies and research. I have gained invaluable

research experience under her supervision.

Working in the Kelley laboratory would not have been the same without its past and present

members whose group discussions and encouraging advice have made my time in the lab

productive and inspirational. It was a pleasure to work alongside a remarkable group of students,

having developed long lasting friendships with all of them.

I would also like to give many thanks to Dr. Frank Merante and Dr. Sandeep Raha, for always

having my best interests at heart. Their constant mentorship in all aspects of life has been

invaluable.

Finally, I would like to give thanks to my family and friends for their continued and unwavering

encouragement, love and moral support.

iv

Table of Contents

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

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

List of Tables ................................................................................................................................................ v

List of Figures ............................................................................................................................................. vi

Preface .......................................................................................................................................................... 1

Chapter 1 ..................................................................................................................................................... 2

1 Literature Review ................................................................................................................................. 2

1.1 Antibodies ...................................................................................................................................... 2

1.2 Antigens .......................................................................................................................................... 2

1.3 Antigen-Antibody Binding ........................................................................................................... 3

1.4 Immunoassays ............................................................................................................................... 4

1.5 Immobilization Strategies ............................................................................................................. 5

1.6 Non-Specific Adsorption (NSA) ................................................................................................... 6

1.7 Immunosensors .............................................................................................................................. 7

1.8 Voltammetry/Amperometry ......................................................................................................... 7

1.9 SU-8 Negative Epoxy-Based Polymer .......................................................................................... 9

1.10 HIV Testing ............................................................................................................................... 11

1.11 Enzyme Labels .......................................................................................................................... 12

1.12 Objective and Rationale ........................................................................................................... 13

Chapter 2 .................................................................................................................................................... 14

2 Accepted Manuscript in Analytical Chemistry ................................................................................ 14

2.1 Author Contributions ................................................................................................................. 14

Chapter 3 .................................................................................................................................................... 28

3 Discussion and Conclusions ............................................................................................................... 28

3.1 Future Directions ........................................................................................................................ 28

References ................................................................................................................................................... 30

v

List of Tables

Table 1: Forces involved in antibody-antigen binding.

Table 2: Substrates for alkaline phosphatase.

Supporting Table S1: Signal to cutoff ratio (s/co) values for the electrochemical ELISA and

Bio-Rad HIV-1/HIV-2 Genetic Systems Kit

vi

List of Figures

Figure 1: Schematic diagram of an IgG antibody.

Figure 2: Diagram showcasing the epitope found on an antigen and the paratope found on an

antibody.

Figure 3: Different immunoassay formats. (a) Homogenous competitive immunoassay, (b)

heterogeneous non-competitive immunoassay, (c) a heterogeneous competitive immunoassay,

and (d) heterogeneous competitive immunoassay.

Figure 4: Voltage applied due to applied voltage shown in Figure 4 for DPV.

Figure 5: Peak current response due to applied voltage for DPV.

Figure 6: Epoxy group (or oxirane) found on the SU-8 surface.

Figure 7: The reaction between the oxirane group of the epoxy resin and a primary amine (A) or

a secondary amine (B).

Figure 8: The reaction between the oxirane group of the epoxy resin and a thiol moiety. Scheme

adapted from (Grazú et al, 2003).

Figure 9: Electrochemical ELISA with proximal reagent generation on the SU-8 substrate. (a)

Schematic illustration of the Au microelectrodes generated by electrodeposition in 5 µm

apertures within the SU-8. (b) Schematic of immunoassay. HIV peptides are covalently

immobilized on the SU-8 layer, after which target HIV antibodies are allowed to bind. Secondary

anti-IgG alkaline phosphatase (ALP) conjugated antibodies bind to the target HIV antibody and

p-aminophenyl phosphate (pAPP) is then added. (c) ALP-labeled antibody converts pAPP to p-

aminophenol which is electrochemically oxidized to generate a current at the electrode that is

proportional to the amount of target antibody bound to the sensor surface. (d) Differential pulse

voltammetry (DPV) reports on the current generated when pAP is oxidized.

vii

Figure 10: Conventional optical indirect ELISA on a 96 well polystyrene plate to ensure (a)

HIV-1 and (b) HIV-2 antigens capture respective antibodies. A dilution series was performed

with varying concentrations of HIV-1/2 antibodies. Absorbance of 4-nitrophenol was measured

at 405nm.

Figure 11: Comparison of the sensitivities of the differently sized microsensors. (a) SEM images

of sensors used for analysis. Scale bar corresponds to 50 microns. (b) Representative DPV curves

obtained with 6.3 µM 4-aminophenol. (c) Quantitation of currents produced with different

concentrations of 4-aminophenol in 50mM Tris-HCl, 10mM NaCl, 10mM MgCl2 were obtained

with 50µm, 100µm, 200µm, and 300µm microsensors. Values represent mean ± SD. Voltage is

measured relative to an Ag/AgCl reference.

Figure 12: Optical ELISA on the SU-8 substrate using HIV-1 antigen. The SU-8 resist layer was

functionalized with HIV-1 antigens and an optical ELISA was completed to ensure the

bioactivity of the antigen was not reduced. The assay was carried out in the same manner as the

electrochemical ELISA with an exception being that 4-nitrophenyl phosphate was replaced for p-

aminophenyl phosphate. The enzymatic product, p-nitrophenol, was pipetted off chip and

measured spectroscopically at 405nm using a NanoDrop ND-1000. Detection of HIV-1

antibodies using functionalization of the SU-8 layer is possible.

Figure 13: Stripping the sensor surface in 1X TBS to recover signal from p-aminophenol.

Stripping the sensor surface in 1X TBS for 6 cycles before the assay is run leads to 25% signal

enhancement. Values represent mean ± SD. * denotes p < 0.05 between stripped and not stripped

surfaces.

Figure 14: Validation of the electrochemical ELISA with the proximal reagent generation

approach. (a) Differential pulse voltammetry data collected as a function of time. (b)

Quantitation of p-aminophenyl phosphate (pAPP) signals as a function of time. HIV-1 peptide

functionalized glass chips with 300µm microsensors were treated with target 1µg mL-1 HIV-1

and non-target 1µg mL-1 HIV-2 antibodies and secondary anti-IgG alkaline phosphatase

conjugated antibodies. The chips were then incubated with 0.5mM pAPP for 1, 5, 10, 15, 20 min.

(c) Electrochemical ELISA for HIV-1 antibody detection using 300µm microsensor SU-8 coated

glass chips. Dotted line represents non-target 1 ug mL-1 HIV-2 current value. r-squared value of

0.9001. (d) Electrochemical ELISA for HIV-2 antibody detection using 300µm microsensor SU-

viii

8 coated glass chips. Dotted line represents non-target 1 ug mL-1 HIV-1 current value. r-squared

value of 0.8668. Values represent mean ± SD. Voltage vs. Ag/AgCl. * denotes p < 0.05 between

HIV-1 antibodies and HIV-2 antibodies.

Figure 15: Testing of clinical samples with the electrochemical ELISA and a gold standard test.

Sera were diluted 1:5 and 20µL was used for the assessment of HIV antibodies. Both HIV-1

positive and HIV-2 positive samples were tested with each HIV peptide. The Bio-Rad test results

were supplied by SeraCare Life Sciences. (a) Detection of HIV-1 antibodies using an SU-8

surface coated with HIV-1 peptide. (b) Detection of HIV-2 antibodies using an SU-8 surface

coated with HIV-2 peptide. Values represent mean expressed as signal to cutoff ratios (s/co).

Ratios ≥ 1.0 are considered positive (dotted line).

Supporting Figure S1: Conventional optical indirect ELISA on a 96-well polystyrene plate to

ensure (a) HIV-1 and (b) HIV-2 antigens capture respective antibodies. A dilution series was

performed with varying concentrations of HIV-1/2 antibodies. Absorbance of p-nitrophenol was

measured at 405nm.

Supporting Figure S2: The SU-8 resist layer was functionalized with HIV-1 antigens and an

optical ELISA was completed to ensure the bioactivity of the antigen was not reduced. The assay

was carried out in the same manner as the electrochemical ELISA with an exception being that

p-nitrophenyl phosphate was replaced for p-aminophenyl phosphate. The enzymatic production,

p-nitrophenol, was pipetted off chip and measured spectroscopically at 405nm using a NanoDrop

ND-1000. Detection of HIV-1 antibodies using functionalization of SU-8 layer is possible.

Supporting Figure S3: Stripping the sensor surface in 1X TBS for 6 cycles before the assay is

run leads to a 25% signal enhancement. Values represent mean ± SD. * denotes p < 0.05 between

stripped and not stripped surfaces.

Supporting Figure S4: Photograph of sensor chip used in this study.

1

Preface

Increasing need for fast, real-time and reliable medical diagnosis has led to growing interest in

new point-of-care biological sensors capable of the sensitive and specific detection of

biomolecules. Biosensors are small devices that utilize biochemical molecular recognition

properties as the basis for a selective analysis. Three major processes are involved in any

biosensor system: analyte recognition, signal transduction, and readout. Due to their specificity,

speed, portability, and low cost, biosensors offer exciting opportunities for numerous

decentralized clinical applications, ranging from testing in a physician’s office, emergency-room

screening, bedside monitoring, or even home self-testing.

Among the various biosensors available today, electrochemical biosensors have received most

attention, as they are simple, inexpensive and yet accurate and sensitive enough for patient

diagnosis. Electrochemical biosensors are capable of transducing a biomolecular recognition

event into an electronic signal. In particular immunosensors exploit the ability of an antibody to

recognize its associated antigen in a complex medium. The interaction between antibodies and

antigens (with usually one of the binding partners immobilized) leads to changes, for example in

refractive index, thickness, and dielectric constant that can be measured by an immunosensor.

Applications of protein analysis are manifold, including the early diagnosis of certain diseases by

the detection of abnormal concentrations of specific peptides and proteins. Proteomics offer an

advantage over genomics, as protein biomarkers are a more accurate sign of a diseased state

since proteins, not gene transcripts, are the actual functional players.1 mRNA levels do not

always reflect protein expression or activity due to a number of posttranslational modifications

such as ubiquitination, protease cleavage, glycosylation, phosphorylation, methylation and

acetylation.2

The aim of the work described in this thesis is to develop electrochemical methods for measuring

proteins (antigens or antibodies) with high sensitivity and reproducibility. This detection can be

performed with methods making use of the specific interaction of proteins/antigens with

antibodies (Ab).

2

Chapter 1

1 Literature Review An immunosensor is a device that is able to detect the

interaction between and antibody (Ab) and an antigen

(Ag). Immunosensors are used in situation where both

high sensitivity and a high selectivity are required.

1.1 Antibodies

Antibodies are immunoglobulins that are capable of

binding specifically to a wide range of natural and

synthetic antigens. Antibodies consist of five general classes

designated as IgG, IgA, IgM, IgD and IgE. IgG, a 150kDa

glycoprotein composed of two heavy chains and light (κ and

λ) chains joined by disulfide bonds (Figure 1), is the most

prevalent antibody in use. The variable amino acid sequence

at the end of each chain determines the antigenic specificity

of the particular antibody. Each unique region of the

antibody that will specifically bind a complementary target is

termed a paratope (Figure 2).3

1.2 Antigens

Antigens are proteins or substances that when introduced

into a foreign host, are capable of inducing an immune

response and antibodies in the host. In addition to proteins

and polypeptides, lipids, nucleic acids, and other compounds

can function as antigens. Each unique region of the antigen

molecule that will specifically bind a complementary

antibody is termed an epitope (Figure 2). On proteins,

epitopes can be continuous/linear (made up of a single

Figure 1 | Schematic diagram of an IgG antibody molecule. Adapted from Tietz.

Figure 2 | Diagram showcasing the epitope found on an

antigen and the paratope found on an antibody.3

3

segment of a polypeptide chain), or discontinuous/conformational (composed of amino acids

from different parts of the polypeptide chain that are brought together by protein folding).3

1.3 Antigen-Antibody Binding

The binding of antibody to antigen is reversible and obeys the law of mass action i.e. the rate of

the reaction is proportional to the concentration of the reactants. The strength or energy of

interaction between an antibody and antigen is described by either affinity or avidity. Affinity

refers to the strength of binding between a single epitope – paratope complex as determined by

the equilibrium constant of the association reaction between antibody (Ab) and antigen (Ag) as

seen below, where Ka is the affinity constant.

The strength of the binding of an antigen (Ag) to an antibody (Ab) depends on several forces

acting together. They include van der Waals, dipole-dipole interactions, hydrophobic

interactions, and ionic binding (Table 1).4 The strength of Ag-Ab binding depends on electrolyte

concentration, pH, temperature, the Ag and Ab types, and the binding affinity of the antibody.

The affinity of an Ag to an Ab is also dependent on the ion species, ionic strength, and polymers.

For example, cationic salts inhibit the binding of an Ab with a cationic Ag.3

4

Avidity refers to the overall strength of binding of an antibody and an antigen, and includes the

sum of the binding affinities of all the individual combining sites, as seen below. For example,

IgG has two affinity-binding sites, whereas IgM has 10 affinity-binding sites per antibody

molecule.

1.4 Immunoassays

Immunoassays are analytical tools used in the clinical and pharmaceutical sciences. Many

different formats are used, as seen in Figure 3, but all rely on a directly or indirectly detectable

label. The label can be a radioactive (RIA, IRMA) or fluorescent molecule (EIA, ELISA,

CEDIA), a particle (PETIA), metal (PENIA), virus (MEIA), latex, or an enzyme (EIA, ELISA,

CEDIA). Whether or not a separation step is necessary classifies an immunoassay as being

heterogeneous or homogenous. In a heterogeneous immunoassay, the bound and unbound

ligands are physically separated prior to detect, whereas with a homogenous immunoassay the

bound and unbound ligands are not separated prior to detection. The separation in a

heterogeneous assay can be achieved after absorbing or binding one of the assay components to a

solid phase. Furthermore an assay can be one-step (ligand and detection molecule are incubated

together) or two-step (ligand is first applied and unbound ligand is washed away prior to addition

of the detection molecule). Based on the interaction of the antibody and antigen, the

immunoassay can be competitive (labeled and unlabeled ligand compete for limited binding) or

non-competitive (excess antibody is present to bind to all antigen). In a competitive

immunoassay the signal is inversely related to the concentration of antigen, while in a non-

competitive immunoassay, the signal is proportional to the concentration of antigen.5

5

1.5 Immobilization Strategies

The selectivity of an immunosensor is provided by the biological recognition system (either

antibody or antigen) immobilized on the sensing surface. To obtain a highly sensitive sensing

surface, it is necessary to orient the recognition molecule in a manner where the corresponding

ligand is able to bind without any steric restriction.6–8

Several groups have studied various protein attachment methods for the development of

immunosensors including physiochemical adsorption, Langmuir-Blodgett methods, and covalent

attachment using glutaraldehyde, carbodiimide, succinimide ester, malein, and periodate, in

addition to using protein linkers such as streptavidin/avidin-biotin and Protein A and G.9–11

In the past, several immunosensors have relied on adsorption to immobilize antibodies/antigens

onto a surface via weak Van der Waals forces.12 Adsorbed surfaces in solution are more

susceptible to instability as desorption may occur due to the reversible nature of non-covalent

attachment. Furthermore, as proteins adsorb it can lead to protein inactivity through modification

or reduction of essential binding sites.10

Covalent binding of proteins to surfaces has been investigated as an alternative to adsorption to

increase stability, prevent aggregation, and control of protein binding site availability. One major

Figure 3 | (a) Homogenous competitive immunoassay, (b) heterogeneous non-competitive immunoassay, (c) a heterogeneous

competitive immunoassay, and (d) heterogeneous competitive immunoassay.6

6

drawback to covalent attachment is that these methods can lead to a loss of protein activity due

to chemical modification of critical residues, environment-induced denaturation, and random

protein orientation which can block the active site.13 These have detrimental effects on the

sensitivity and reproducibility of immunosensors.14

Construction of an ideal biosensor surface and immobilization of recognition elements present

many challenges. Proteins have multiple functional groups, which are most convenient for

attachment to sensor surfaces - the two dominant functional groups being amino and carboxyl

groups. Furthermore, surface-bound proteins can lose their native conformation and thus

biological activity due to multiple interactions of different side chains of the proteins with both

hydrophilic and hydrophobic structures on the sensor surface.15,16

1.6 Non-Specific Adsorption (NSA)

Reduction of nonspecific adsorption (NSA), or distinguishing between specific and nonspecific

adsorption, is an important topic in the development of immunosensors. In sensor applications,

the target molecule should bind to the surface by specific interactions in order to obtain selective

detection. Nonspecific interactions cause adsorption of unwanted molecules that induce an

interfering signal, which is an important origin of background signal (noise).

The NSA of proteins is a complex event that often leads to failure or loss of activity of a

biosensor. The process of NSA is governed by: the nature of the protein (structure, size, and

distribution of charge and polarity); the properties of the biomaterial surface (charge,

hydrophobicity, and state of surface energy); environmental conditions (pH, ionic strength, and

temperature); and the kinetics of the adsorption process (transport to the surface via diffusion or

convection, adsorption reaction, structural changes).15,17

A wide variety of approaches have been developed to render sensor surfaces protein repellent

and reduce the effects of NSA. Whitesides et al. (1991) have determined four molecular level

characteristics a functional group or polymer must possess to inhibit protein adsorption:

a) They should be hydrophilic

b) They should contain hydrogen bond acceptors

c) They should not contain hydrogen bond donors

d) Their overall electrical charge should be neutral

7

A number of non-fouling coating materials have been explored to decrease NSA. The two major

classes of blocking reagents are: 1) proteins (ex. bovine serum albumin, non-fat dry milk or

casein, whole normal serum, gelatin), and 2) detergents (ex. Tween-20, Triton X-100). Ideal

blocking reagents should inhibit NSA, inhibit non-specific protein-protein interactions, exhibit

no cross-reactivity with assay components, not disrupt the bonds that immobilize the specific

protein/biomolecule to the surface, and exhibit consistent, reproducible performance with every

lot. Biomimetic materials including phosphorylcholine (PC), oligo/polysaccharides

(dextran/heparin), and synthetic polymers such as oligo/poly(ethylene glycol) (OEG/PEG),

acrylates (PHEMA, PMEA), polyurethanes, and other hydrophilic synthetic polymers and

hydrogels have also been developed.18,19

1.7 Immunosensors

Immunosensors are compact analytical devices that are capable of detecting the formation of

antigen-antibody complexes and converting the response (ex. change in refractive index,

thickness, dielectric constant, mass, electrochemical, optical) to an electrical signal, which can be

processed, recorded and displayed. Transduction has been completed using optical (surface

plasmon resonance), piezoelectrical (quartz crystal microbalance), surface scanning (atomic

force microscopy), scanning electron microscopy, and other electrochemical techniques. Of these

techniques, electrochemical readout offers several potential advantages including increased

sensitivity, low cost, miniaturization, small volumes, and lack of interferences caused by turbid

or colored samples.20–25 Furthermore, electrochemical detection of antibody-antigen interactions

can be performed with and without labeling. Detection can be performed by cyclic voltammetry,

voltammetry/amperometry, and impedimetry. These methods are able to detect a change in the

capacitance and/or resistance of the electrode induced by binding of a target. Voltammetric and

amperometric techniques are among the most sensitive and widely applicable of all

electroanalytical methods.

1.8 Voltammetry/Amperometry

In voltammetric immunosensing, the analyte-concentration-dependent current is measured by

applying a constant and/or varying potential to the working electrode. Voltammetric techniques

involve the application of a potential (E) to an electrode and the monitoring of the resulting

current (i) flowing through the electrochemical cell. The applied potential can be varied and the

8

current can be monitored over a period of time (t). The applied potential forces a change in the

concentration of an electroactive species at the electrode surface by electrochemically reducing

or oxidizing it. The electrochemical cell consists of a working electrode, a reference electrode,

and a counter/auxiliary electrode. The reduction or oxidation of a substance occurs at the surface

of a working electrode, which leads to the mass transport of new material to the electrode surface

and the generation of current.26

As described by the Nernst equation, the applied potential at the working electrode controls the

concentrations of the redox species at the electrode surface (CO0 and CR0) and the rate of the

reaction (k0):

For a reversible electrochemical reaction (i.e.

equilibrium is always reestablished), the application of

a potential E forces the concentrations of CO0 and CR0

to a ratio in compliance with the Nernst equation,

where R is the molar gas constant (8.3144 J mol-1K-1),

T is the absolute temperature (K), n is the number of

electrons transferred, F = Faraday constant (96,485

C/equiv), and E0 is the standard reduction potential for

the redox couple. If the potential is made more

negative, the ratio become larger as CO0 is reduced,

and if the potential is made more positive, the ratio

becomes smaller as CR0 is oxidized. The current

produced is a quantitative measurement of how fast a

species is being reduced or oxidized at the electrode

surface. It is affect by many factors including the

concentration of the redox species, the size, shape and

material of the electrode, the solution resistance, the

cell volume, and the number of electrons transferred.26

To increase speed and sensitivity, many forms of

Figure 4 | Voltage applied during a typical DPV scan.27

Figure 5 | Peak current response due to applied voltage shown in Figure 4 for

DPV. 27

9

potential modulation have been tried over the years including normal pulse (NPV), differential

pulse (DPV), and square-wave (SWV). The method most commonly used in sensors is

differential pulse voltammetry (DPV). This technique uses a series of potential pulses of

increasing small amplitude in which the current is measured just before the application of the

pulse and near the end of each pulse, allowing time for the charging (non-faradaic) current to

decay (Figure 4 and 5).

Voltammetry provides excellent sensitivities with a large linear concentration range for both

inorganic and organic species (10-12 to 10-1 M), use of many solvents and electrolytes, a wide

range of temperatures, rapid analysis times, simultaneous detection of several analytes, ability to

determine kinetic and mechanistic parameters and an ease in generating waveforms and

measuring small currents.

1.9 SU-8 Negative Epoxy-Based Polymer

Microsystems for bioanalytical applications require immobilization of biomolecules on the

sensor element or the surface of the microsystem to allow for sensing. Immobilization has been

traditionally completed using organosilane functionalization of silicon dioxide and silicon nitride

surfaces or thiol derivitization on gold surfaces.27–29 Typically, covalent immobilization of

biomolecules on polymer surfaces requires modification of the surface to have at least one

functional group such as CHO, NH2, SH, COOH, which binds to biological molecules. Grafting

of amine groups (NH2) has also been achieved using wet chemical surface modification,30

however the use of strong oxidizing/hydrolyzing agents can potentially cause damage to the

surfaces adjacent to the sensor. Most of these processes also require multiple steps, in addition to

careful control of pH, concentration, temperature, which add to the complexity and are thus time

consuming. Dry surface modification techniques which graft hydroxyl or amine groups on the

polymer surface using oxygen or ammonia plasma treatment respectively,

can also allow for the immobilization of biomolecules.31 As various new

polymers such as SU-8 emerge, easier methods to immobilize

biomolecules on these polymers have assumed importance.

SU-8 is a negative epoxy-based polymer developed at IBM research often

used for the production of high aspect ratio structures.32 SU-8 photoresist Figure 6 | Epoxy group (or oxirane) found on the SU-8 surface.

10

has excellent mechanical properties, thermal stability, etching resistance and is chemically stable

against several acids and bases.33 SU-8 is also highly transparent under near UV and visible

light, making it useful as an optical waveguide.34 SU-8 has great potential for the fabrication of

microelectromechanical systems (MEMS) based sensors and microfluidic devices due to its

unique high-aspect-ratio (20:1). Therefore, it is of great interest to find new methods for SU-8

surface functionalization especially for bioanalytical applications.

Studies have shown that single stranded DNA can be passively absorbed onto a cured SU-8

surface. Probe densities of about 10 fmol/mm2 can be obtained using this simple immobilization

method.35 It is hypothesized that the exposed epoxy groups can be involved in creating the link

of SU-8 to DNA. Recently, Wang et al. were able to control protein attachment and cell growth

on patterned SU-8 surfaces using a surface modified with poly(acrylic) acid and other water-

soluble monomers.36 It is assumed that SU-8, being an epoxy-based polymer, may offer reactive

epoxy groups (also known as oxiranes) on its surface (Figure 6) that can also allow proteins to

bind to SU-8 surface. Amine-epoxy addition reaction is a common strategy for curing epoxy

resins and thus immobilizing probes onto an epoxy-modified substrate (Figure 7). Primary

amines undergo an addition reaction with the epoxy group to form a hydroxyl group and a

secondary amine. The secondary amine can further react with an epoxy group. Kinetic studies

have shown that the reactivity of the primary amine is approximately double that of the

secondary amine.37

Thiols (also known as mercaptans) found on the surface of proteins can also react with epoxy

groups via a simple nucleophilic ring-opening (SN2 ring opening) reaction (Figure 8).38

Figure 7 | The reaction between the oxirane group of the epoxy resin and a primary amine (A) or a secondary amine (B).

11

The above properties of SU-8 epoxy groups can allow for functionalization of the surface with

proteins. Blagoi et al. (2008) reported a functional fluorescent sandwich immunoassay detecting

C-reactive protein with a detection limit of 30ng/mL on bare SU-8 using passive absorption of

proteins onto the surface.32 No electrochemical immunoassays have been reported using SU-8

functionalization.

1.10 HIV Testing

An arsenal of laboratory methods is available to screen blood, diagnose infection, and monitor

disease progression in individuals infected by HIV. These techniques identify HIV infection

either by the detection of HIV-specific antibodies in serum or plasma, identification of the p24

antigen, detection of viral nucleic acids, or by growing virus in cell culture. Antibody testing,

typically using enzyme immunoassays (EIAs), is the most commonly used method to diagnose

HIV infection. Seroconversion can be detected in most individuals as early as two to three weeks

after infection using current EIAs.39

The high cost of anti-HIV EIA kits becomes prohibitive for routine use in many developing

countries, precluding early detection and prevention of new infections.40,41 Several EIA-based

diagnostic kits use synthetic peptides and/or recombinant proteins mainly from the envelope gp

of HIV-1 group M, HIV-1 group O, and HIV-2. The genes of HIV are located in the central

region of the proviral DNA and encode at least nine proteins. These proteins are divided into

three classes: the major structural proteins (Gag, Pol, and Env), the regulatory proteins (Tat and

Rev) and the accessory proteins (Vpu, Vpr, Vif, and Nef).42 The envelope glycoproteins (gp),

gp41 of HIV-1 and gp36 of the closely related HIV-2, are highly immunogenic and important

diagnostic intermediates for the detection of antibodies to these viruses in human sera.43

Figure 8 | The reaction between the oxirane group of the epoxy resin and a

thiol moiety.

12

A 35 amino acid peptide from the gp41 immunodominant region of HIV-1 group M was

synthesized with the following sequence:

CCSGGGSGGGLAVERYLKDQQLLGIWGCSGKLICT. This region shows 100% positivity

with patient sera.44 A 28 amino acid peptide from the gp36 immunodominant region of HIV-2

was synthesized with the following sequence: CCSGGGSGGGQDQARLNSWGCAFRQVCH.

1.11 Enzyme Labels

Different enzymes (ex. horseradish peroxidase, alkaline phosphatase, glucose oxidase, urease,

catalase, laccase, galactosidase, and acetyl cholinesterase) are often employed as labels in

ELISA. Of these, alkaline phosphatase, horseradish peroxidase, and glucose oxidase are the most

common due to the ease in which they can be found from commercial sources. While with

optical ELISA the enzyme label should catalyze the production of a coloured species, in an

electrochemical ELISA, the enzymatic product should be electroactive so that it can be easily

measured through voltammetric or amperometric techniques.45

For optical ELISAs, horseradish peroxidase is the preferred choice due to better sensitivity.

However with electrochemical immunosensors, alkaline phosphatase is often the preferred

choice. Alkaline phosphatase (E.C.3.1.3.1) (ALP) is a homodimeric 160kDa hydrolase that

converts orthophosphoric monoesters into alcohols with an optimum activity around pH 8-10

(see Table 2 for common electrochemical substrates for ALP).46 Kulys et al. have reported p-

aminophenyl phosphate (pAPP) as a suitable substrate for alkaline phosphatase.47 The product of

the enzymatic reaction, p-aminophenol, has favourable electrochemical properties for application

in biosensors. It can be detected at low redox potentials that are well separated from the redox

potential of the parent compound.48,49

13

Table 2. Substrates for Alkaline Phosphatase.

Substrate Eox (mV)a Km (mM) Kcat (s-1) Productb Eo

(mV)a

t1/2 (h)

4-APP

(4-aminophenyl

phosphate)

480 (pH 9.0) 0.23 2270 4-AP -25 0.5

4-ANP

(4-amino-1-

naphthylphosphate)

300 (pH 9.0) 2.5 1050 4-AN -200 1.0

4-HNP

(4-hydroxynaphthyl

phosphate)

235 (pH 9.0) NDb ND DHN -309 >4

a Vs. Ag/AgCl reference electrode in 0.1M Tris. b Not determined. Adapted from (Masson et al. 2004).50

1.12 Objective and Rationale

The objective of this thesis is to develop a new platform and assay for the detection of HIV-1/2

antibodies in serum using synthesized peptides from the immunodominant region of gp41 or

gp36. Using the scheme found in Figure 1 of the manuscript, we want to detect concentrations as

low as 1 ng mL-1 of antibody, which is clinically relevant.

14

Chapter 2

2 Accepted Manuscript in Analytical Chemistry Reproduced with permission from Bhimji, A.; Zaragoza, A. A.; Live, L. S.; Kelley, S.O.

Analytical Chemistry, 2013, in press. Copyright 2013 American Chemical Society.

2.1 Author Contributions

A.B. and A.Z. designed the study; A.Z. designed the peptides; A.B. performed all experiments;

A.Z. performed clinical sample experiments; A.B. and L.S.L. collected and analyzed data, and

created the figures; A.B. prepared the manuscript; S.O.K. edited the manuscript; L.S.L. and

S.O.K. gave technical support and conceptual advice.

15

16

17

18

19

20

21

22

23

24

25

26

27

28

Chapter 3

3 Discussion and Conclusions Biosensor technology is aimed at developing simple and economical devices that can be used

directly at point-of-care. In these devices, the biochemical reaction is performed in a confined

space on the device and directly linked to the transducer that will convert the biochemical signal

to an electronic signal. Electrochemical transducers offer the advantage of low detection limits,

fast response, simple design, and ease of miniaturization. Yet, electrochemical reactions are not

often used for immunoassays due to a limited number of suitable labeling molecules and

substrates that can be electrochemically activated. Kulys et al. have shown however, that p-

aminophenyl phosphate is a suitable substrate when alkaline phosphatase is used as a labeling

enzyme. The product of the enzymatic reaction, p-aminophenol, has favourable electrochemical

properties for application in biosensors including low redox potential. Numerous studies have

used this substrate for immunoassay applications.

This study aimed to develop an electrochemical immunosensor for the detection of HIV-1/2

antibodies. Based on the results provided, electrochemical detection based on the surface

functionalization of SU-8 and oxidation current of p-aminophenol is feasible and practical.

Because of the heterogeneous nature of the assay, there is no interference by electroactive

substances or electrode fouling. The high sensitivity of the electrochemical detection provides a

fast procedure and allows for determination of analyte in the ng mL-1 range. Differentiation

between HIV-1 and HIV-2 infection is also possible in clinical samples, indicating that this assay

exhibits the needed specificity to be used with clinical samples.

3.1 Future Directions

Multiplex analysis is intended to simultaneously look for multiple targets in one sample. This

approach has been largely adopted in genomics and is expanding to other areas of laboratory

investigation, including proteomics. In protein analysis, multiplexing and miniaturization of

immunoassays is of great applicability to both basic and applied research. In this present assay

design, the lack of multiplexing is a limitation, as often patterns of several biomarkers have

better predictive value compared to detection of a single analyte. Furthermore, incorporation of

29

multiplexing would also provide a cost-effective solution, for example HIV and Hepatitis C

Virus (HCV) co-infection detection. Individuals with HIV infections are often affected by viral

hepatitis; about one-third are co-infected with HCV. Individuals with a co-infection are at

increased risk for serious, life-threatening complications including liver disease, liver failure, and

liver-related death.51 As such, anyone living with HIV should also be tested for HCV.

In addition, while reagent consumption has been minimized to only 20 µL, it is still a fair amount

of volume. It would be ideal to assay a single sample of a patient’s serum from a pinprick for

many different disease markers.

A potential solution to these drawbacks is the miniaturization of the immunoassay using

microfluidics. Digital microfluidics (DMF) is a fluid-handling platform that enables the

manipulation of micro-volumes of liquid. The smaller dimensions of microfluidics allows for

faster diffusion times and less reagent consumption, resulting in faster analysis and lower cost

per assay. Furthermore, handing of reagents in microfluidics can be automated with simple, easy,

compact instrumentation. Fluids are electrostatically controlled as discrete droplets (picoliters to

microliters) on an array of insulated electrodes, making it easy to merge, mix, split and dispense

droplets, as well as control multiple droplets simultaneously. Analytical techniques coupled with

DMF enable higher sensitivities, multiplexing and process automation.52,53 Integration of the

HIV antibody detection assay onto the DMF platform has potential for automation and

multiplexing.

30

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