BIOCONJUGATION TECHNIQUES and EXPERIMENTAL PROCESSING of MYELOPEROXIDASE DETECTION SYSTEM by DANIEL NING-ENN WANG Submitted In

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BIOCONJUGATION TECHNIQUES AND EXPERIMENTAL PROCESSING

OF MYELOPEROXIDASE DETECTION SYSTEM

DANIEL NING-ENN WANG

Submitted in partial fulfillment of the requirements

for the degree of Master of Science

Thesis Advisor: Professor Chung-Chiun Liu

Department of Chemical and Biomolecular Engineering

CASE WESTERN RESERVE UNIVERSITY

May, 2020

CASE WESTERN RESERVE UNIVERSITY

SCHOOL OF GRADUATE STUDIES

We hereby approve the thesis/dissertation of

Daniel Ning-Enn Wang

candidate for the degree of Master of Science

Committee Chair

Chung-Chiun Liu

Committee Member

Julie Renner

Committee Member

Heidi Martin

Date of Defense

March 17, 2020

*We also certify that written approval has been obtained

for any proprietary material contained therein.

Table of Contents

page

List of Figures iv

Abstract vi

1. Introduction 1

1.1. Background on MPO 1 1.2. Introduction to Biosensor System for Detection 2

1.3. Scope of Work 5

2. Methods and Materials 6 2.1. Biosensor Prototype Fabrication 6

2.2. Bioconjugation 7

2.2.1. Traut’s Reagent 10 2.2.2. SATA Reagent 10

2.2.3. BDT Reagent 11 2.3. Sensor Preparation 11

2.4. Electrochemical Detection 13

2.4.1. Differential Pulse Voltammetry 13

2.4.2. Electrochemical Impedance Spectroscopy 13

3. Results and Discussion 14 3.1. Sensor Surface Treatment 14

3.2. Antibody Bioconjugation 15

3.3. MPO Detection 20 4. Conclusions and Recommendations 23

References 25

iii

List of Figures

page

2. Introduction Figure 1. Illustration of HOCl formation through MPO enzyme 1 Figure 2. Schematic of several pathways in cardiovascular disease that MPO affects 2 3. Methods and Materials Figure 3. Structure and dimensions of the biosensor prototype 7 Figure 4. Reaction of Traut’s reagent with amine containing molecule 9 Figure 5. Reaction of amine-containing molecule with SATA reagent 10 4. Results and Discussion Figure 6. DPV measurements of a group of five sensors demonstrating the success of the cleaning procedure of the sensor 16 Figure 7. DPV measurements of bioconjugated gold electrode using Traut’s Reagent for antibody immobilization over a concentration range of 0.15 µg/ml and 15 µg/ml 17 Figure 8. Calibration curve of biosensor using Traut’s Reagent for the bioconjugation of MPO antibody over a concentration range of 0.15 µg/ml and 15 µg/ml 18 Figure 9. DPV measurements of bioconjugated gold electrode using SATA for the bioconjugation of MPO antibody over MPO antibody concentration range of 0.115 µg/ml and 11.5 µg/ml 19 Figure 10. Calibration curve of biosensor using SATA for the bioconjugation of MPO antibody over MPO antibody concentration range of 0.115 µg/ml and 11.5 µg/ml 20 Figure 11. DPV measurements of MPO antibody immobilized to the electrode using BDT 21 Figure 12. Electrochemical Impedance Spectroscopy of MPO antibody conjugated using SATA reagent onto electrode surface compared to blank sensor 22 Figure 13. DPV measurements of MPO on sensor conjugated with MPO antibody over an MPO concentration range of 0.133 µg/ml to 4 ug/ml at an MPO antibody concentration of 7.67 ug/ml 23 Figure 14. DPV measurements of MPO on sensor conjugated with MPO antibody over an MPO concentration range of 0.08 ng/ml to 0.8 ug/ml at an MPO antibody concentration of 18.75 ug/ml 24

Acknowledgements

I am grateful for the support of my advisor, Professor Chung-Chiun Liu, whose guidance and insights kept me optimistic throughout the progression of this work, despite the many challenges encountered along the way. I would like to thank Professors Heidi Martin and Julie

Renner for serving on my committee, and for their recommendations to clarify this document.

I would like to thank Xiaowei Wu and Yifan Dai for their patience and for providing both mentorship and their technical expertise. I would also like to thank Derek Li, for donating many hours of his time during his summer vacation to preparing samples and testing various one-off measurements that would come up.

Finally, I am grateful for the support I received from my parents and Michelle Chang when

I decided to leave a perfectly stable job in pursuit of higher education. I am certain this decision is worthwhile and sincerely hope that the value of my decision will become apparent in the near future.

Bioconjugation Techniques and Experimental Processing

of Myeloperoxidase Detection System

Abstract

DANIEL N. WANG

Cardiovascular disease (CVD) is the leading cause of death in the United States since the mid-20th century and has many well-established biomarkers such as C-reactive protein and

N-terminal pro-8 type peptide. Recent studies suggest that detection of a specific enzyme, myeloperoxidase (MPO) can be used for improved risk stratification in CVD, independent of other more established biomarkers. Myeloperoxidase is an enzyme produced by leukocytes, and functions as a catalyst for the creation of reactive oxidants and radical species. The pathways utilizing MPO have been determined to be an important process in phagocytosis. However, these same pathways are identified as potentially proatherogenic biological activities at various stages of CVD development.

Measurement of MPO appears to be a valuable tool in the assessment of early stages of

CVD, and this study investigated the viability of an electrochemical sensor system to detect MPO.

Specifically, this system used a single-use electrochemical sensor prototype, with a bio-recognition mechanism using MPO antibody. The fabrication and preparation of the sensor system explored two separate bioconjugation techniques, 2-iminothiolane (Traut’s reagent) and N-succinimidyl

S-acetylthioacetate (SATA), to immobilize the MPO antibody to the gold working electrode.

Bioconjugation procedures for the immobilization of the MPO antibody were established, and the results of performance of these bioconjugated MPO antibody sensors were presented and discussed. Electrochemical impedance spectroscopy over a frequency range of 0.01 Hz –

10,000 Hz for a bioconjugated MPO antibody electrode was carried out to assess the surface coverage of the electrode element by the antibody. Preliminary measurements of MPO enzyme using this bioconjugated antibody-sensor appeared to be feasible, and a lower detection limit at a concentration of 0.008 µg/ml MPO was observed in this study.

vii 1. Introduction

1.1. Background on MPO

Myeloperoxidase (MPO) is a leukocyte-derived enzyme. MPO catalyzes the formation a number of reactive oxidants and radical species1. The potent oxidants formed are capable of chlorinating and nitrating phenolic compounds1-4. The chlorinating species include hypochlorous acid (HOCl)5, which processes potent bacterial and viricidal activities6,7. MPO is an essential enzyme to anti-microbial activity and phagocytosis, as the innate ability of the leukocytes to neutralize microbes is slowed significantly in MPO-deficient subjects8. The pathway for the generation of the anti-microbial agent HOCl mediated by MPO is shown in Figure 1.

Figure 1. Illustration of HOCl formation through MPO enzyme (from Ref. [8]).

However, the reactive species HOCl interacts with electron-rich moieties of a large range of biomolecules2. Thus, MPO, its reactive oxidants, and its chlorinating and nitrating processes have been implicated in tissue injury during inflammatory condition1-3, 9-11. Consequently, MPO and its oxidative pathway have been attributed to potentially proatherogenic biological activities throughout various stages of cardiovascular disease (CVD), including the initiation, propagation,

1 2 and acute complication phases of the atherosclerotic process, examples of which are shown in

Figure 2.12,13. At early stages, MPO can oxidize low density lipoprotein (LDL), which promotes the accumulation of cholesterol on arterial walls, and in later stages, can target and oxidize high density lipoproteins (HDL), shutting down the beneficial antioxidative and anti-inflammatory pathways of HDL13. Furthermore, the release of MPO both reduces the bioavailability of nitric oxide, resulting in endothelial dysfunction, and destabilizes atherosclerotic plaques, contributing to an increased risk of cardiovascular disease13.

Figure 2. Schematic of several pathways in cardiovascular disease that MPO affects (from Ref. [13]).

Cardiovascular disease (CVD) is known as the leading cause of death in the United States.

On a global scale, deaths from CVD have increased by 41% between 1990 and 201314,15. These statistical estimates underscore its severity and the importance of combating CVD in order to improve the quality of health care provided. Examination of CVD suggests that a sequence of events occur leading to CVD, including endothelial dysfunction, atherosclerotic plaque formation,

3 and rupture. Furthermore, a key event in CVD development is based on the inflammation and oxidative stress through the stages of the events. The MPO enzyme is linked to both inflammation and oxidative stress and thus further implicated in each event leading to CVD. Consequently, the detection and monitoring of MPO are very desirable as a meaningful biomarker in negating CVD, especially at the early and propagating stages of the CVD development. In recent years, measurement of MPO in plasma has been associated with improved CVD risk stratification.

Higher concentrations of MPO are associated with increased CVD risk, independent of classical

CVD risk factor markers such as C-reactive protein (CRP), N-terminal pro-8 type peptide and others16-28. Therefore, detection and quantification of MPO provide a valuable assessment of

CVD, especially for early stages of CVD development.

However, it is recognized that MPO concentration depends on the assay method, sampling material, pre-analytical and analytical procedures. Several studies by independent groups showed that higher risk of major adverse events was more likely above MPO levels ranging from 0.257 nM to 0.557 nM29. In this research, our overall goal is to develop a technique for MPO detection and quantification in an aqueous test medium as a base for further advancement for MPO detection.

1.2. Introduction to Biosensor System for Detection

Biosensors are recognized as an effective and practical device for the detection and monitoring of biochemical species and biomarkers of disease. Fundamentally, a biosensor contains two major mechanisms: a bio-recognition mechanism and a transduction mechanism30.

The bio-recognition mechanism often employs a bio-receptor, derived from the molecules of a biological system to interact with the specific analyte. This bio-receptor can be an enzyme, antibody, microorganism, nucleic acid, among others, which interact, bind, or recognize the

4 specific analyte of interest. The transduction mechanism translates the result from the bio- recognition mechanism into a practical and readable signal. This signal can be fluorescent color change, electrical potential, current, impedance, or other various forms of signal output. Therefore, optical and electrochemical sensors are commonly used devices to achieve the transduction mechanism of a biosensor.

Optical systems measure an optical signal through fluorescent, colorimetric, or luminescent methods, enabling rapid, sensitive, and direct detection of analyte of interest. Electrochemical systems are also ideal for the development of point-of-care devices due to their simplicity, portability, rapidness, cost-effectiveness and sensitivity31. Detection of target biomarkers directly using human sera samples has been achieved using an electrochemical detection method32,33.

Therefore, we chose to select an electrochemical-based biosensor for the detection and quantification of MPO, a biomarker of CVD, in this study. Specifically, a three-electrode configuration sensor prototype with thin gold-film working and counter electrodes and Ag/AgCl reference electrode is chosen. More details of this sensor prototype will be given later.

Interaction between the antibody and the antigen is the result of prior expression of the corresponding disease-related genome. Therefore, nature provides a high-affinity binding between the antibody and the antigen used as the recognition element for the antigen detection, simplifying the efforts for specific design of the recognition elements for biosensing systems.

Myeloperoxidase (MPO) is one of the major autoantigens recognized by anti-neutrophil cytoplasm antibodies34. The primary function of the MPO antigen is to destroy phagocytosed microorganisms by generating highly reactive oxidative species within the phagosome. Therefore, in this research we will target detection and quantification of MPO using an MPO antibody to provide the bio-recognition mechanism.

Currently, the conventional methods for detection of such biomarkers including MPO, such as ELISA and Western Blot, require exhaustive processing steps and multiple reagents, which are expensive, difficult to operate, and time-consuming35. These methods are not ideal for the robust demands of a rapid, simple, cost-effective point-of-care system. In order to overcome this concern, a self-assembled monolayer (SAM) is a promising platform technology for biosensor applications36-40. Typically, SAM is formed by an alkane-linked thiol molecule producing a gold−sulfur (Au−S) bond with the gold electrode surface of a biosensor. Subsequent activation of the terminal functional group allows for immobilization of the antigen binding molecule, such as antibody, aptamer, or other specific receptors. The formation of the gold working and counter electrode elements of the biosensor can be accomplished by various techniques and in different dimensions. Thus, the general procedures for preparing a commonly used biosensor are complex and require both days of preparation and consumption of excess chemicals.

Several challenges, however, do exist in the use of biosensors produced with SAMs, including relatively low sensitivity and poor reproducibility. Monolayer defects such as pinholes, inhomogeneity of surface coverage, and others are quite common and lead to poor reproducibility.

This research explored an alternative technique for the preparation of a biosensor, through bioconjugation. The bioconjugation mechanism conjugates two or more molecules, forming a novel complex that incorporates the combined properties of its individual components41. This method makes a zero-length linkage between the protein and electrode elements of the biosensor possible. Bioconjugation techniques will also shorten the preparation process and enhance the coverage of the biosensor surface by minimizing the pinhole effect, consequently improving the practical clinical application. The interaction between antibody and antigen remained to be the bio-recognition mechanism in this research endeavor.

1.3. Scope of Work

The objectives of this research focus on three specific aims:

(1) Exploring the effectiveness of different bio-conjugation techniques to immobilize the

MPO antibody on to the gold electrode surface of the biosensor.

(2) Assessing experimentally the effectiveness of using the interaction of the

bioconjugated MPO antibody for the detection of MPO.

(3) Establishing an experimental testing procedure using an aqueous test medium

providing a test procedure for further MPO detection research.

The results of these specific aims will lead to the overall goal of this research, namely detecting and quantification of MPO as a biomarker for the minimization of CVD risk, including at the early and propagating stages of the disease.

2. Methods and Materials

2.1. Biosensor Prototype Fabrication

A single-use biosensor prototype was fabricated using both thick- and thin-film manufacturing processes. This biosensor prototype was a three-electrode configuration electrochemical-based system, containing thin gold film working and counter electrodes and ta thick-film printed silver/silver chloride (Ag/AgCl) reference electrode. Sputtering physical vapor deposition was used to deposit the thin gold film of the working and counter electrodes with a thickness of 10 nm on a polyethylene terephthalate (PET) substrate of 355 x 280 mm2. Laser ablation was employed to create four rows of 25 electrodes that were reproducible and uniform.

Thick-film printing was used to create the Ag/AgCl reference electrode with DuPont #5870

Ag/AgCl ink. Thick-film printing was also used for the insulation layer using Nazdar APL 34 silicone-free dielectric ink, creating a total sensing area of 1.54 mm2 with a diameter of 1.4 mm for each biosensor. Thick film printed silver electrical connections were also printed for all electrodes. A more detailed description of the fabrication process has been provided in other previous publications42,43. Figure 3 shows the details and actual dimensions of this biosensor prototype. This biosensor prototype can be manufactured on an industrial scale using a cost- effective process with excellent reproducibility.

Figure 3. Structure and dimensions of the biosensor prototype (from Ref. [43]).

7 8

2.2. Bioconjugation

The immobilization of the antibody to the gold film working electrode surface is an important step in the biosensor development in this research. As mentioned above, self-assembled monolayer (SAM) techniques for bonding the antibody to the gold film electrode are complex and time-consuming. Furthermore, the biosensors prepared using SAMs had relatively low sensitivity and poor reproducibility, because of common monolayer defects. Alternative immobilization techniques of binding the antibody to the gold electrode are explored and used in this study.

Bioconjugation is a process by which antibodies or proteins are immobilized to the surface of an electrochemical sensor. In this research, specifically, two separate bioconjugation techniques and BDT with gold nanoparticles were used and assessed. It is well established that thiol groups have a high affinity for gold. For our single-use sensor prototype, the primary binding mechanism of the antibody to the gold electrode surface is through thiolation of the antibody. Hence, the first bioconjugation technique employed in this research was using Traut’s reagent. Traut’s reagent, or

2-iminothiolane, and its mechanism of action is illustrated in Figure 4. The cyclic structure forms an intermediate at the amine location of the antibody and the ring opens to produce a thiol group.

Figure 4. Reaction of Traut’s reagent with amine-containing molecule (from Ref. [44]).

The second bioconjugation technique generally follows the bioconjugation processes detailed by Hermanson and Dai et al.44,45. This process employs the reagent N-succinimidyl

S-acetylthioacetate (SATA), a known protein modifier. The chemical reaction of this bioconjugation process, illustrated in Figure 5, indicates that the products generated are a modified protein with a protected sulfhydryl group and N-hydroxysuccinimide. The modified protein must then be deacetylated in order to expose the thiol group, which will allow the molecule to bind to the gold surface of the sensor.

Figure 5. Reaction of amine-containing molecule with SATA reagent (from Ref. [44]).

The third bioconjugation technique used employs a strategy using a combination of SATA conjugated antibody and 1,4 butanedithiol (BDT). Previously published research demonstrated that the use of BDT and gold nanoparticles allowed for significantly enhanced selective recognition of 3,4-dihydroxy-phenylalanine between its D- and L- enantiomer forms46. The specific aim of using this bioconjugation method is to determine not only the effectiveness of antibody binding but also the potential enhancement to the binding selectivity to the gold electrode surface using BDT. The linking of gold nanoparticles with a bi-functional monomer, such as BDT, is generally referred to as a miniemulsion process. Thus, the size of the nanoparticles and the quantity of the monomer used will directly affect the results of this linking. Furthermore, a base

11 catalyst as well as elevated temperature may employed to enhance the miniemulsion process. With limited knowledge of this particular miniemulsion process and time to assess this technical approach, we limit our evaluation of this process on a qualitative basis to provide guidance for further studies of this bioconjugation process.

2.2.1. Traut’s Reagent

One mg of 2-iminothiolane (26101, ThermoFisher Scientific) was dissolved in 13.3 ml of

0.1M PBS with 10 mM ethylenediaminetetraacetic acid (EDTA) and 150 mM NaCl. Thirty µl of stock MPO antibody (0.5 mg/ml, H00004353-M01, Abnova) was added to 4 µl of Traut’s reagent solution to create a 25x molar ratio between Traut’s reagent and the MPO antibody. Antibody was incubated with the reagent for 30 minutes at room temperature, vortexing every 10 minutes.

Contents were placed into a 0.5 ml centrifuge tube with a 3k filter (Amicon) and filled with 0.1M

PBS to a total volume of 500 ml. The sample was centrifuged at 10,000 rpm for 15 min at 5 °C.

The collected liquid that did not pass through the filter was collected and aliquots of 10 µl per vial were stored at -20°C until needed.

2.2.2. SATA Reagent

The second conjugation reagent used was N-succinimidyl S-acetylthioacetate (SATA,

26102, ThermoFisher Scientific). One mg of SATA was dissolved in 1 ml of DMSO, and 1.98 µl of this solution was added to 30 µl of stock antibody solution to create a 100x molar ratio of SATA to antibody. Antibody and SATA were incubated for 30 minutes at room temperature, vortexing every 10 minutes. The mixture was placed into a 0.5 ml centrifuge tube with a 3k filter and 0.1M

PBS was added to reach a total volume of 500 ml. The sample was centrifuged at 10,000 rpm for

15 min at 5 °C. Deacetylation solution of 0.5 M hydroxylamine and 25 mM EDTA was prepared

12 by adding a target of 6.95 mg hydroxylamine hydrochloride (255580-100G, Sigma-Aldrich) and

1.46 mg EDTA (EDS-100G, Sigma-Aldrich), respectively, to 200 µl 0.1M PBS. Remaining liquid that did not pass through the filter was placed in a new vial and 5 µl of the hydroxylamine/EDTA solution were added for deacetylation of the protected sulfhydryl group. The solution was incubated for 2 hours, vortexing every 10 minutes. Contents were moved back to a centrifuge tube with a 3k filter and 0.1 M PBS was added to reach a total volume of 500 ml. The sample was centrifuged again at 10,000 rpm for 15 minutes at 5 °C. The collected liquid that did not pass through the filter was collected and aliquots of 10 µl per vial were stored at -20 °C until needed.

2.2.3. BDT Reagent

Five µl of BDT solution (B85404-5G, Sigma-Aldrich) were diluted in ethanol to achieve a concentration of 2 mM. Electrodes were incubated with 10 µl of BDT solution for 2 hours.

Incubation of 5 µl of aliquoted SATA conjugated antibody was performed with 50 µl gold nanoparticles (50 nm diameter, OD 1, stabilized in 0.1 mM PBS, 753645-25ml, Sigma-Aldrich).

Gold nanoparticle linked antibody was then incubated on the BDT-electrode system for 30 minutes before rinsing with 0.1 M PBS and gently drying with nitrogen.

2.3. Sensor Preparation

The sensors were first cleaned by rinsing with deionized (DI) water, followed by ethanol, and again with DI water. Initial treatment of the biosensor surface was performed using solutions of KOH, H2SO4, and HNO3 to remove any particulates or oxidation that may have formed on the sensor surface. KOH pellets were dissolved in DI water to achieve concentration of 2.0 M, while

H2SO4 and HNO3 were diluted at a 1:20 volumetric ratio in DI water. One row of six sensors was most commonly used for each set of experiments, and the sensors were immersed in each treatment

solution for 10 minutes - first in KOH, followed by H2SO4, and finally HNO3. Sensors were rinsed with DI water between each immersion and dried with nitrogen after immersion in the HNO3 solution. Cleaning and surface preparation of the sensors were evaluated using differential pulse voltammetry (DPV).

Bioconjugation was performed for mouse monoclonal MPO antibody, using Traut’s reagent or SATA conjugating agents. Conjugated antibody solution was thawed and serially diluted to 5 concentrations of 0.15, 1.5, 3, 5, and 15 µg/ml for Traut’s reagent with 0.1M

PBS/10 mM EDTA/150 mM NaCl. Concentrations used for SATA conjugated antibody were

0.115, 1.15, 2.3, 3.83, and 11.5 µg/ml. SATA conjugated antibody was also used at a concentration of 9.677 µg/ml after incubation with gold nanoparticles for binding to BDT. Twenty ml of the corresponding conjugated antibody solution was incubated on the cleaned biosensors for

2 hours. Sensors were then rinsed with 0.1 M PBS and gently dried with nitrogen. Binding of the antibody to the biosensor surface was first evaluated using DPV and electrical impedance spectroscopy to ensure that MPO antibody had indeed bound to the surface of the sensor before proceeding to the next phase.

Antibody binding at concentrations of 7.67 and 18.75 µg/ml was performed on cleaned sensors, followed by incubation of MPO on the biosensor surface. MPO test solutions were prepared by serially diluting stock recombinant MPO protein (0.02 mg/ml, H00004353-Q01,

Abnova) with 0.1 M PBS to 5 concentrations of 0.133, 0.4, 0.8, 1.33, and 4 µg/ml, and 0.08 ng/ml,

0.8 ng/ml, 0.008 µg/ml, 0.08 µg/ml, and 0.8 µg/ml for antibody concentrations of 7.67 and

18.75 µg/ml, respectively. MPO was incubated on the biosensor surface for 1 hour and the sensors were rinsed with PBS and gently dried with nitrogen before characterization using DPV.

2.4. Electrochemical Detection

2.4.1. Differential Pulse Voltammetry

Electrochemical detection was performed using differential pulse voltammetry (DPV).

DPV allows for highly sensitive measurements of the current, as charging current effects are minimized. Redox solutions such as methylene blue, Prussian blue, and ferricyanide are all commonly used in electrochemical sensing, and we selected the redox solution of 5mM potassium hexacyanoferrate(III) (K3Fe(CN)6) and 5mM potassium hexacyanoferrate(II) (K4Fe(CN)6) to be used as the transduction mechanism. The solution was prepared by dissolving 0.069 g of

K3Fe(CN)6 (244023-100G, Sigma-Aldrich) and 0.0845 g K4Fe(CN)6 (P3289-100G, Sigma-

Aldrich) in 40 ml 0.1 M PBS. Sensors were placed in the cradle connected to an electrochemical workstation (CH Instruments). Twenty ml of the redox solution were added to the working area of the sensor. The scan was set up for potentials from -0.3 V to +0.5 V in increments of 0.004 V.

The potential amplitude was set at 0.05 V, pulse period at 0.2 s, and pulse width at 0.05 s. Data of the sweep was collected and plotted.

Oxidation and reduction processes occur simultaneously and cannot happen independently of one another. The oxidation and reduction are each defined as a half-reaction, and both half- reactions form the whole redox reaction. The oxidation is defined as an increase in oxidation state, and reduction as a decrease in oxidation state. In practice, the transfer of electrons will cause a change in oxidation state, but there are reactions that are classified as “redox” even though no electron transfer occurs, such as those involving covalent bonds. In redox processes, the reducing agent loses electrons and is oxidized, and the oxidizing agent gains electrons and is reduced. While the pair of oxidizing and reducing agents involved in a particular reaction is called a redox pair,

3- 4- the reducing species and its corresponding oxidizing form, e.g. Fe(CN)6 / Fe(CN)6 , are

15 commonly used redox couples in electrochemical sensing based on their own electron transfer. In this case, the redox coupling reaction is FeCNeFeCN34−−+→− and in our testing medium ( )66( ) ,

3- 4- Fe(CN)6 ions are more stable and the reduction yields Fe(CN)6 ions; however, as stated above, there is no electron transfer of this redox couple with the surrounding system. The current output is the direct result of the MPO antibodies and the corresponding MPO antigens.

2.4.2. Electrochemical Impedance Spectroscopy

Electrochemical impedance spectroscopy (EIS) determines the dielectric properties of materials. In this study, we used EIS for the assessment of the coverage of the gold working electrode surface by the conjugated antibody comparing to that of the bare gold electrode. Again, redox solution of 5mM K3Fe(CN)6/K4Fe(CN)6 in 0.1 M PBS was used as the transduction mechanism. Sensors were connected to the electrochemical workstation and 20 ml of redox solution were added to the working area of the sensor. The scan was performed with an amplitude of 0.01 V at a frequency range of 0.01-10,000 Hz.

3. Results and Discussion

3.1. Sensor Surface Treatment

Biosensor cleaning and preliminary surface treatment of the sensor surface at the electrodes were tested using DPV to assess that the solutions had indeed removed unwanted particulates from the surface of the sensor. As mentioned, the cleaning procedure involved the sequential use of

KOH, H2SO4, and HNO3 solutions, and any oversoaking in any of the solutions as well as contaminants in the solution would affect the DPV measurement of the cleaning procedure.

Therefore, this cleaning procedure must be handled carefully. Figure 6 shows the DPV measurements of 5 individually cleaned sensors with one sensor tested twice. The peak currents of the DPV measurements of these sensors occurred at +0.14 V versus the thick-film printed

Ag/AgCl reference electrode and appeared to be reproducible and consistent, indicating the cleaning procedure of theses electrodes was successful and the sensor was ready to be used for further study.

Figure 6. DPV measurements of a group of five sensors demonstrating the success of the cleaning procedure of the sensor.

16 17

3.2. Antibody Bioconjugation

The bioconjugation of the antibodies for potential MPO detection was assessed using DPV measurement. MPO antibodies were bioconjugated onto the gold electrode surface using different bioconjugating chemicals. Figure 7 shows the experimental results of the sensor system which employed Traut’s reagent for the bioconjugation of MPO antibody. This test was performed on one set of sensors at 5 different antibody concentration ranging from 0.15 µg/ml to 15 µg/ml.

Using the experimental data obtained from Figure 7, simple linear regression was performed, and the corresponding calibration curve is given in Figure 8. Linear regression of the data indicated a trend of decreasing current output with increasing MPO antibody concentration, with an r2 value of 0.9133 and a p-value of 0.01113, indicating that the linear regression can be used to fit the data and that we can reject the null hypothesis that the coefficient is equal to zero, respectively.

0.15 µg/ml

1.5 µg/ml

3 µg/ml

5 µg/ml 15 µg/ml

Figure 7. DPV measurements of bioconjugated gold electrode using Traut’s Reagent for antibody immobilization over a concentration range of 0.15 µg/ml and 15 µg/ml.

5 y = -0.1325x + 6.5512

R² = 0.9133 5 A) 5 - 4

3 Current (1e Current 2

0 0 2 4 6 8 10 12 14 16 Antibody Concentration (µg/ml)

Figure 8. Calibration curve of biosensor using Traut’s Reagent for the bioconjugation of MPO antibody over a concentration range of 0.15 µg/ml and 15 µg/ml.

Similarly, Figure 9 shows the experimental results from DPV measurement of the sensor system which used SATA for the bioconjugation of MPO antibody over an antibody concentration range of 0.115 µg/ml to 11.5 µg/ml. Figure 10 is the corresponding calibration curve based on mean values of the experimental data obtained from Figure 9 and two additional repeats for a total of 3 sets, with error bars indicating standard error. As shown, the trend of the DPV current output decreased as the MPO antibody concentration increased. An examination of the experimental results demonstrated that the SATA bioconjugation of the MPO antibody may have limitations at higher MPO antibody concentration, namely, above 6 µg/ml in our study. At the MPO antibody concentration of less than 6 µg/ml, a good linear trend of the current output as a function of the

MPO antibody concentration was observed. Further experimental evaluation of SATA

19 bioconjugation to the MPO antibody concentration over the range of 6.0 µg/ml to 11.5 µg/ml will be necessary.

As expected, both bioconjugated sensor systems demonstrated the trend that at higher MPO antibody concentration resulted in lower current output of the DPV measurement indicating the coverage of the antibody on the electrode was quantitatively more leading to a higher dielectric value. One interesting observation in this study was that the current output of the DPV measurement of the SATA conjugated sensor was higher than that of the cleaned bare gold electrode as shown in our experimental results in Figure 9. SATA is a short-chain (2.8 angstrom spacer arm) reagent for covalent modification of primary amines and the addition of a protected yet exposable sulfhydryl group, -SH group, may provide additional electronic charges resulting in higher current output.

0.115 µg/ml 1.15 µg/ml 2.3 µg/ml

3.83 µg/ml 11.5 µg/ml Blank Sensor

Figure 9. DPV measurements of bioconjugated gold electrode using SATA for the bioconjugation of MPO antibody over MPO antibody concentration range of 0.115 µg/ml and 11.5 µg/ml.

6.2

6.1

6 y = -0.0068x + 5.9572

R² = 0.1084 5 A) 5 - 5.9

5.8

Current (1e Current y = -0.0678x + 6.0578 R² = 0.9887 5.7

5.6

5.5 0 2 4 6 8 10 12 14 Antibody Concentration (µg/ml)

Figure 10. Calibration curve of biosensor using SATA for the bioconjugation of MPO antibody over MPO antibody concentration range of 0.115 µg/ml and 11.5 µg/ml.

Figure 11 shows the DPV measurements obtained using BDT and gold nanoparticle bound to bioconjugated antibody. As mentioned, the size of the gold nanoparticles affects the sensitivity of the sensing system. For this study, only one size of the gold nanoparticles was used. In the

DPV measurements, the peak currents decreased as expected at the MPO antibody concentration of 9.677 µg/ml. Multiple experimental runs at this concentration were carried out, and minute variations in the current outputs can be seen in Figure 11. It was very positive that the peak current of the DPV measurement of this gold nanoparticle and bioconjugated MPO antibody system showed a decrease of about 3.5 x 10-5 A compared to that of the blank electrode, suggesting a high sensitivity in this detection system. Additional characterization of the electrode system is needed to analyze the molecular system that has been bound the electrode system using BDT, and further refinement of the experimental process will be required.

Figure 11. DPV measurements of MPO antibody immobilized to the electrode using BDT.

Electrochemical impedance spectroscopy was performed on this sensing system with antibody bioconjugated with SATA. Two different frequency ranges were employed in this study.

The frequency range was initially applied from 0.1 Hz – 10,000 Hz. However, the experimental results were incomplete and inconclusive, and comprehensive assessment of the coverage was not possible. Therefore, a wider frequency range was employed in the EIS study, extending from

0.01 Hz – 10,000 Hz. Figure 12 shows the experimental results obtained from EIS measurements in this frequency range. The results showed that the imaginary impedance, the -Z’’ (y-axis) value, for the SATA conjugated MPO antibody was much higher, indicating that the combined resistance and capacitance of the SATA conjugated MPO antibody was larger than that of the blank gold electrode. This demonstrated that MPO antibody coverage on the gold electrode was successful,

22 even though the actual quantity of MPO coverage was not evaluated at different MPO antibody concentrations. The concentration of MPO antibody used in this study was 1.765 µg/ml.

Figure 12. Electrochemical Impedance Spectroscopy of MPO antibody conjugated using SATA reagent onto electrode surface compared to blank sensor.

3.3. MPO Detection

Preliminary measurements of MPO using the bioconjugated sensor system were carried out in this study. These results are shown in Figure 13 and Figure 14. Figure 13 is made up of one set of 5 different MPO concentrations ranging from 0.133 µg/ml to 4 µg/ml and indicated a decrease in current as the concentration of MPO increases. A lower, but significantly wider MPO concentration range of 0.08 ng/ml to 0.8 µg/ml was then assessed, provided in Figure 14. This test was run twice at these concentrations. In Figure 14, it was apparent that the peak current decreased noticeably between MPO concentrations of 0.008 µg/ml and 0.08 µg/ml; however, the sensitivity

23 of the measurements at the three lowest concentrations appeared to be limited. This observation suggested that the lower limitation of MPO that could be detected by this sensing system had been reached. Additionally, resolution in the DPV did not appear to be consistent at higher concentrations of MPO above 0.08 µg/ml. One explanation for this could be that MPO at this high concentration may have saturated the MPO antibody on the sensor. Higher MPO antibody concentration may need to be assessed to accommodate higher MPO concentrations.

Abconcentrationof 7.67 µg/ml [MPO] 0.133 µg/ml [MPO] 0.4 µg/ml [MPO] 0.8 µg/ml

[MPO] 1.33 µg/ml [MPO] 4 µg/ml Blank Sensor

Figure 13. DPV measurements of MPO on sensor conjugated with MPO antibody over an MPO concentration range of 0.133 µg/ml to 4 ug/ml at an MPO antibody concentration of 7.67 ug/ml.

Abconcentration of 18.75 µg/ml [MPO] 0.08 ng/ml

[MPO] 0.8 ng/ml [MPO] 0.008 µg/ml [MPO] 0.08 µg/ml [MPO] 0.8 µg/ml

Ab only 18.75 µg/ml

Abconcentration of 18.75 µg/ml [MPO] 0.08 ng/ml

[MPO] 0.8 ng/ml [MPO] 0.008 µg/ml [MPO] 0.08 µg/ml [MPO] 0.8 µg/ml

Ab only 18.75 µg/ml

Figure 14. DPV measurements of MPO on sensor conjugated with MPO antibody over an MPO concentration range of 0.08 ng/ml to 0.8 ug/ml at an MPO antibody concentration of 18.75 ug/ml.

4. Conclusions and Recommendations

This study focuses on investigating the fundamental approach of detecting myeloperoxidase (MPO), a leukocyte-derived enzyme considered to be an important biomarker of cardiovascular disease. Specifically, we aim to use MPO antibody as the base for the bio- recognition mechanism and electrochemical analytical technique as the transduction mechanism in this detection approach. A platform sensor prototype was designed, fabricated, and used, and the first phase of this study was to establish an experimental procedure to prepare the platform senor prototype for the immobilization of MPO antibody onto the gold based electrochemical sensor elements. Self-Assembled Monolayer (SAM) was the commonly used technique for the linking of biomolecules, in this case MPO antibody, to the metallic based electrode. However,

SAM techniques are elaborate and have time-consuming processes. Therefore, this study aimed to employ a simpler bioconjugation procedure to immobilize MPO antibody onto the electrode, replacing the SAM immobilization technique.

Two potential bioconjugation reagents, Traut’s reagent and N-succinimidyl

S-acetylthioacetate (SATA), were investigated in this bioconjugation study. MPO antibody concentration ranges of approximately 0.15 µg/ml to 15 µg/ml for Traut’s reagent and 0.115 µg/ml to 11.5 µg/ml for SATA were used, respectively. The calibration curve for each bioconjugation reagent showed the expected trend of decreased current output of the sensor as a result of higher

MPO antibody concentration, indicating the quantity of the MPO antibody was related to the coverage of the electrode surface area. Differential pulse voltammetry (DPV) was the primary electrochemical analytical technique used in this study. In addition, electrochemical impedance spectroscopy (EIS) was carried out with SATA bioconjugated sensor and blank gold electrode. As expected, the imaginary impedance for the SATA conjugated MPO antibody was much higher

25 26 than that of the bare gold sensor indicating that the combined resistance and capacitance of the

SATA conjugated MPO antibody was larger than that of a blank gold electrode. This result indicated the MPO antibody did produce coverage on the electrode element. At present, we are only able to use the initial concentration of the MPO antibody to describe the coverage of the electrode surface, and this initial concentration applied is the maximum actual MPO antibody coverage on the electrode. Actual coverage of the MPO antibody will require further and future study.

In limited preliminary experimental results, we observed that detection levels of MPO on this bioconjugated MPO antibody sensor is limited to 0.008 µg/ml, or 0.219 nM. While future studies will have to quantitatively evaluate more optimal ranges of MPO for which this MPO antibody sensor can be applied, our sensor system was already able to detect MPO at concentrations below the level indicated by other studies as increased risk, ranging from 0.257 to

0.557 nM, and several orders of magnitude lower than similar antibody and MPO systems. Further study is also needed in the characterization of the binding pattern of the antibody on the sensor surface and quantification of antibody binding for these bioconjugation techniques, which can be achieved through methods such as quartz crystal microbalance. Selectivity of this conjugated MPO antibody sensor system also needs to be investigated through detection of the target molecule MPO in other media such as human sera in order to be a viable point-of-care system. The effects of molecules such as H2O2 and other proteins could have on the redox couple response has not been extensively investigated, and these interference studies would allow for better understanding of the interactions of these molecules with the conjugated antibody sensor system and MPO. In summary, we established experimental processes for the immobilization of MPO antibody using bioconjugation techniques and assessed the detection of MPO using this system in an aqueous

27 medium. This fundamental study provided a base for future advancement on the development of an applicable MPO test system with a detection limit at the nanomolar level.

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