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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 by DANIEL NING-ENN WANG Submitted In BIOCONJUGATION TECHNIQUES AND EXPERIMENTAL PROCESSING OF MYELOPEROXIDASE DETECTION SYSTEM by 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 iv 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. v Bioconjugation Techniques and Experimental Processing of Myeloperoxidase Detection System Abstract by 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. vi 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.
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