Electrochemical Studies of Clinically Important Enzyme/substrate Systems

Bekhit, Michael Yemane
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The rapid and reliable determination of clinical biomarkers such as enzymes and their substrates play an important role in the diagnosis and monitoring of human diseases. In this work, the new electrochemical methods for the determination of four enzyme biomarkers in human serum, urine, saliva, and synovial (joint) fluid were designed, developed, and characterized. The selected biomarkers include myeloperoxidase (MPO, cardiovascular disease marker), leukocyte esterase (LE, infection), sorbitol dehydrogenase (SDH, drug-induced liver injury), and alkaline phosphatase (ALP, liver disease). The electrochemical methods have the advantage of simplicity, sensitivity, ability to analyze color/turbid samples, and the potential for miniaturization and easy portability. The classical example of electroanalysis is the measurement of blood glucose at a commercial glucose test strip to monitor diabetes by millions of people every day. The glucose test strips (a technology perfected for decades) are adapted here for the determination of enzymes by sensing non-glucose analytes. Chapter 1 of this Dissertation provides background information about the selected enzymes and experimental techniques that were used to study them. Chapter 2 presents new electrochemical assays and immunoassays that were developed for the quantification of picomolar levels of MPO. Chapter 3 introduces the concept of biosensing with MPO as a recognition element for electrochemical biosensors and describes the first amperometric biosensor for one of its substrates, the thiocyanate ion (SCN–, cystic fibrosis marker). In Chapter 4, the discovery of LE–triggered hydrolysis of methyl pyruvate is explored in the development of coupled-enzyme assays for LE. Chapter 5 describes the synthesis of new glucosyl esters (collaboration with Dr. McHardy's group) and their use for the determination of LE at a carbon nanotube electrode and commercial glucose test strips integrated with a laboratory potentiostat instead of a glucometer. Chapter 6 pioneers the use of glucose test strips for the determination of LE by sensing the hydroquinone discharged from a hydroquinone releasing LE substrate, which was recently synthesized by Dr. McHardy's group. Chapter 7 presents a method for the attomole detection of SDH at glucose test strips based on the NADH/fructose system. Chapter 8 demonstrates the use of glucose test strips for the determination of ALP by sensing the ALP-triggered release of hydroquinone from a commercially available hydroquinone diphosphate.
Chapter 9 shows how to quickly screen nine esters as prospective new substrates for LE. Chapter 10 provides the final conclusions and potential future directions for the continuation of this type of research.

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Biomarkers, Biosensors, Electroanalysis, Enzymes, Sensors, Substrates