Peroxidase-based electrochemical detection using osmium complexes as mediators: application to sensing and immunoassays
Mandal, Tanushree
Mandal, Tanushree
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Publication Date
2023-05-16
Type
Thesis
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Abstract
Horseradish peroxidase (HRP) is an oxidoreductase enzyme that has been used in many bio-electrochemical applications such as biosensors, immunoassays, fuel cells etc. Osmium polypyridyl complexes are advantageous as mediators as they are stable, have low and tunable redox potentials. The objective of this thesis is to study application of osmium polypyridyl complex as mediators for immunoassays and sensors. A range of osmium polypyridyl complexes with general formula [Os(N-N)2Cl2]/[Os(N-N)2Cl2]+ and [Os(N-N)2(L)Cl]2+ (where N-N = bipyridine and its derivatives; L = 4-(aminomethyl)pyridine, 1-(3-aminopropyl)imidazole, and 1-methylimidazole) were synthesized and characterized. Additionally, osmium redox polymers with general formula [Os(N-N)2(polyvinylimidazole)10Cl]+ were also synthesized and characterized. The redox potentials of these complexes and polymers were manipulated by substituting the 4 and 4' positions of bipyridine ligand with electron withdrawing groups (EWGs) and electron donating groups (EDGs). These complexes and polymers were characterized using various analytical techniques such as cyclic voltammetry, mass spectroscopy (MS), and single-crystal X-ray diffraction (SCXRD). Furthermore, a comprehensive report for synthesis of osmium complexes and polymers was prepared with various synthetic approaches and extraction procedures. These complexes and polymers were utilized as mediators in applications such as immunoassay and peroxide sensor. The kinetics of HRP-mediator interaction in solution phase have been evaluated for hydroquinone and the osmium polypyridyl complexes synthesized in the earlier chapter. The objective of this study was finding an alternative mediator that could be used in place of hydroquinone. Initially, the stability of aqueous solutions of hydroquinone and osmium complexes were studied using UV-VIS spectroscopy. Finally, the relative rate constants of HRP-mediator interactions were estimated using Nicholson-Shain approach. The [Os(2,2’-bipyridine)2Cl2]+ complex was estimated to be the best mediator for HRP among the osmium polypyridyl complexes studied in this study. The stability studies and the rate constant calculations can be used to screen mediators for any enzyme. Screen-printed electrodes (SPEs) are a versatile choice for electrodes for sensors. However, the variability in surface area of electrodes will cause variability in current output. This variability can interfere with the repeatability and accuracy of any sensor. Therefore, normalization of current output is required to improve the accuracy of a sensor. Two in-situ normalization methods, capacitive currents, and faradaic currents were studied for their applicability as normalization methods for current outputs from electrodes. These normalization methods were further employed to normalize the current output from bio-electrochemical applications such as immunoassay and sensors. This study showed that capacitive currents produce high standard deviations and could not be used to normalize current output. A peroxide sensor was designed using [Os(2,2’-bipyridine)2(polyvinylimidazole)10Cl]+ (OsbpyPVI) crosslinked to HRP using poly(propylene glycol)diglycidylether (PEGDGE). The amounts of the individual components were optimized and validated using a Box-Behnken design (BBD) to maximize the current density produced by the enzyme electrode. This is a short study on application of a chemometric tool such as BBD to optimize components of an enzyme electrode. A multiplex electrochemical immunoassay for diagnosis of prostate cancer (PCa) is designed. This immunoassay builds up on the previously designed immunoassays where an oxygen-sensitive mediator (hydroquinone) was used. An oxygen-insensitive osmium polypyridyl complex is used as an alternate mediator in the current immunoassay which eliminates the need to deoxygenate mediator solutions throughout the experimentation time. Moreover, 3D printing was used to design microfluidic chips which eliminates bulky setup and promotes ease of use. Overall, this thesis studies various aspects of designing an electrochemical sensor. The synthesis and characterization of osmium-based mediators is studied. Subsequently, these osmium-based mediators were screened as suitable mediators for HRP enzyme. The solution phase kinetics study is the fundamental aspect of studying the enzyme-mediator interaction. The electrodes play an important role in any electrochemical sensor. Variability in current output arising from use of electrodes with irregular surface area will interfere with the accuracy of any sensor. Therefore, normalization methods were studied to supress the electrode-to-electrode surface area variability. A short study for application of BBD to a peroxide sensor was performed. Finally, an osmium-based mediator, 3D printing, and microfluidics were integrated into design of a rapid and sensitive multiplex electrochemical immunoassay for PCa.
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NUI Galway