The Direct Electrochemistry Of Redox Proteins And The Application Of Their Mimics In Antioxidant Assay

Direct electrochemistry of redox proteins and enzymes has attracted great interest in bioelectrochemical and biological fields. Studies on direct electrochemistry of redox proteins/enzymes have been used to measure essential physicochemical data concerning the kinetics and energetics of protein redox reactions, providing mechanistic studies of electron exchange among proteins in biological systems. More importantly, direct electron transfer between immobilized enzyme and underlying electrode can establish a foundation for fabricating novel mediator-free biosensors, biofuel cells, bio-reactors. Furthermore, antioxidants have been proven as effective free radical scavangers, which play a key role in preventing the damage of radical reactions to human beings. It is therefore important to study antioxidants' radical scavenging processes and antioxidant capabilities. The main work of this thesis is summarized as follows:1. A novel electrochemical biosensing platform based on amino acid ionic liquids (AAILs)/carbon nanotubes (CNTs) composites was fabricated. AAILs were used as a novel solvent for glucose oxidase (GOD) and the GOD-AAILs/CNTs electrode was conveniently prepared by immersing carbon nanotubes (CNTs) modified glassy carbon (GC) electrode into AAILs containing GOD. The direct electrochemistry of GOD on the GOD-AAILs/CNTs/GC electrode has been investigated and a pair of reversible peaks was obtained by cyclic voltammetry. The immobilized glucose oxidase could retain bioactivity and catalyze the reduction of dissolved oxygen. Due to the synergic effect of AAILs and CNTs, the GOD-AAILs/CNTs/GC electrode shows excellent electrocatalytic activity towards glucose with a linear range form 0.05 to 0.8 mM and a detection limit of 5.5μM (S/N = 3). Furthermore, the biosensor exhibits good stability and ability to exclude the interference of commonly coexisted uric and ascorbic acid.2. Based on the layer-by-layer self-assembly of positively charged cetyltrimethylammonium bromide (CTAB) wrapped gold nanorods (AuNRs) and negatively charged superoxide dismutase (SOD), a third generation biosensor ((SOD/AuNRs)2/Cys/Au) for superoxide anion (O2?-) was developed. Two layers assembly of SOD/AuNRs was demonstrated, which significantly enhance the direct electron transfer between SOD and electrode. Bifunctional enzymatic catalytic activities of the SOD offers a dual electrochemical approach to determination of O2?-, in which O2?- can be detected both anodically and cathodically. In both the oxidation and reduction regions, (SOD/AuNRs)2/Cys/Au electrode displays excellent analytical performance, such as wide linear range, low detection limit, quick response time, good stability and reproducibility.3. A novel, simple, colorimetric DNAzyme-based method was reported to detect the radical-scavenging capacity of antioxidant. In this new strategy, horseradish peroxidase (HRP) mimicking DNAzyme catalyzes the oxidation of ABTS2- (2, 2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)) by H2O2 to generate blue/green ABTS?- radical, which can be scavenged by antioxidants resulting in color change. The typical kinetic curve of antioxidant-inhibited generation of ABTS?- shows distinct biphasic pattern, involving a lag phase (stage I) and a linear increase phase (stage II). kt value, the product of lag time (t) and the slope of the curve in stage II (k), was used as the parameter for antioxidant capacity determination. This DNAzyme-based antioxidant assay has been effectively used to quantitatively detect the concentrations of antioxidants and evaluate the antioxidant capabilities of a variety of antioxidants and some real samples.