Development And Applications Of Electrochemical Biosensor Based On Electron Mediator Immobilization

There are three parts in this thesis. Two new and simple strategies were established for fabricating of electrochemical biosensor based on immobilization of electron mediators (Fe(CN)63? and Thionine) by electrostatic interaction of polyelectrolyte-calcium carbonate microsphere (the first chapter) and electrochemically pretreatment (the second chapter). A novel H2O2 biosensor was also fabricated based on in-situ covalent immobilization of horseradish peroxidase (HRP) through sol–gel process in the third part.1. A novel electro-active material was successfully prepared with the Fe(CN)63? ions loaded by electrostatic interaction onto the layer of poly(allylamine) hydrochloride (PAH), which was firstly assembled on the prepared poly(Sodium 4-styrenesulfonate) (PSS)-doped porous calcium carbonate microspheres (CaCO3). Further, an electrochemical sensor toward ascorbic acid (AA) detection was constructed with the use of above electro-active materials embedded into a chitosan (CS) sol-gel matrix as electron mediators. The electrocatalytic oxidation of AA by ferricyanide was observed at the potential of 0.27 V, which was evidently negative-shifted compared with that by direct electrochemical oxidation of AA on the glassy carbon electrode at the potential of 0.4 V. The experimental parameters including pH value of testing solution and applied potential for detection of AA were optimized. The current electrochemical sensor not only exhibited a good reproducibility and storage stability but also showed a fast amperometric response to AA in a linear range (1.0×10?6 to 2.143×10?3 M), a low detection limit (7.0×10?7 M).2. A convenient and effective strategy based on biospecific binding affinity of Concanavalin A (Con A) through polydopamine (PDA) mussel-inspired coatings on Thionine (Th) modified glassy carbon electrode was used to develop a non-enzyme-based glucose biosensor for the detection of glucose. In the proposed electrodeposition strategy, PDA was applied as functional"Double-sided adhesive-type"for inspired incorporating of Th and Con A due to its adhesive mechanisms and Schiff base reaction. With the peak current response of the Th moiety changed by biospecific binding Con A-glucose complex, quantitative measurement of glucose could be achieved. The current electrochemical sensor not only exhibited a excellent reproducibility, storage stability and high anti-interferent ability, but also showed broader linear response from 1.0×10?6 to 1×10?4 M with a low detection limit of 7.5×10?7 M to glucose. As a result, the proposed economical, efficient methodology was potentially attractive for evaluating clinical health risk.3. A simple and reliable one-pot approach was established for the development of a novel hydrogen peroxide (H2O2) biosensor based on in-situ covalent immobilization of horseradish peroxidase (HRP) into biocompatible material through polysaccharide incorporated sol–gel process. Siloxane with epoxide ring and trimethoxy anchor groups was applied as the bifunctional cross-linker and the inorganic resource for organic-inorganic hybridization. The reactivity between amine groups and epoxy groups allowed the covalent incorporation of HRP and the functional biopolymer, chitosan (CS) into the inorganic polysiloxane network. Some experimental variables, such as mass ratio of siloxane to CS, pH of measuring solution and applied potential for detection were optimized. HRP covalently immobilized in the hybrid matrix possessed high electrocatalytic activity to H2O2 and provided a fast amperometric response. The linear response of the as-prepared biosensor for the determination of H2O2 ranged from 2.0×10-7 to 4.6×10-5 M with a detection limit of 8.1×10-8 M. The apparent Michaelis-Menten constant was determined to be 45.18μM. Performance of the biosensor was also evaluated with respect to possible interferences. The fabricated biosensor exhibited high reproducibility and storage stability. The ease of the one-pot covalent immobilization and the biocompatible hybrid matrix serve as a versatile platform for enzyme immobilization and biosensor fabricating.