Fabrication And Applications Of New Electrochemical Biosensor Based On Bacteria Surface Disply Dehydrogenase Enzyme System

There are three parts in this thesis. Two new and simple strategies were established for fabricating of monosaccharides electrochemical sensor based on immobilization of electron mediators (multi-walled carbon nanotubes) based on bacterial surface display xylose dehydrogenase(part1) and bacterial surface display glucose dehydrogenase(part2);The third part through co-immobilization of bacterial surface display xylose dehydrogenase and glucose oxidase fabricate dual-enzyme biosensors for simultaneous voltammetric detection of D-glucose and D-xylose.1. A selective and sensitive d-xylose electrochemical biosensor based on xylose dehydrogenase displayed on the surface of bacteria (XDH-bacteria) was established. The electrochemical characterization of biosensor were cyclic voltammetry, differential pulse voltammetry and current analysis means. The XDH-bacteria could catalyze the oxidization of xylose to xylonolactone with coenzyme NAD+, where NAD+(nicotinamide adenine dinucleotide) is reduced to NADH (the reduced form of nicotinamide adenine dinucleotide). The resultant NADH is further electrocatalytically oxidized by MWNTs on the electrode, resulting in an obvious oxidation peak around0.50V (vs. Ag/AgCl). Developed electrochemical biosensor exhibited good analytical performance such as long-term stability, a wide dynamic range of0.6-100μM and a low detection limit of0.5μM d-xylose (S/N=3). The proposed microbial biosensor is stable, specific, sensitive, reproducible, simple, rapid and cost-effective, which holds great potential in real applications.2. A novel electrochemical sensing platform by modification of glucose dehydrogenase displayed on the surface of bacteria (GDH-bacteria) onto glassy carbon electrode surface was constructed, Nafion-MWNTs film has a good electrical conductivity and high catalytic performance, which may be selectively sensitive detection of d-glucose.Optimized experimental conditions, the glucose electrochemical biosensor could show a rapid response to d-glucose within3s and a linear calibration plot ranged from50-800μM with a detection limit of4μM (S/N=3). The proposed microbial biosensor exhibited good reproducibility, long-term stability and no interference from saccharides. Due to its excellent performance, this nano-biocomposite electrode is expected to find possible application in anodic biocatalysts, biofuel cells and other bioelectrochemical devices.3. The dual-enzyme biosensor was firstly prepared with the addition of glucose oxidase (GOD) onto the surface of xylose dehydrogenase displayed bacteria (XDH-bacteria). The optimal conditions for the immobilized enzymes were established. Both enzymes retained their good stability and activities. For the proposed biosensor, anodic peak current at+0.55V(vs. saturated calomel electrode, SCE) was linear with the concentration of d-xylose in the range of0.25-4mM with a low detection limit of0.1mM d-xylose (S/N=3), and the cathode cpeak current at-0.5V(vs. SCE) was linear with the concentration of d-glucose with in the range of0.25-6mM with a low detection limit of0.1mM d-glucose (S/N=3). Further, d-xylose and d-glucose did not interfere with each other. The proposed biosensor is stable, specific, reproducible, simple, rapid and cost-effective. Therefore, it is envisioned that this bienzyme based electrochemical biosensor will be found promising applications.