| In the general introduction, biosensors including the history, fundamental principle, immobilization strategies of the biocomponent in the biosensor methodology are introduced. The methods of realizing direct electrochemistry of enzyme and the technology in direct electrochemistry are reviewed, especially in electroanlytical applications of carbon nanotubes.Electrochemical sensor is quite unique, because it combines the enzyme specificity with the sensitivity and convenience of electroanalytical techniques in a compact form to facilitate analysis. Mediators were employed to improve the electron transfer between enzyme and electrode. However, they usually polluted electrodes. Much attention has been paid to the construction of the third generation biosensor, which is based on the direct electron transfer between the enzyme and the electrode. It is of great importance for studying the biological redox process, which could help us understand the energy transform and material metaboly in life action and develop the mediatorless biosensors.Since the discovery of carbon nanotubes in 1991, there has been an explosion of research into the physical and chemical properties of this novel material. It could catalyze many biological molecules and help to direct electrochemistry of enzymes on modifying electrodes.This thesis concerns three research sections. In first research section, the three of novel third-generation hydrogen peroxide biosensors were fabricated incorporating HRP and MWNT at aiming to overcome leakage of enzyme from electrode. We explored the feasibility of direct electron transfer of HRP on MWNT modified electrode with different immobilization strategies. HRP was immobilized on MWNT-modified electrodes by three different approaches including glutaraldehyde cross-linking, sol-gel and gold nanoparticle to construct three third-generation hydrogen peroxide biosensors. The three biosensors all displayed excellent electrocatalytic response to the reduction of hydrogen peroxide without the aid of an electron transfer mediator. The resulting biosensors exhibited fast response (< 3 s) to H2O2.(1) The direct electrochemistry of HRP immobilized on a MWNT modified GCE bythe cross-linking method was investigated and the resulting biosensor was estimated. HRP immobilized on a MWNT modified electrode by the cross-linking method in 0.10 mol L-1 PBS (pH =7) displayed a cathodic peak at -373 mV and an anodic peak at -57 mV. The reduction peak current of the HRP increased linearly with an increase of scan rate from 10 to 150 mV s-1, thus the electrode reaction was typical of surface-controlled quasi-reversible process. The apparent heterogeneous electron transfer rate constant of the HRP was 1.0 s-1. The average surface coverage of HRP immobilized on MWNT/GCE was calculated to be 5.61×10-10mol.cm-2. The apparent Michaelis-Menten constant was 0.057 mmol L-1. The H2O2 biosensor exhibited linear relation to the concentration of H2O2 in the range from 9.5×10-3 mol L-1 to 9.5×10-7mol L-1 at an applied potential of-400 mV with a relate standard deviation of 3.2 % (n=11).(2) A H2O2 sensor was fabricated by immobilizing HRP on a MWNT modified GCE by the sol-gel method and examined. The fabricated biosensor for the analysis of H2O2 showed a wide linear response range from 70 μmol L-1 to 3 mmol L-1 at -300 mV. When the biosensor was stored dry at 4 癈 for 60 day, the current decrease was less than 5%.(3) A H2O2 sensor was fabricated by immobilizing HRP on Pt electrode modified by gold nanoparticles. The sensor responded to H2O2 in the concentration range from 10 μmol L-1 to 1 mmol L-1 at -300 mV.In second research section, a novel disposable H2O2 biosensor using screen-printed technology with MWNT acted as paste materials was designed and fabricated at aiming to explore possibility of disposable third-generation biosensors using MWNT acted as paste materials. The biosensors constructed exhibited excellent electrocatalytic response to the reduction of hydrogen peroxide without the aid of an electron transfer mediator...
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