Studies On Amperometric Enzyme Biosensors Based On New Kinds Of Nanomaterial

Electrochemical enzyme biosensors based on the recognition of specific substrates have the advantages of high-sensitivity, nice-selectivity as well as easy miniaturization and automation. In the design and fabrication of the electrochemical biosensor the crucial step is how to develop a simple and effective strategy for the construction of sensitive membrane on or into the electrode. This research is aimed to develop new immobilization strategies of enzyme for the purpose of improving the performance and long-term stability of biosensor. The detailed materials are summarized as following: 1. Considering chitosan's excellent film-forming ability and good adhesion properties, we used it to form a pre-coated film on the surface of the glassy carbon electrode (GCE). The plenty amounts of amino groups of chitosan film on the surface of GCE facilitated the formation of nano-Au film on its surface through strong electrostatic interaction. Cysteamine contains mercapto groups which make it contact with nano-Au tightly. By immobilizing tyrosinase on the positively-charged amino groups of cysteamine, a catechol biosensor can be fabricated. The biosensor possessed excellent responses to the substrate with the presence of negatively-charged nano-Au which can effectively retain activity of tyrosinase. The linear range of detection for catechol is 1.0×10-7 ~ 1.0×10-4 mol/L and the lifetime of the biosensor was over 1 month. 2. C/Fe nanocomposite was synthesized by intercalating Fe3+ into graphite oxide (GO) layers under a reducing H2 atmosphere. Its structure and electrochemical characteristics were studied and the results showed that the C/Fe nanocomposite possesses excellent ability of electron-transfer. By immobilizing GOx on the surface of C/Fe nanocomposite paste electrode, a mediatorless glucose biosensor was fabricated. In the absence of mediator, the activity and stability of GOx on the surface of biosensor could be effectively retained. The linear range of detection for glucose is 6.67×10-6 ~ 1.0×10-2 mol/L. The biosensor has no responses to ascorbic acid and uric acid which often interfere with the detection of glucose. 3. Carbon nanotubes have attracted comprehensive attentions in application of the fabrication of biosensors because of their unique physical and chemical characteristics and their excellent ability of electron-transfer. We modified multi-wall carbon nanotubes (MWCNTs) with nano-Au in the presence of chitosan, then sol-gel was applied to embed the nano-Au modified MWCNTs to fabricate a submicroelectrode array. The negative-charged nano-Au on the surface of MWCNTs can immobilize HRP through electrostatic interation to prepare the H2O2 biosensor. Results displayed that HRP could be stably immobilized on nano-Au monolayer and the MWCNTs could accelerate the electron-transfer; Compared to normal dimentional biosensor, the submicroelectrode array responses to substrate more sensitively and rapidly with the linear range for H2O2 is 6.67×10-7 ~ 1.33×10-4 mol/L. 4. A tyrosinase biosensor based on a boron-doped diamond (BDD) electrode as the base electrode has been developed. The enzyme biosensor catalyzes phenolic compounds to get quinines which can be reduced to produce reduction current. The catalytic effect with respect to phenols of such biosensor is better than the one which was based on the glassy carbon electrode as the base electrode. The enzyme electrode provided a linear response to catechol over a concentration range of 1.0×10-8 to 1.0×10-5 mol/L with a detection limit of 5.2×10-9 mol/L. The apparent Michaelis-Menten constant ( K mapp ) for the sensor was calculated to be 33.65 μmol/L. The enzyme biosensor has good responses to phenol and p-cresol, the linear calibration ranges to them are 5.0×10-8 ~ 2.0×10-5 mol/L and 5.0×10-8 ~ 5.0×10-6 mol/L respectively. The biosensor was evaluated with satisfactory analytical performance in terms of the repeatability and stability.