Study On Preparation And Property Characterization Of Novel Oxidase Eletrodes

Amperometric enzyme biosensor is the most important and sensitive biosensor in the biosensor area. It has also been paid more attention and has more useful potential. A new approach is described that prepare the amperometric oxidase biosensors based on high sensitive and selective electrocatalysis of Prussian Blue(PB)-modified electrode for H2O2 combined with Si02 sol-gel immobilization in a 'sandwich' configuration. The glucose oxidase(GOD) electrode and the galactose oxidase(GAO) electrode have been developed. The optimum preparing conditions, experimental conditions as well as the response characteristics of the enzyme electrodes have been investigated. Attempts have been made to extend the response linear range of more conventional glucose electrode by the use of external membranes to restrict diffusion. The results show that the developing sensors exhibit high sensitivity and selectivity due to high sensitive and selective electroreduction of PB for H2O2 at low potential and fast response and higher stability of the sensors are attributed to biocompatible microenviroment provided by the silica sol-gel and the 'sandwich' construction maintaining the enzyme activity. The linear range of the improved GOD electrode for glucose is extended to 2-3 times as wide as that of the enzyme electrode without the outer membrane. Based on localization of enzyme in the histochemistry and cytochemistry combined with X-ray microanalysis, The microanalysis method of enzyme activity on the enzyme electrode has been established. Ce(NO3)3 serves as capturer. Substrate is catalysed by immobilized oxidase on the electrode to produceH2O2 and the H2O2 is captured by Ce(NO3)3 to form precipitate. The precipitation deposited the active site of immobilized oxidase. Then X-ray microanalysis is employed to locate active site of immobilized enzyme on the electrode. The X-ray microanalysis exhibites the properties of the enzyme electrode depend on the distribution of enzyme activity on its surface in microscopic view. The distribution of activated enzyme on the enzyme electrodes prepared by different methods has been obtained. The results show that the form of carrier membrane and the method of immobilized enzyme effect on enzyme activity and its distribution on the enzyme electrode surface. This analysis method is helpful for preparation of the excellent enzyme biosensor. The novel GOD electrode has been applied for clinical assay of diabetic blood glucose. The recovery was found to be in the 95.5%-109.8%. The measuring results are in satisfactory agreement with those of the spectrophotometric enzyme-coupling method. The developing GOD electrode has also been used to determinate the glucose content in juices and beverages. In this experiment, the BSA electrode made similarly taking BSA instead of GOD, which serves as the reference electrode, has been employed to study the interference of matrix in real sample. The testing results show that matrix and interfering species do not produce any observable interference for the assay. The reproducibility is satisfactory and the recovery is found to be in the 102.1 %-104.2%.