Fabrication of Biosensors with the use of Electrodes Immobilizing Plural Kinds of Enzymes

(1)固定化胆固醇氧化酶和胆固醇酯酶的金电极制备胆固醇传感器氨乙硫醇在金电极上的化学吸附提供了致密的自组装膜。以戊二醛为交联剂，将胆固醇氧化酶和胆固醇酯酶共价固定在电极上。所得电极在含有硫代素作为电子介体的电解液中，在0Vvs.SCE下极化。发现注射游离胆固醇或胆固醇酯酶可产生氧化电流，且随着底物浓度的增加，氧化电流逐渐增大。根据这些实验结果，我们建立了一种实用的方法，利用所制备的胆固醇传感器精确地测定游离胆固醇和胆固醇酯酶的浓度。(2)用金电极固定尿酸酶和电子介体制作尿酸传感器也用上述方法制备了固定尿酸酶(尿酸氧化酶)的金电极。如果使用含有适当电子介体的电解质溶液，该电极可用作尿酸传感器。利用氧化还原聚合物作为尿酸酶的电子介体，制备了尿酸酶和电子介体固定的金电极。在这种情况下，无需在溶液中添加任何其他物质即可测定溶液中的尿酸浓度。此外，还利用烷硫醇和磷脂自组装的单层膜自组织融合形成的人工双层与电子介体一起固定化膜蛋白之一的尿酸酶。双层电极作为尿酸传感器工作良好，具有极高的底物选择性。

(1) Fabrication of Cholesterol Sensors using Au electrodes immobilizing Cholesterol Oxidase and Cholesterol EsteraseChemical adsorption of aminoethanethiol on a gold electrode provided its densely packed self-assembled monolayer. Cholesterol oxidase and cholesterol esterase were covalently immobilized on the electrode with use of glutaraldehyde as a cross-linking agent. The resulting electrode was polarized at 0 V vs. SCE in an electrolyte solution containing thionin as an electron mediator. It was found that injection of free cholesterol or cholesterol esterase gave generation of oxidation currents and the value increased steadily with an increase in the substrate. From these experimental results, we established a practical methodology of precise determination of concentration of free cholesterol and cholesterol esterase with the use of the prepared cholesterol sensors.(2) Fabrication of Uric Acid Sensors using Au electrodes immobilizing Uricase and Electron MediatorAn Au electrode immobilizing uricase (uric acid oxidase) was also prepared by the same method as that mentioned above. The electrode worked as a uric acid sensor if an electrolyte solution containing an appropriate electron mediator was used. Preparation of Au electrode on which uricase and electron mediator were immobilized was achieved by using a redox polymer which can work as an electron mediator for uricase. In this case, concentration of uric acid in solution was able to be determined without addition of any other substances in the solution. Furthermore, an artificial bilayer formed by self-organized fusion of a self-assembled monolayer of alkanethiol and phospholipid was used to immobilize uricase that is one of membrane proteins together with electron mediators. The bilayer electrodes worked well as the uric acid sensor possessing an extremely high substrate selectivity.