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

使用固定多种酶的电极制造生物传感器

• 批准号: 10555305
• 负责人: KUWABATA Susumu
• 金额: $ 3.14万
• 依托单位: Osaka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10555305/
• 关键词: biosensor; cholesterol sensor; uric acid sensor; cholesterol oxidase; uricase; electron mediator; artificial bilayer; self-assembled monolayer; コレストロールエステラーゼ; コレステロールエステラーゼ

(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.

(1)使用固定胆固醇氧化酶和胆固醇酯酶的金电极制造胆固醇传感器氨基乙硫醇在金电极上的化学吸附提供了其致密堆积的自组装单层。使用戊二醛作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。所得电极在含有硫堇作为电子介体的电解质溶液中以0V vs. SCE 极化。结果发现，注射游离胆固醇或胆固醇酯酶会产生氧化电流，且氧化电流值随着底物的增加而稳定增加。根据这些实验结果，我们建立了一种使用制备的胆固醇传感器精确测定游离胆固醇和胆固醇酯酶浓度的实用方法。(2)使用固定尿酸酶的金电极和电子介体制备尿酸传感器固定尿酸酶的金电极(尿酸氧化酶)也通过与上述相同的方法制备。如果使用含有适当电子介体的电解质溶液，则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，制备了固定有尿酸酶和电子介体的Au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，通过链烷硫醇和磷脂的自组装单层的自组织融合形成的人工双层被用来将作为膜蛋白之一的尿酸酶与电子介体一起固定。双层电极作为尿酸传感器效果很好，具有极高的底物选择性。

项目成果

Takahiro Nakaminami, Shin-ichiro Ito, Susumu Kuwabata and Hiroshi Yoneyama: "Uricase-catalyzed oxidation of uric acid using an artificial electron acceptor and fabrication of amperometric uric acid sensors with use of a redox ladder polymer"Anal. Chem.. 7

Takahiro Nakaminami、Shin-ichiro Ito、Susumu Kuwabata 和 Hiroshi Yoneyama：“使用人工电子受体进行尿酸酶催化的尿酸氧化，并使用氧化还原梯聚合物制造安培尿酸传感器”。

Susumu Kuwabata, Takahiro Nakaminami, Shin-ichiro Ito and Hiroshi Yoneyama: "Preparation and properties of amperometric uric acid sensors"Sens. Actuators, B. B52(1-2). 72-77 (1998)

Susumu Kuwabata、Takahiro Nakaminami、Shin-ichiro Ito 和 Hiroshi Yoneyama：“电流式尿酸传感器的制备和性能”Sens。

Takahiro Nakaminami: "Uricase-catalyzed oxidation of uric acid usin an artificial electron acceptor and fabrication of amperometric uric acid sensors with use of a redox ladder polymer"Analytical Chemistry. 71(10). 1928-1934 (1999)

Takahiro Nakaminami：“使用人工电子受体进行尿酸酶催化的尿酸氧化，并使用氧化还原梯聚合物制造电流型尿酸传感器”分析化学。

Takahiro Nakaminami: "A biomimetic phospholipid/alkanethiolate bilayer immobilizing uricase and an electron mediator on on an Au electrode for amperometire determination of uric acid"Analytical Chemistry. 71(19). 4278-4283 (1999)

Takahiro Nakaminami：“仿生磷脂/烷硫醇双层将尿酸酶和电子介体固定在金电极上，用于安培法测定尿酸”分析化学。

Takahiro Nakaminami: "A biomimetic phospholipid/alkanethiolate bilayer immobilizing uricase and an electron mediator on an Au electrode for amperometirc determination of uric acid"Analytical Chemistry. 71(19). 4278-4283 (1999)

Takahiro Nakaminami：“仿生磷脂/烷硫醇双层固定尿酸酶和金电极上的电子介体，用于安培测定尿酸”分析化学。