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Study on Enzyme Electrodes Involving Amplification by Substrate Recycling

底物回收放大酶电极的研究

基本信息

批准号：
01540491
负责人：
YAO Toshio
金额：
$ 0.83万
依托单位：
University of Osaka Prefecture
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1989
资助国家：
日本
起止时间：
1989 至 1990
项目状态：
已结题

项目摘要

Substrate recycling is an interesting approach for achieving amplification for the enzymatic determination of small amounts of substrate. The substrate is recycled by choosing a combination of coupled enzymes such that the product of the first enzyme-catalysed reaction is the substrate for the second enzyme. Such a principle was applied to produce highly sensitive enzyme electrodes, i. e., recycling of L-glutamate by coimmobilizing L-glutamate oxidase and glutamic-pyruvic transaminase, recycling of phosphate by coimmobilizing alkaline phosphatase, nucleotide phosphorylase and xanthine oxidase, and electrochemical-enzymatic recycling of ferrocene by immobilized glucose oxidase. In particular, L-glutamate electrode was operated with ca. 1000-fold increased sensitivity compared with the amplified enzyme electrode. However, these amplified electrodes were generally less stable, because of limited amounts of enzymes immobilized on the electrode. In contrast the enzyme reactors generally consist of a relatively large amount of immobilized enzymes. This characteristics are very favorable for substrate recycling. Some works were done for highly sensitive detection of L-glutamate, phosphate, L-lactate/pyruvate couple, and NAD coenzymes, by using the enzyme reactors involving amplification by substrate recycling. These substrates could be determined with ca. 400-fold increase in sensitivity compared to the unamplified responses, by using the proposed flow-system with the reactors involving amplification. The detection limits were 0.02 and 0.1 pmol for L-lactate/pyruvate couple and NAD/NADH couple, respectively. Also, the proposed systems were successfully used as the specific post-column detector system of h. p. l. c. to detect these substrates high-sensitively and simultaneously.

底物回收是一种有趣的方法，用于实现扩增酶测定少量底物。通过选择偶联酶的组合使第一酶催化反应的产物成为第二酶的底物来回收底物。利用这一原理制备了高灵敏度的酶电极，即利用l-谷氨酸氧化酶和谷丙转氨酶共固定化l-谷氨酸再循环，利用碱性磷酸酶、核苷酸磷酸化酶和黄嘌呤氧化酶共固定化磷酸盐再循环，利用葡萄糖氧化酶固定化二茂铁电化学-酶再循环。特别是，与扩增酶电极相比，l -谷氨酸电极的灵敏度提高了约1000倍。然而，这些放大的电极通常不太稳定，因为固定在电极上的酶数量有限。相反，酶反应器通常由相对大量的固定化酶组成。这一特性对基材回收利用非常有利。利用底物循环扩增的酶反应器，对l -谷氨酸、磷酸、l -乳酸/丙酮酸偶联酶和NAD辅酶进行了高灵敏度检测。与未放大的响应相比，通过使用涉及放大的反应器的拟议流动系统，可以以大约400倍的灵敏度确定这些底物。l -乳酸/丙酮酸偶对和NAD/NADH偶对的检出限分别为0.02和0.1 pmol。此外，所提出的系统成功地作为hp - l -c的特异柱后检测系统，对这些底物进行了高灵敏度的同时检测。