Study on Enzyme Electrodes Involving Amplification by Substrate Recycling

底物回收是实现酶法测定微量底物的一种有趣的方法。通过选择偶联酶的组合来回收底物，使得第一酶催化反应的产物是第二酶的底物。应用这一原理制作了高灵敏的酶电极，即通过共固定化L谷氨酸酶和谷丙转氨酶来回收谷氨酸，通过共固定化碱性磷酸酶、核苷酸磷酸化酶和黄嘌呤氧化酶来回收磷酸盐，通过固定化葡萄糖氧化酶来电化学酶循环二茂铁。特别是，L-谷氨酸电极的灵敏度比放大酶电极提高了约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。这一特性非常有利于底物的回收利用。利用底物循环扩增的酶反应器，对L-谷氨酸、磷酸、L-乳酸/丙酮酸对和NAD辅酶进行了高灵敏的检测。与未放大的反应相比，使用所提出的带有放大反应的反应器的流动系统，这些底物的测定灵敏度可提高约400倍。L-乳酸/丙酮酸对和NAD/NADH对的检出限分别为0.02pmol和0.1pmol。此外，所提出的系统还被成功地用作H.P.L的特定柱后检测器系统，以高灵敏地同时检测这些底物。

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.