Study on Enzyme Electrodes Involving Amplification by Substrate Recycling

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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.