The enzyme couples horseradish peroxidase/glucose dehydrogenase, glucose oxidase/glucose dehydrogenase, and cytochrome b,/lactate dehydrogenase are applied in enzyme electrodes. Based on amplification by the recyclization reactions catalyzed by these two-enzyme systems, NADH, NAD', glucose, lactate and pyruvate, are determined with 8-40-fold increased sensitivity compared to the unamplified reactions. Detection limits are 1.0 X lo* M NADH, 1.2 x 10d M NAD', 8 X lo-' M glucose, and 3 X lo-' M lactate or pyruvate.
Coupled enzyme reactions in enzyme electrodes may involve sequential [l-3] or competitive [3, 41 coupling. In the latter, competition of two enzymes for the same substrate is utilized; in the former, the product of the first enzyme is the substrate of the second, which catalyzes the electrochemitally-monitored conversion. If the enzymes for such a sequence are chosen so that the product of the second enzyme is the substrate for the first, substrate recycling takes place, which leads to amplification of the sensor response. This type of coupling can therefore be called enzymatic amplification. The effect has been observed in a previous study in which glucose was recycled by co-immobilized glucose oxidase and glucose dehydrogenase [5]. Malinauskas and Kulys [6] studied the recycling of the NAD'--NADH couple between phenazine methosulfate and immobilized alcohol dehydrogenase attached to an oxygen electrode. This chemical-enzymatic amplification permits micromolar concentrations of the cofactor to be determined. Recently, lactate oxidase and lactate dehydrogenase have been used for lactatepyruvate recycling in an enzyme electrode [ 71. This communication reports some different approaches to enzyme amplification in biosensors by co-enzyme and substrate recycling based on the enzyme couples horseradish peroxidase (HRP)/glucose dehydrogenase (GDH), glucose oxidase/GDH and cytochrome b,/lactate dehydrogenase (LDH), as depicted in the following scheme