Chemically amplified current responses of FAD-enzyme electrodes based on the intermediate regeneration induced by L-ascorbate

The amounts of enzymatic oxygen consumption in the oxidase reaction catalyzed by flavo-proteins (glucose oxidase, cholesterol oxidase and lactate oxidase) exceeded the amount of substrates added to the enzyme solution containing a relatively high concentration of L-ascorbate (> 5 mid), although the final enzymatic reaction products are not reduced to the original substrate by L-ascorbic acid (AsA). The oxygen consumed during the amplification is revealed to be finally converted to hydrogen peroxide. The electron spin resonance (ESR) signal of the mondehydroascorbate radical (MDA, the one-electron reduced form of ascorbate) was apparently increased during the amplification reaction. These observations suggest that AsA reacts with the enzymatic intermediate complex and the amplification of the consumed oxygen is due to the recycling of substrates via an intermediate complex accumulating hydrogen peroxide. These intermediate regeneration induced by AsA enables signal amplification of immobilized oxidase-based electrodes when AsA coexists in the sample solution. The amplification factor of L-lactate was increased up to ca. 20 and the detection limit (2 nA) was shifted from 2 x 10(-6) M to 1 x 10(-7) M in the presence of 5 mM AsA in the buffer solution (pH 7.5) at 35 degrees C.