CHEMICALLY AMPLIFIED CURRENT RESPONSES OF FAD-ENZYME ELECTRODES BASEDON THE INTERMEDIATE REGENERATION INDUCED BY L-ASCORBATE

The amounts of enzymatic oxygen consumption in the oxidase reaction ca talyzed by flavo-proteins (glucose oxidase, cholesterol oxidase and la ctate oxidase) exceeded the amount of substrates added to the enzyme s olution 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 consume d during the amplification is revealed to be finally converted to hydr ogen peroxide. The electron spin resonance (ESR) signal of the mondehy droascorbate radical (MDA, the one-electron reduced form of ascorbate) was apparently increased during the amplification reaction. These obs ervations suggest that AsA reacts with the enzymatic intermediate comp lex and the amplification of the consumed oxygen is due to the recycli ng of substrates via an intermediate complex accumulating hydrogen per oxide. These intermediate regeneration induced by AsA enables signal a mplification of immobilized oxidase-based electrodes when AsA coexists in the sample solution. The amplification factor of L-lactate was inc reased 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 s olution (pH 7.5) at 35 degrees C.