Chemically amplified xanthine and hypoxanthine sensors based on substrate recycling between the enzyme-substrate complex and substrate

The amount of consumed oxygen in the xanthine oxidase reaction exceeded the initial amounts of xanthine added when the enzymatic reaction was performed in the presence of reducing agents such as L-ascorbic acid (AsA) or dithiothreithol (DTT). Significantly amplified signals of the immobilized xanthine oxidase electrode were observed in a buffer solution containing AsA or DTT. The amplification factor increased with increasing concentration of the reducing agents, and the amplification factor for 1× 10−6 M xanthine was increased up to 38 when 5 × 10−3 M AsA coexisted in the phosphate buffer (pH 8.5) at 25 °C. The calibration curves of the amplified responses were shifted towards lower concentration ranges compared with those of a non-amplified response, and the detection limits of xanthine and hypoxanthine were lowered to 5 × 10−7 M. The electron spin resonance signal of the monodehydroascorbate radical (MDA) was significantly increased during the amplification reaction. The addition of uric acid, a final product of the xanthine oxidase reaction, produced no oxygen consumption. These results suggest that reducing agents react with the enzyme-substrate (E-S) complex intermediate, and recycling between the substrate and the E-S complex takes place accompanied by amplified consumption of oxygen.