ENZYME ELECTRODES FOR ADP ATP WITH ENHANCED SENSITIVITY DUE TO CHEMICAL AMPLIFICATION AND INTERMEDIATE ACCUMULATION

Substrate recycling is used for making a highly sensitive enzyme electrode for the determination of ADP/ATP. A graphite electrode is modified with an adsorbed bis(benzophenoxazinyl) derivative of terephthaloic acid and covered with a gel containing entrapped glucose-6-phosphate dehydrogenase, pyruvate kinase, and hexokinase, all behind a dialysis membrane. The recycling produces NADH, which is detected by oxidation at the modified electrode. The cycle is driven by conversion of phosphoenolpyruvate to pyruvate and by phosphorylation of glucose to glucose-6-phosphate, which is oxidized to the corresponding gluconic acid with the simultaneous production of NADH. The calibration curves for ADP and ATP were linear to about 0.4-mu-M and the detection limit was 1 nM. A steady-state response was obtained after 2-10 min. Sensitivity toward ADP can be increased further by the accumulation of ATP inside the membrane.