Enzyme electrodes for ADP/ATP with enhanced sensitivity due to chemical amplification and intermediate accumulation

Abstract

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(benzophenoxaziny1) 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 p 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.

Third Generation ATP Sensor with Enzymatic Analyte Recycling

“…The sensor reaches a steady state signal since the concentration of ATP approaches a constant value in the enzyme layer due to its regeneration by PK. Stationary signals for a given ATP concentration have been reported in earlier publications on recycling sensors 9, 21, 25. This finding is in accordance with mathematical models of the recycling sensors 16.…”

“…Coupling the HK/PK system with the detection of pyruvate resulted in a 220‐fold amplification of the ATP signal 9. The sensitivity for ATP detection has been further amplified by accumulation of an intermediate 21 and recycling of pyruvate in the pyruvate oxidase/lactate dehydrogenase enzyme pair respectively 9. This “double amplification” resulted in a subnanomolar limit of detection of ATP.…”

Mediated, Amperometric Biosensor for Glucose-6-Phosphate Monitoring Based on Entrapped Glucose-6-Phosphate Dehydrogenase, Mg2+Ions, Tetracyanoquinodimethane, and Nicotinamide Adenine Dinucleotide Phosphate in Carbon Paste

“…G6PDH in soluble form has an optimal pH of 7.5 (9). The pH optimum for the carbonpaste-immobilized enzyme was also similar to that reported by other researchers (10). The effect of temperature is seen in Fig.…”

Scanning Electrochemical Microscopy Inside the Biocatalytic Layer of Biosensors, Investigation of a Double Function Complex Multienzyme Reaction Layer

“…In the simplest case the reaction steps catalyzed by the different enzymes followed each other sequentially (sequential enzyme sensors). While enzyme catalyzed reactions could be used for interference elimination [8,9], or chemical amplification [10]. Competition of enzymes catalyzing different enzyme reaction routes could be used to prepare competitive enzyme sensors [7].…”