Amperometric trienzyme ATP biosensors based on the coimmobilization of salicylate hydroxylase, glucose-6-phosphate dehydrogenase, and hexokinase

Two types of amperometric ATP biosensors were developed by using the coimmobilization of salicylate hydroxylase (SHL, EC 1.14.13.1), glucose-6-phosphate dehydrogenase (G6PDH, EC1.1.1.49), and hexokinase (HEX, EC 2.7.1.1) on a Clark-type oxygen electrode and on a screen-printed electrode. The principles of the determination schemes are as follows: HEX transfers the phosphate group from ATP to glucose to form glucose-6-phosphate. G6PDH catalyzes the specific dehydrogenation of glucose-6-phosphate by consuming NAD+. The product, NADH initiates the irreversible decarboxylation and hydroxylation of salicylate by SHL to consume dissolved oxygen and generate catechol. This results in a detectable signal on a Clark-type electrode due to the SHL-enzymatic consumption of oxygen, or a detectable signal on a screen-printed electrode due to the SHL-enzymatic generation of catechol in the measurement of ATP. Both sensors show high performance characteristics with broad detection ranges, short measuring times, and good specificities.