Step-by-Step Avidin−Biotin Construction of Bienzyme Electrodes. Kinetic Analysis of the Coupling between the Catalytic Activities of Immobilized Monomolecular Layers of Glucose Oxidase and Hexokinase

Abstract

Avidin−biotin technology is used to achieve the step-by-step construction of electrode coatings in which two monomolecular layers of biotinylated hexokinase are immobilized on top of five monomolecular layers of biotinylated glucose oxidase. The two enzymes compete for the consumption of glucose. Because the reaction of hexokinase with glucose depends on the presence and concentration of ATP in the solution, the electrochemical response is sensitive to the ATP concentration. Such a system illustrates the possibility of translating the catalytic activity of a nonredox enzyme into an electrical signal within a spatially ordered structure. Full kinetic analysis of the electrochemical responses allowed a description of the diffusion controlled communication between the two enzymes over distances that are comparable to those involved in enzyme coupling in cell cytoplasmic systems.