Glucose and Acetylcholine Sensing Multilayer Enzyme Electrodes of Controlled Enzyme Layer Thickness

Abstract

Multilayer enzyme networks assembled by a stepwise synthesis onto Au electrodes provide the basis for glucose, choline, and acetylcholine amperometric biosensors. Glucose oxidase is assembled as a multilayer array on smooth and rough Au electrodes. Electrical communication of the GOx layers with the electrode is established by the presence of ferrocenecarboxylic acid as diffusional electron mediator or by covalent linkage of [(ferrocenylmethyl)amino]caproic acid to the enzyme components. The resulting enzyme electrodes are applied as amperometric biosensors for glucose. The sensitivity of the resulting enzyme electrode is controlled by the number of enzyme layers assembled onto the electrode and by the roughness factor of the electrode surface. A multilayer enzyme array of choline oxidase (ChO) immobilized onto a rough Au electrode provides an amperometric biosensor for the amperometric detection of choline in the presence of 2,6-dichloroindophenol (DIP) as diffusional electron mediator. Stepwise organization of a multilayer biocatalytic array consisting of four layers of ChO and three layers of acetylcholine esterase (AChE) provides a bifunctional enzyme electrode for the amperometric detection of acetylcholine. In this system, choline generated by AChE hydrolysis of acetylcholine is amperometrically detected by the ChO layers in the presence of DIP acting as diffusional electron mediator. Here we report on novel means to enhance the sensitivity of multilayer enzyme electrodes by the application of rough Au electrodes as the surface for assembling the enzyme network.