Avidin-Biotin System-Based Enzyme Multilayer Membranes for Biosensor Applications: Optimization of Loading of Choline Esterase and Choline Oxidase in the Bienzyme Membrane for Acetylcholine Biosensors

Abstract

The loading of choline esterase (ChE) and choline oxidase (ChOx) in the enzyme membrane of an acetylcholine biosensor was optimized based on a layer-by-layer construction of the bienzyme layers on the surface of a platinum (Pt) and a Pt-black electrode. To this goal, ChE and ChOx were tagged with biotin residues, so that the enzymes could be built into the multilayer assemblies composed of monomolecular layers of the enzymes and avidin. The ChE/ChOx bienzyme multilayer-modified electrodes showed amperometric response to acetylcholine in solution, showing that ChE and ChOx catalyzed hydrolysis reaction of acetylcholine and an oxidation reaction of resulting choline, respectively, successively in the bienzyme layer. It was found that the acetylcholine sensor which is modified with 10-layer ChOx exhibited its maximum response to acetylcholine when additional 2 layers of ChE were added to the surface of the ChOx layer. This optimum ratio of ChE to ChOx in the bienzyme layer was reasonably explained in terms of the relative values of catalytic activity of the enzymes. The usefulness of the layer-by-layer deposition technique in optimizing the enzyme loading in bienzyme system is discussed in detail.

Layer-by-layer Thin Films and Microcapsules for Biosensors and Controlled Release

“…78 The amperometric response of all the sensors increased with the number of enzyme layers in the LbL film, confirming that the enzymes retain their catalytic activity and that enzyme substrates penetrate the LbL film. The facile preparation of bi-enzyme biosensors through the avidin-biotin protocol has also been demonstrated; these include enzyme sensors composed of GOx and ascorbate oxidase, 79 GOx and hexokinase, 80 GOx and HRP, 81 choline esterase and choline oxidase, 82 and polyphenol oxidase and alkaline phosphatase. 83 Ferrocene-attached avidin and streptavidin have also been used for the preparation of protein assemblies on an electrode surface to study the electron-transfer mechanism across LbL films.…”

Biosensor for organophosphorus pesticides based on the acetylcholine esterase inhibition mediated by choline oxidase bioelectrocatalysis

“…There have been a number of methods for ChOx immobilization on biosensor surfaces 16,17 . As an example, enzyme immobilization based on an avidin-biotin system has been developed 18 . The avidinbiotin interaction was very specific, so ChOx could be effectively immobilized into a multilayer format.…”

Glucose and Lactate Biosensors Prepared by a Layer-by-Layer Deposition of Concanavalin A and Mannose-Labeled Enzymes: Electrochemical Response in the Presence of Electron Mediators.

“…1,2) In this context, we have developed high-performance glucose and lactate sensors using enzyme multilayer films which were prepared by a layer-by-layer deposition of monomolecular layers of avidin and biotin-labeled glucose oxidase (GOx) and lactate oxidase (LOx). [3][4][5] The output current of the multilayer film-modified biosensors can be controlled precisely by regulating the number of enzyme layers in the multilayer films; the size of the response current is proportional to the number of enzyme layers. [3][4][5] It was also possible to eliminate ascorbate interference by immobilizing ascorbate oxidase simultaneously in the GOx and LOx multilayer films.…”

“…[3][4][5] The output current of the multilayer film-modified biosensors can be controlled precisely by regulating the number of enzyme layers in the multilayer films; the size of the response current is proportional to the number of enzyme layers. [3][4][5] It was also possible to eliminate ascorbate interference by immobilizing ascorbate oxidase simultaneously in the GOx and LOx multilayer films.6) Thus, the enzyme multilayer films prepared by taking advantage of a strong affinity between avidin and biotin were quite useful for improving performance characteristics of enzyme biosensors.We recently reported another protocol to prepare enzyme multilayer films by means of the biological interaction between concanavalin A (Con A) and mannose. 7,8) Con A is a lectin protein (molecular weight; ca.…”

“…[3][4][5] It was also possible to eliminate ascorbate interference by immobilizing ascorbate oxidase simultaneously in the GOx and LOx multilayer films. 6) Thus, the enzyme multilayer films prepared by taking advantage of a strong affinity between avidin and biotin were quite useful for improving performance characteristics of enzyme biosensors.…”