Preparation and optimization of bienzyme multilayer films using lectin andglyco-enzymes for biosensor applications

Enzyme multilayer films were prepared on the surface of a glassy carbon ele ctrode by a layer-by-layer deposition of concanavalin A (Con A) and horsera dish peroxidase (HRP) or glucose oxidase (GOx) alternately. The enzyme thin films were formed through biological affinity between Con A and sugar chai ns intrinsically located on the surface of the enzymes. Both HRP and GOx re tained their catalytic activity in the multilayer film and the enzyme-modif ied electrodes served as enzyme biosensors. For the glucose sensors prepare d using HRP-GOx bienzyme multilayer films, the loading and geometry of the enzymes in the multilayer film were optimized by varying the number of enzy me layers and the sequence of immobilization in the multilayer film. The ma gnitude of response of the glucose sensors increased with the increasing nu mber of Cox layers, while the bilayers of HRP sufficed for attaining maximu m response. It was found that the electrode surface should be first coated with HRP layers and then the GOx layer should be immobilized as an outer la yer to obtain higher performance sensors. The results were explained reason ably based on the relative activity of the enzymes and the reaction scheme. (C) 2001 Elsevier Science B.V. All rights reserved.