Integration of enzymes and electrodes: spectroscopic and electrochemical studies of chitosan-enzyme films.

A new film-forming solution was developed for the efficient immobilization of enzymes on solid substrates. The solution consisted of a biopolymer, chitosan (CHIT), that was chemically modified with a permeability-controlling agent, Acetyl Yellow 9 (AY9), using glutaric dialdehyde (GDI) as a molecular tether. A model enzyme, glucose oxidase (GOx), was mixed with the CHIT-GDI-AY9 solution and cast on the surface of platinum electrodes to form robust CHIT-GDI-AY9-GOx films for glucose biosensing. UV-visible and infrared spectroscopies were used to determine the composition of the films. The optimized films contained on average 1 molecule of AY9/3 glucosamine units of chitosan and 25 free GDI tethers/1 molecule of GOx. The electrochemical assays of the films indicated both a very high efficiency of enzyme immobilization (approximately 99%) and large enzyme activity (60 units cm(-2)). The latter translated into a high sensitivity (42 mA M(-1) cm(-2)) of the Pt/CHIT-GDI-AY9-GOx biosensor toward glucose. The biosensor operated at 0.450 V, had a fast response time (t90% < or = 3 s), and was free of typical interferences, and its dynamic range covered 3 orders of magnitude of glucose concentrations. The lowest actually detectable concentration was 10 microM glucose. In addition, the biosensor displayed a practical shelf life and excellent operational stability, e.g. its response was stable during 24-h testing under continuous polarization and continuous flow of 5.0 mM glucose solution. The proposed approach to enzyme immobilization is simple, efficient, and cost-effective and should be of importance in the development of biosensors based on other enzymes that are more expensive than glucose oxidase.