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 (∼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% ≤ 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 μM 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.