Preparation of chitosan/glucose oxidase nanolayered films for electrode modification by the technique of layer-by-layer self-assembly

Abstract

Enzyme immobilization onto the electrode surface is a critical step in assembling amperometric biosensors. Recently, layer-by-layer (LBL) techniques have been developed for the immobilization of enzyme on various matrices [1, 2]. The multilayer films of various proteins alternating with synthetic or natural polyanions and polycations were prepared to fabricate biosensors by this novel technique [3–10]. The glucose oxidase is widely used for the detection of glucose in body fluids and in removing residual glucose and oxygen from beverages and foodstuffs. The glucose oxidase (β-D-glucose: oxygen 1-oxidoreductase, EC1.1.3.4) catalyses the oxidation of β-D-glucose to D-glucono1,5-lactone and hydrogen peroxide, using molecular oxygen as the electron acceptor (as Fig. 1 shows). The chitosan, an N-deacetylated derivative of chitin, is a naturally occurring biopolymer found in the exoskeleton of crustaceans, in fungal cell walls, and in other biological materials [11]. It has an unusual combination of properties [12], which includes excellent membrane-forming ability towards water, good adhesion, nontoxicity, high mechanical strength, especially good biocompatibility and susceptibility to chemical modification due to the presence of reactive amino and hydroxyl functional groups. In recent years, the artificial polyelectrolytes have mainly been used to immobilize various enzymes by LBL techniques [13]. Chitosan was generally used to immobilize various enzymes by techniques such as sol-gel [14], crosslinking [15] etc. The LBL techniques could control the enzyme loading by changing the adsorption cycles with fewer side effects on glucose oxidase while the chitosan was used for preparing LBL films, which was expected to be an alternative method for the chitosan to immobilize glucose oxidase. In this paper, we report the first attempt to prepare chitosan/GOx nanolayered films for electrode modification by the technique of layer-by-layer selfassembly. In situ gravimetric measurement of quartz crystal microbalance (QCM, QCA917, EG&G) was applied to investigate the assembly process. An AFM (Nanoscope IIIa, Digital Instruments) was used to characterize the surface morphology. The electrochemical