Chinese Journal of Chemistry, 2005, 23, 275—279 Full Paper A Glucose Biosensor Based on Immobilization of Glucose Oxidase in Chitosan Network Matrix YAO, Hui(姚慧) LI, Nan(李楠) XU, Jing-Zhong(徐锦忠) ZHU, Jun-Jie*(朱俊杰) Key Laboratory of Analytical Chemistry for Life Science, Department of Chemistry, Nanjing University, Nanjing, Jiangsu 210093, China A simple and promising glucose biosensor was constructed by successful entrapment of glucose oxidase (GOD) into chitosan matrix, which was cross-linked with glutaraldehyde (GA). This material provided good biocompatibil- ity and the stabilizing microenvironment around the enzyme. The morphologies and properties of chitosan and chi- tosan/GOD were characterized by FTIR, UV-Vis and SEM techniques. This biosensor exhibited a fast amperometric response to glucose. The linear range for glucose determination was from 1×10－5 to 3.4×10－3 mol•L－1, with a de- tection limit of 5×10－6 mol•L－1 based on S/N＝3. The biosensor could retain ca. 90% of its original activity after two weeks of storage under dry conditions at 4 ℃. Keywords chitosan, glucose oxidase, biosensor, glucose, crosslink Introduction tionality. As an attractive structural material, chitosan has good biocompatibility and has been studied as a Chitosan obtained by N-deacetylation of chitin, has support for immobilization of enzyme, and it can retain attracted some attention since chitin is the secondly enzyme activity and improve the performance of bio- most abundant polysaccharide. As a natural product, sensor. chitosan has the inherent properties of biocompatibility, Chitosan is a functional structural material and has biodegradability, chemical inertness, low toxicity, high been studied as a support for the immobilization of en- mechanical strength and hydrophilicity, and good film zyme and cells. There are some papers reporting on the forming ability. Due to these special properties, chitosan application of the chitosan modified biosensors, e.g. has been used over a wide range of applications, such as 1 2 3 sol-gel/chitosan membrane modified glucose biosen- drug carriers, wound-healing agents, chelating agents, 8,9 4 sor, tyrosinase biosensor based on immobilizing ty- membrane filter for water treatment, biodegradable rosinase in chitosan-silica gel membrane,10 and glucose coating or film for food packaging,5 and enzyme immo- 6 biosensor based on premixing chitosan modified glu- bilization. cose oxidase into carbon paste containing platinum.11 Enzyme immobilization technology has attracted Most of these articles based on the sol-gel technique much attention in connection with the design, fabrica- constructing the biosensors have achieved great success, tion and applications of biosensor. The catalytic proper- but at the same time, to our knowledge, there are also ties of immobilized enzymes as well as the life-time and some disadvantages using the method to construct bio- stability are influenced by immobilization method and 7 sensor, e.g. slow leaching of enzyme from the electrode support materials. The immobilization technique can surface via the porosity of the thin sol-gel matrix and involve either a physical (e.g., absorption or entrapment) the biosensor has a fairly slow response, complicated or chemical method (e.g., covalent bonding) that allows procedures and fragility, and the enzyme is exposed to relatively simple, sensitive and selective electrochemi- some harsh conditions during the sol-gel process, i.e., cal detection of the trace components of biological, partial denaturation of enzyme by the catalyst (either clinical, industrial and environmental systems. Indeed, basic or acidic) or the solvent used (generally methanol investigation on suitable, low cost and effective enzyme or ethanol). immobilization methodology is still of wide research To overcome these problems, a novel biosensor is interest. To retain the specific biological function of described in this paper. We constructed a simple am- enzyme, the immobilization on solid matrix is a key perometric glucose biosensor, based on glucose oxidase factor in preparation of biosensors. That is, the structure directly entrapped in chitosan film cross-linked with and thickness of materials used to immobilize enzyme glutaraldehyde on the Pt electrode. This low cost design should not induce a severe decrease of enzyme func- simplifies the fabrication procedure to obtain a stabile * E-mail: [email protected]; Fax and Tel.: ＋86-25-83594976 Received July 14, 2004; revised October 25, 2004; accepted November 19, 2004. Project supported by the National Natural Science Foundation of China (No. 20325516) and Science and Technology Foundation of Jiangsu (No. BK 2004210). © 2005 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 276 Chin. J. Chem., 2005, Vol. 23, No. 3 YAO et al. － and sensitive biosensor compared to the sol-gel/chitosan chitosan flake in hot 0.05 mol•L 1 acetic acid. The solu- modified GOX biosensor. Such devices are of consid- tion was cooled to room temperature and stirred at 250 erable interest due to their tremendous promise for ob- rpm for 3 h. taining sequence-specific information in a faster, sim- A platinum disk electrode (1 mm diameter) was used pler, and cheaper manner than sol-gel method. The op- as the base electrode for the preparation of the chito- timized conditions of the biosensor were studied, and san/glucose oxidase biosensor. The Pt electrode was the analytical performance of the biosensor was evalu- polished with 1700 diamond paper, followed by 0.3, 0.2 ated. µm alumina particles on chamois leather, then washed successively with 1∶1 nitric acid, alcohol and doubly Experimental distilled water in an ultrasonic bath, and dried in air be- fore use. Due to the weak solubility of chitosan, under Reagents our conditions, the film would be very stable after their Glucose oxidase (GOD, EC 1.1.3.4, Type VII-S, cross-linking. 196.000 U/g, Aspergillus niger) and glucose were ob- 0.2 mg of GOD was dissolved in 0.5 mL of chitosan tained from Sigma. Chitosan (MW 1.68×105, 91.7% solution (1%), and then 10 µL of glutaraldehyde solu- deacetylation) was purchased from Nantong Shuanglin tion (2.5%) was added into the GOD-chitosan solution. Co. The mixture was hand-mixed completely. The Pt elec- Glucose stock solutions were allowed to mutarotate trode was coated with a drop of 5 µL of the resulting at room temperature overnight before use. Other re- mixture, and then was left for at least 24 h at 4 ℃. Thus, agents were of analytical grade. All solutions were pre- GOD was entrapped in the membrane. The enzyme pared in doubly distilled water. biosensor was stored at 4 ℃ in a refrigerator when not in use. Apparatus and measurements All electrochemical experiments were carried out in Results and discussion a three-electrode cell controlled by a CHI 660 electro- chemical workstation (CH Instruments, USA). A chito- Morphologies of chitosan and chitosan/GOD films san glucose sensor was used as the working electrode. SEM images as shown in Figure 1 characterized the Reference and counter electrodes were an SCE and micrographs of the chitosan and chitosan/GOD films. platinum wire, respectively. Obviously, the membrane of chitosan is in homogene- Fourier transform infrared (FTIR) spectra by KBr ous structure (Figure 1a) and the surface is symmetri- pellets containing the chitosan films or chitosan/GOD cally flat. Compared to the chitosan film, the blend － were obtained in the range of 4000—500 cm 1 on a membrane of chitosan/GOD has a bump structure (Fig- Bruker 22 FT-IR spectrometer at room temperature. ure 1b). The bump, the sizes and shapes of which are UV-visible absorption spectra were obtained with a regular and uniform, was considered glucose oxidase to UV-3100 spectrophotometer (Shimadzu) at room tem- be immobilized in the chitosan network film. perature. Scanning electron micrographs (SEM) of chitosan Intermolecular interaction between chitosan and and chitosan/GOD film were taken on a SEM glucose oxidase (JSM-5900) instrument. The interactions between chitosan and glucose oxi- dase were studied by infrared spectra and UV-visible Preparation of glucose biosensor spectra. The FITR spectra of chitosan, GOD and A 1.0% chitosan solution was prepared by dissolving GOD/chitosan are shown in Figure 2(a). Figure 1 SEM photos of chitosan film before (a) and after (b) immobilized enzyme. © 2005 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Biosensor Chin. J. Chem., 2005 Vol. 23 No. 3 277 Figure 2 FT-IR (a) and UV-Vis (b) spectra of chitosan and chitosan/GOD. The IR spectrum of chitosan showed three strong H2O2 was liberated from the enzyme base reaction cor- － peaks at 1024, 1086 and 1150 cm 1, which were char- respondingly, and the oxidation current (under air satu- acteristic peaks of the saccharide structure. There were ration) decreased with the increase of H2O2 concentra- － two weak absorption peaks of amide at 1654 cm 1 and a tion at electrode surface. According to the results in － middle strong peak of 1597 cm 1, indicating that chito- Figure 3, a working potential of ＋0.6 V was chosen for san had a very high deacetylation degree.12 In the case the amperometric determination of glucose. of GOD, the most important features are the amide І － band (around 1650 cm 1) and amide II band (around － 1540 cm 1) of the amide group. The IR spectra of blend film are almost identical and similar to those of chitosan. However, there were some differences between the IR spectra of chitosan and GOD/chitosan. The adsorption － － band at 1654 cm 1 of chitosan shifted to 1657 cm 1 and the intensity increased, and in this spectrum a new weak － band appeared at 1537 cm 1, which was assigned to the absorption bands of glucose oxidase. Another feature at － about 1151 cm 1 could be clearly observed and the in- tensity decreased.