Biosensors and Bioelectronics 22 (2007) 816–821 Amperometric phenol biosensor based on laponite clay–chitosan nanocomposite matrix Quan Fan a , Dan Shan a , Huaiguo Xue a,∗ , Yuanyuan He a , Serge Cosnier b a School of Chemistry and Chemical Engineering, Yangzhou University, Yangzhou 225002, China b Laboratoire d’Electrochimie Organique et de Photochimie R´ edox, UMR CNRS 5630, Institut de Chimie Mol´ eculaire de Grenoble (FR CNRS 2607), Universit´ e Joseph Fourier, Grenoble, France Received 28 October 2005; received in revised form 21 February 2006; accepted 1 March 2006 Available online 19 April 2006 Abstract A novel strategy to fabricate an amperometric biosensor for phenol determination based on chitosan/laponite nanocomposite matrix was described. The composite film was used to immobilize PPO on the surface of a glassy carbon electrode. Chitosan was utilized to improve the analytical performance of the pure clay-modified bioelectrode. The biosensor exhibited a series of properties: good affinity to its substrate (the apparent Michaelis–Menten constant for the sensor was found to be 0.16 mM), high sensitivity (674 mA M -1 cm -2 for catechol) and remarkable long-term stability in storage (it retains 88% of the original activity after 60 days). In addition, optimization of the biosensor construction as well as effects of experimental variables such as pH, operating potential and temperature on the amperometric response of the sensor were discussed. © 2006 Elsevier B.V. All rights reserved. Keywords: Phenol biosensor; Nanocomposite material; Laponite; Clay; Chitosan 1. Introduction Inorganic clay has been widely exploited for the designs of biosensors, especially in the group of Cosnier (Poyard et al., 1998; Cosnier et al., 2000; De Melo et al., 2002; Shan et al., 2002, 2003, 2004). Clays exhibit many advantageous features as enzyme immobilization matrix. The unusual intercalation properties of clays, as well as their porosity due to a swelling phenomenon in water, allows enzyme immobilization without covalent binding and provides host matrices which exhibit a high hydrophilic character (Mousty, 2004). The entrapment of biomolecules in inorganic clays can also effectively constitute a cheap, fast and easy method for the elaboration of enzyme elec- trodes by adsorption. This procedure consists of the adsorption of an enzyme/clay aqueous colloidal mixture on an electrode surface. Glutaraldehyde is generally used as a chemical cross- linking agent to prevent the release of the encapsulated enzyme molecules from the clay film. ∗ Corresponding author. Tel.: +86 514 7975520; fax: +86 514 7975590/8410. E-mail address: chhgxue@yzu.edu.cn (H. Xue). Inorganic clay films may crack when kept dry for storage, leading to serious problems, such as a short lifetime of the biosensor. In some extreme cases it can lead to the removal of the film from the electrode surface. Concerning the swelling property of the inorganic gel, it may limit the practical appli- cation of these biosensors in some cases (Wang et al., 1998; Tan et al., 2005). Glutaraldehyde contains complicated chemi- cal species of documented cytotoxic nature and can cause the denaturation of the immobilized enzyme to some extent (Shan et al., 2004; Wang et al., 2003). Therefore, particular efforts should be devoted to overcome the weakness of the pure inor- ganic clay-modified biosensors and to improve their analytical performances. The determination of phenolic compounds is of great impor- tance due to their toxicity and persistency in the environ- ment. With these aims in view, the present article describes a novel biosensor for monitoring phenol based on a polyphenol oxidase (PPO) entrapped in a inorganic–organic hybrid film: laponite/chitosan. Laponite is a synthetic cationic clay of formula of (Mg 5.5 Li 0.5 )Si 4 O 10 (OH) 2 (Na + 0.73 ·nH 2 O) (Tanabe et al., 1989). When suspended in deionized water, at a concentration <10 g l -1 , a complete delamination process of the elementary 0956-5663/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2006.03.002