Full Paper Electrochemical Cholesterol Sensor Based on Tin Oxide-Chitosan Nanobiocomposite Film Anees A. Ansari,* Ajeet Kaushik, Pratima R. Solanki, B. D. Malhotra* Department of Science & Technology Centre on Biomolecular Electronics, National Physical Laboratory, Dr. K.S. Krishnan Marg, New Delhi-110012, India *e-mail: bansi.malhotra@gmail.com; aneesaansari@gmail.com Received: October 24, 2008 Accepted: December 26, 2008 Abstract A chitosan (CS)-tin oxide (SnO 2 ) nanobiocomposite film has been deposited onto an indium-tin-oxide glass plate to immobilize cholesterol oxidase (ChOx) for cholesterol detection. The value of the Michaelis – Menten constant (K m ) obtained as 3.8 mM for ChOx/CS-SnO 2 /ITO is lower (8 mM) than that of a ChOx/CS/ITO bioelectrode revealing enhancement in affinity and/or activity of ChOx towards cholesterol and also revealing strong binding of ChOx onto CS-SnO 2 /ITO electrode. This ChOx/CS-SnO 2 /ITO cholesterol sensor retains 95% of enzyme activity after 4 – 6 weeks at 4 8C with response time of 5 s, sensitivity of 34.7 mA/mg dL 1 cm 2 and detection limit of 5 mg/dL. Keywords: Chitosan, SnO 2 nanoparticles, Cholesterol biosensor, Nanobiocomposite DOI: 10.1002/elan.200804499 1. Introduction Estimation of cholesterol by a rapid, cost-effective and reliable method is considered as very important for clinical diagnosis since its level in blood is closely related to human health [1 – 5]. Normal concentration of cholesterol in human serum is in the range of 1.3 – 2.6 mg mL 1 of which 30% is present as sterol and 70% is esterified with fatty acids [3, 5]. The high cholesterol level in blood increases the risk of developing clinical disorders such as hypertension, arterio- sclerosis and myocardial infarction [3 – 5]. Among the analytical methods reported in literature, enzymatic meth- ods offer improvements in terms of specificity and selectiv- ity [1, 2]. The immobilization of proteins, enzymes and DNA on desired substrates plays an important role for rapid, sensitive and selective detection of desired analytes [1 – 5]. Chitosan (CS) is a biopolymer that has recently been used for biosensor application due to its biocompatibility, biode- gradability, nontoxicity, nonimmunogenicity, excellent film forming ability with good adhesion, mechanical strength, higher permeability and cost effectiveness [6 – 10]. More- over, CS has found applications in many fields such as biomedicine, waste water treatment, functional membranes and flocculation. Xu et al. have developed an electrochem- ical method of DNA sequence-specific using a DNA probe labeled with aminoferrocene and CS modified electrode immobilized with single stranded DNA [7]. Miao et al. have prepared amperometric hydrogen peroxide sensor based on immobilization of horseradish peroxidase (HRP) in CS cross-linked with glutaraldehyde [8]. Wei et al. have fab- ricated acetyl yellow-9-modified film of chitosan for immo- bilization of glucose oxidase [9]. Cruz et al. have studied electrochemical properties of CS films fabricated on glassy carbon electrodes [10]. Metal oxide nanoparticles due to high surface-to-volume ratio for enzyme loading, high electron communication to obtain low detection limit and better adsorption capability for high stability have been used for application to biosensors. And sensing characteristics of CS can perhaps be improved by incorporating metal oxide nanoparticles such as zinc oxide (ZnO), iron oxide (Fe 3 O 4 ), cerium oxide (CeO 2 ), zirconium oxide (ZrO 2 ) and titanium oxide (TiO 2 ) etc. [6, 11 – 15]. Among the metal oxide nanoparticles, tin oxide (SnO 2 ) based nanoparticles have recently been used as an interesting matrix for immobilization of desired biomolecules [16, 17]. SnO 2 nanoparticles having a large band gap (330 nm), isoelectric point (IEP ~ 5.0) and higher conductivity compared to that of TiO 2 and SiO 2 has found applications in microelectronics, photoelectronics, solar cells, sensing devices etc [18 –19]. Topoglidis et al. have immobilized proteins (cytochrome c and hemoglobin) on nanostructured SnO 2 film [17]. The incorporation of nano- structured SnO 2 greatly enhances the active surface area available for protein immobilization. Jia et al. have inves- tigated direct electrochemistry and electrocatalysis of horseradish peroxidase (HRP) immobilized on sol-gel- derived SnO 2 /gelatin composite film [16]. We report results of the studies relating to preparation of the CS-SnO 2 /ITO nanobiocomposite film for immobiliza- tion of ChOx for application to cholesterol sensor. 965  2009 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim Electroanalysis 2009, 21, No. 8, 965 – 972