Analytica Chimica Acta 502 (2004) 229–234 Amperometric cholesterol biosensor based on immobilized cholesterol esterase and cholesterol oxidase on conducting polypyrrole films Suman Singh, Asha Chaubey, B.D. Malhotra ∗ Biomolecular Electronics and Conducting Polymer Research Group, National Physical Laboratory, Dr. K.S. Krishnan Marg, New Delhi 110012, India Received 10 June 2003; received in revised form 22 September 2003; accepted 30 September 2003 Abstract Fabrication of an amperometric cholesterol biosensor by co-immobilization of cholesterol esterase (ChEt) and cholesterol oxidase (ChOx) onto conducting polypyrrole (PPY) films using electrochemical entrapment technique is described. Electrochemical polymerization was carried out using a two-electrode cell configuration at 0.8V. Characterization of resulting amperometric biosensor for the estimation of cholesterol has been experimentally determined in terms of linear response range, optimum pH, applied potential, temperature, and shelf-life. These PPY/ChEt/ChOx electrodes can be used for cholesterol ester estimation from 1 to 8 mM and have shelf-life of about 4 weeks at 4 ◦ C during which about 15 estimations of cholesterol ester could be made. The sensitivity of PPY/ChEt/ChOx electrode has been found to be 0.15 A/mM and the apparent K m value for this electrode is 9.8 mM. Conductivity of the polymer films found to be about 3 × 10 -3 S/cm. © 2003 Published by Elsevier B.V. Keywords: Immobilization; Biosensor; Cholesterol esterase; Cholesterol oxidase; Polypyrrole 1. Introduction There is increased demand for developing fast and sen- sitive methods for the determination of various analytes present in the whole blood. Biosensors have been con- sidered to be one of the most suitable devices due to selectivity, fast response, miniature size, reliable and re- producible results. Biosensors using immobilized enzymes as the bio-recognition element are among the most widely investigated devices for both fundamental research and ap- plications point of view [1–4]. In this context, design and study of new amperometric biosensors for the estimation of concentration of various analytes of clinical interest such as glucose, lactate, and urea including cholesterol have received considerable attention [5–10]. Cholesterol and its fatty acid esters are important com- pounds for human beings because they are the components of nerve and brain cells and are precursors of other bio- logical materials such as bile acid and steroid hormones [11,12]. The estimation of cholesterol in blood is an impor- tant parameter for the diagnosis and prevention of several ∗ Corresponding author. Fax: +91-11-25726938. E-mail address: bansi@csnpl.ren.nic.in (B.D. Malhotra). heart diseases, cerebral thrombosis, and arteriosclerosis [13–15]. Several cholesterol biosensors including enzymatic and non-enzymatic methods have been reported [16–20]. However, these often have certain difficulties like lack of specificity and selectivity due to the presence of various interfering reactions and the use of unstable and corrosive reagents. Organic conducting polymers have recently emerged as a new class of electroactive materials and are interesting subjects for research and development [21–24]. This is due to the fact that they possess the ability to bind oppo- sitely charged complex entities in their oxidized conducting states and release them in their neutral insulating states [25]. Polypyrrole (PPY) is found to be of particular interest among various conducting polymers [26–28]. Its low ox- idation potential enables a conducting polymer film to be grown from aqueous solutions that is compatible with most of the biological elements [29]. Moreover, its easy poly- merization, high electrical conductivity, chemical stability and ability to form freestanding films are added advantages for its application to biosensors. Reports are available for determination of free choles- terol using cholesterol oxidase (ChOx) bound to conducting polymer matrix (polypyrrole or polyaniline) [28,30]. Vidal et al. [31] have recently prepared a cholesterol amperometric 0003-2670/$ – see front matter © 2003 Published by Elsevier B.V. doi:10.1016/j.aca.2003.09.064