Fabricating an Amperometric Cholesterol Biosensor by a Covalent Linkage between Poly(3-thiopheneacetic acid) and Cholesterol Oxidase.docVIP

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Fabricating an Amperometric Cholesterol Biosensor by a Covalent Linkage between Poly(3-thiopheneacetic acid) and Cholesterol Oxidase.doc

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Fabricating an Amperometric Cholesterol Biosensor by a Covalent Linkage between Poly(3-thiopheneacetic acid) and Cholesterol Oxidase

Sensors 2009, 9, 1794-1806; doi:10.3390/OPEN ACCESS sensors ISSN 1424-8220 /journal/sensors Article Fabricating an Amperometric Cholesterol Biosensor by a Covalent Linkage between Poly(3-thiopheneacetic acid) and Cholesterol Oxidase Po-Chin Nien 1, Po-Yen Chen 1 and Kuo-Chuan Ho 1,2,* 1 Department of Chemical Engineering, National Taiwan University/No. 1, Sec. 4, Roosevelt Rd., Taipei 10617, Taiwan; E-Mails: .tw (P.C.N.); .tw (P.Y.C.) 2 Institute of Polymer Science and Engineering, National Taiwan University/No. 1, Sec. 4, Roosevelt Rd., Taipei 10617, Taiwan. * Author to whom correspondence should be addressed; E-Mail: kcho@.tw (K.C.H.); Tel.: +886-2-2366-0739; Fax: +886-2-2362-3040 Received: 16 December 2008; in revised version: 2 March 2009 / Accepted: 13 March 2009 / Published: 13 March 2009 Abstract: In this study, use of the covalent enzyme immobilization method was proposed to attach cholesterol oxidase (ChO) on a conducting polymer, poly(3-thiopheneacetic acid), [poly(3-TPAA)]. Three red-orange poly(3-TPAA) films, named electrodes A, B and C, were electropolymerized on a platinum electrode by applying a constant current of 1.5 mA, for 5, 20 and 100 s, respectively. Further, 1-ethyl-3-(3-dimethylamiopropyl)carbodiimide hydrochloride (EDC HCl) and N-hydroxysuccinimide (NHS) were used to activate the free ? carboxylic groups of the conducting polymer. Afterwards, the amino groups of the cholesterol oxidase were linked on the activated groups to form peptide bonds. The best sensitivity obtained for electrode B is 4.49 mA M-1 cm-2, with a linear concentration ranging from 0 to 8 mM, which is suitable for the analysis of cholesterol in humans. The response time (t95) is between 70 and 90 s and the limit of detection is 0.42 mM, based on the signal to noise ratio equal to 3. The interference of species such as ascorbic acid and uric acid increased to 5.2 and 10.3% of the original