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 current response, respectively, based on the current response of cholesterol (100%). With respect to the long-term stability, the sensing response retains 88% of the original current after 13 days.
Highlights
In clinical diagnosis, cholesterol is an important indicator in human blood for hypertension, myocardial infarction and arteriosclerosis
Based on the above studies, an amperometric biosensor with poly(3-thiopheneacetic acid) as a matrix has been fabricated in this study for linking cholesterol oxidase, in order to carry out the detection of cholesterol
Pt/poly(3-TPPA) electrode linked with cholesterol oxidase) film is assigned to the N-H bend of the cholesterol oxidase, which confirms the formation of covalent bonds between enzyme and polymer
Summary
Cholesterol is an important indicator in human blood for hypertension, myocardial infarction and arteriosclerosis. Enzyme-immobilization methods can be divided into four types; adsorption, entrapment, cross-linking and covalent attachment [4]. In covalent attachment, chemical bonds are formed between the enzyme and the matrix through functional groups, such as amino or carboxylic groups. Among the four methods mentioned, the covalent attachment and cross-linking methods provide better immobilizing performance than the others, which may be related to the presence of strong attractive forces formed by chemical bonds. In previous work on lactate biosensors [15], the carboxylic groups of poly(3-thiopheneacetic acid), which is one of the functional polythiophenes, were linked to the amino groups of lactate oxidase by two reagents, namely, 1-ethyl-3-(3-dimethylamiopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide. Based on the above studies, an amperometric biosensor with poly(3-thiopheneacetic acid) as a matrix has been fabricated in this study for linking cholesterol oxidase, in order to carry out the detection of cholesterol.
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