Bienzymatic amperometric biosensor for choline based on mediator thionine in situ electropolymerized within a carbon paste electrode

An amperometric enzyme biosensor for the determination of choline utilizing two enzymes, choline oxidase (CHOD) and horseradish peroxidase (HRP), is described. The biosensor consisted of CHOD cross-linked onto a HRP-immobilized carbon paste electrode. The biosensor was prepared by in situ electropolymerization of poly(thionine) within a carbon paste containing the enzyme HRP and thionine monomer and then CHOD was immobilized by using chitosan film through cross-linking with glutaraldehyde. The in situ electrogenerated poly(thionine) displays excellent electron transform efficiency between the enzyme HRP and the electrode surface, and the polymer enables improvement in enzyme immobilization within the paste. Several parameters such as the amount of thionine and enzyme, the applied potential, the pH, etc. have been Studied. Amperometric detection of choline was realized at an applied potential of -0.2 V vs saturated calomel electrode in 1/15 M phosphate buffer solution (pH 7.4) with a linear response range between 5.0 x 10(-6) and 6.0 x 10(-4) M choline and a response time of 15 s. When applied to the analysis of phosphatidylcholine in serum samples, a 0.997 correlation was obtained between the biosensor results and those obtained by a hospital method. (C) 2004 Elsevier Inc. All rights reserved.