Bioelectrochemical characteristics of cholesterol oxidase immobilized in a polyaniline film

Cholesterol oxidase has been immobilized in polyaniline film by the electrochemical doping method. The measurements of the response current are carried out in the solutions containing cholesterol and Triton X-100 of 5% and 1%, respectively. The latter is necessary for solubilizing cholesterol. The response current of the polyaniline cholesterol oxidase electrode increases with increasing potential from 0.35 to 0.60 V (vs. SCE). A maximum response current occurs at 0.60 V, which is independent of the concentration of Triton X-100. The response current of the enzyme electrode at Triton X-100 of 1% is about twice as high as that at Triton X-100 of 5%. The optimum pH and the optimum temperature of the immobilized cholesterol oxidase are 7.26 and 35°C, respectively. The activation energy of the enzyme-catalyzed reaction is 71.1 kJ mol−1 at Triton X-100 of 5% and 67.4 kJ mol−1 at Triton X-100 of 1%. The apparent Michaelis–Menten constant Km′ is 2.72 mmol dm−3 at 0.45 V and 3.26 mmol dm−3 at 0.60 V for Triton X-100 of 5%, and 0.51 mmol dm−3 at 0.45 V and 0.56 mmol dm−3 at 0.60 V for Triton X-100 of 1%. The response current of the enzyme electrode increases linearly with increasing concentration of cholesterol in the range 0.05–0.5 mmol dm−3 at 0.45 V and 0.05 to 0.2 mmol dm−3 at 0.60 V for Triton X-100 of 5%, and 0.01 to 0.1 mmol dm−3 at both potentials of 0.45 and 0.60 V for Triton X-100 of 1%. The response current of the enzyme electrode drops to 51% of its initial value after 11 days. During this period, this enzyme electrode is employed to determine the substrate solved in Triton X-100 of 1% at different conditions for 230 times. Thus, the polyaniline cholesterol oxidase electrode can be used to determine cholesterol concentration.