Amperometric determination of total cholesterol at gold electrodes covalently modified with cholesterol oxidase and cholesterol esterase with use of thionin as an electron mediator

Immobilization of cholesterol oxidase (EC 1.1.3.6) (ChOx) on a gold electrode was attempted by cross-linking using glutaraldehyde between ChOx molecules and a self-assembled monolayer of 2-aminoethanethiolate. The resulting electrode (ChOx/Au) exhibits an amperometric response to free cholesterol in the presence of thionin as an electron mediator, and a steady-state response is obtained similar to 60 s after injection of cholesterol into the electrolyte solution. Coimmobilization of cholesterol esterase (EC 3.1.1.13) (ChE) and ChOx (ChE/ChOx/Au) allows the amperometric determination of both esterified cholesterol and free cholesterol. Cyclic voltammetry of the ChE/ChOx/Au and the dependence of the amperometric response to cholesterol on the concentration of thionin suggest that thionin is encapsulated in the enzyme film on the electrode surface. Apparent Michaelis constants of the ChOx/Au and the ChE/ChOx/Au electrodes suggest that the amperometric response was controlled by penetration of the reaction substrate into the films of the enzyme(s). The concentration of total (free and esterified) cholesterol in human serum samples, determined by using the techniques developed in the present study, is in good agreement with that determined by the well-established technique using colorimetry.