Amperometric assays of total and free cholesterols in serum by the combined use of immobilized cholesterol esterase and cholesterol oxidase reactors and peroxidase electrode in a flow injection system

Abstract

A flow injection system for assays of total cholesterol and free cholesterol was described. The total cholesterol assay system included an amperometric peroxidase electrode to measure hexacyanoferrate(III) converted from hydrogen peroxide, which was generated by injecting a 2-μl sample into the packed-bed reactors of immobilized cholesterol esterase and cholesterol oxidase covalently bound to silica. The free cholesterol was assayed with the same system without the cholesterol esterase reactor. The peak current was linearly related to cholesterol in the range 2–160 mg/dl and to total cholesterol in the range 3–300 mg/dl; the assay speed was about 80 samples/h for free cholesterol and 40 samples/h for total cholesterol. Reliable results were obtained in the assays of free cholesterol and total cholesterol in human sera. Both the reactors and the peroxidase electrode retained over 90% of their original activities, even after repetitive use for 4 and 2 months, respectively.