Factors concerning the design and calibration of an amperometric enzyme electrode system for the flow injection analysis of cholesterol

Abstract

An amperometric enzyme electrode in a flow injection analysis system is described, based on cholesterol oxidase immobilised on nylon mesh and held over a platinum electrode in a modified three-electrode amperometric Stelte cell. The electrode is operated at +600 mV versus a silver-silver chloride reference electrode, using a carrier stream (2.3 cm³ min^–1) of phosphate buffer (0.1 M, pH 7.0) containing Triton X-100 (1%). Owing to electrode interference by the solublising surfactant, aqueous cholesterol calibrating standards should be confined to ca. 1%V/V Triton X-100 when the calibration range used is 10 µM–1.035 mM of cholesterol. The response times of <60 s and a wash time (time between samples) of <90 s permit a fast throughput of samples.

The enzyme electrode lifetime (ca. 6 weeks) is governed by the deleterious effect of the surfactant used for solubilising cholesterol and by cholesterol oxidase activity. As regards enzyme quality, isoelectric focusing and steric exclusion chromatography indicate the cholesterol oxidase to be more heterogeneous than the other enzymes (glucose oxidase, xanthine oxidase and peroxidase) examined.