Preparation of a reusable enzyme strip for determination of serum cholesterol

Abstract

A reusable enzyme strip for the determination of cholesterol was prepared by co-immobilizing cholesterol esterase (CEase), cholesterol oxidase (COD) and peroxidase (POD) onto alkylamine glass beads af­fixed on a plastic strip by a non-reactive fixative. The co-immobilized enzymes retained 71.42% of initial activity of free enzymes with a conjugation yield of 11.0 mg g^-1 beads and showed optimum activity at <i style="">p</i>H 6.6, when incubated at 32°C for 6 min. The strip was employed for direct determination of total cholesterol in serum. The minimum detection limit of the method was 2.14 mg dL^-1 reaction mixture. The within batch and between batch coefficient of variation (CV) were <7.0% and <19.0%, respectively. The serum total cholesterol values as determined by the strip, were in good correlation (<i style="">r</i>=0.885) with those by commercial enzo-kit method employing free enzymes. The method is characterized by reuse of co-immobilized enzymes with great ease and unaffected by various serum substances.