Co-immobilization of lipase, glycerol kinase, glycerol-3-phosphate oxidase and peroxidase on to aryl amine glass beads affixed on plastic strip for determination of triglycerides in serum

Abstract

Commercial lipase, glycerol kinase (GK), glycerol-3-phosphate oxidase (GPO) and peroxidase (POD) have been co-immobilized covalently on to arylamine glass beads affixed on a plastic strip through diazotization with a conjugation yield of 89.1 mg/g support and 64.1% retention of specific activity. The co-immobilized enzymes showed maximum activity at pH 7.5, when incubated at 40°C for 20 min. The strip was employed for determination of serum triglycerides (Tgs). The minimum detection limit of the method was 0.20 mM/L. The recovery of added Tgs was 88.0%. Within day and between day coefficient of variations were <7.0 % and <11.0%, respectively. A good correlation (r = 0.982) was observed between total serum Tgs values obtained by present method and the most commonly used enzymic colorimetric method, employing free enzymes. Among the various serum substances tested at their physiological concentrations, only cholesterol, ascorbic acid and bilirubin caused 30%, 15%, and 20% inhibition of strip-bound enzymes, respectively. The strip lost 50% of its activity after 150 regular uses over a period of 33 days, when stored in reaction buffer at 4°C. The method reported here has the advantage over other existing methods, as it provides higher sensitivity, better stability and reusability of co-immobilized enzymes and is also economical.