Determination of serum triglyceride by enzyme electrode using covalently immobilized enzyme on egg shell membrane

摘要

A mixture of commercial lipase, glycerol kinase and glycerol-3-phosphate oxidase was co-immobilized onto egg shell membrane through covalent coupling. A method is described for fabrication of a triglyceride (TG) biosensor using egg shell membrane bound enzymes. The biosensor measured current, i.e. flow of electrons generated from H_2O_2, maximally when polarized at 0.4V. The biosensor showed optimum response within 10sec at pH 7.0 and 35°C. The current was in proportion to concentration of TG in the range 0.56-2.25mM. An amperometric method was developed for determination of TG employing this enzyme electrode. The minimum detection limit of the method was 0.28mM. The analytic recovery of added TG was 95.00% and 96.50%. Within batch and between batch coefficients of variations (CV) were <2.14% and <3.48% respectively. A good correlation (r=0.985) was obtained between serum TG level by standard enzymic colorimetric method and the present method. Serum substances such as urea, uric acid, glucose, cholesterol, ascorbic acid and pyruvic acid had no interference. The enzyme electrode was used 200 times over a period of 70 days without any considerable loss of activity, when stored at 4°C.