Construction of an amperometric enzymic sensor for triglyceride determination

An amperometric enzyme sensor for determination of triglycerides (TGs) was constructed by mounting cellulose acetate (CA) membrane-bound commercial lipase (E.C.3.1.1.3), glycerol kinase (GK) (E.C.2.7.1.30), and glycerol-3-phosphate oxidase (GPO) (E.C.1.1.3.21), on a platinum electrode (working electrode), then connecting it to potentiostat along with Ag/AgCl reference electrode and Ag auxillary electrode. The biosensor measures the electrons generated from H2O2 under a potential of 0.4 V which in turn formed from triolein/triglycerides by co-immobilized lipase, GK and GPO. The concentration of triolein/TG was directly proportional to the current measured. The enzyme electrode showed optimum response when operated at 25 °C in 0.1 M sodium phosphate buffer, pH 6.5 for 40 s. A linear relationship was obtained between triolein concentration ranging from 0.2 to 3.5 mM and amount of current (mA). The minimum detection limit of the method was 0.2 mM. The level of TG in serum of healthy and persons suffering from cardiovascular disease and pancreatitis was in the range 53.2–174.5 and 189–990 mg/dl, respectively. The analytical recovery of added triolein was 89.0%. Within batch and between batches CV (coefficient of variation) were <8 and <4%, respectively. A good correlation (r = 0.91) was found between serum TG values obtained by commercial enzymic colorimetric and present method. Among the various serum substances tested only cholesterol and ascorbic acid caused 18 and 16% inhibition, respectively. The enzyme electrode was used 150 times over 25 days without any significant loss of activity, when stored at 4 °C.