Fabrication of an Amperometric Triglyceride Biosensor based on PVC Membrane

Abstract

An amperometric enzyme sensor for determination of triglycerides (TGs) was constructed by mounting polyvinyl chloride (PVC) membrane bound lipase, glycerol kinase (GK), glycerol-3-phosphate oxidase (GPO), and horseradish peroxidase (HRP) on a platinum electrode (working electrode), then connecting it to electrometer along with Ag/AgCl reference electrode and Cu auxiliary electrode. The biosensor measures the electrons generated from H₂O₂ at 0.4 V, which is formed from triolein/triglycerides by co-immobilized lipase, GK, GPO, and HRP. 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 7.5, for 30 s. A linear relationship was obtained between triolein concentration ranging from 0.56 to 2.25 mM and amount of current (mA). The minimum detection limit of the method was 0.11 mM. The levels of TG in serum of apparently healthy persons and persons suffering from cardiovascular disease and pancreatitis, as measured by this sensor, were in the range 50–216 mg/dl and 200–499 mg/dl respectively. The analytical recovery of added triolein was 85.2%. Within-batch and between-batch coefficients of variation were <2.53% and <3.21% respectively. A good correlation (r = 0.91) was found between serum TG values obtained by commercial enzymic colorimetric and the present method. Among the various serum substances tested, only cholesterol and bilirubin caused inhibition, 15% and 8% respectively. The enzyme electrode was used 100 times over 40 days without any considerable loss of activity when stored at 4 °C.

Keywords:

• Co-immobilization
• GK
• GPO
• HRP
• Lipase
• PVC
• Serum
• TG biosensor
• Triglycerides
• Triolein

Acknowledgments

This work was supported by a grant to one of the authors (C. S. P.) from Department of Biotechnology, Government of India, New Delhi.