An improved amperometric triglyceride biosensor based on co-immobilization of nanoparticles of lipase, glycerol kinase and glycerol 3-phosphate oxidase onto pencil graphite electrode

• Published in: Enzyme and microbial technology Vol. 100; pp. 11 - 16
• Main Authors: Narwal, Vinay, Pundir, C.S.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.05.2017
• Subjects: Adult; Aerococcus viridans; Biosensing Techniques - methods; biosensors; Blood Chemical Analysis - methods; blood serum; Candida rugosa; carbon electrodes; carboxylic ester hydrolases; Case-Control Studies; Cellulomonas; colorimetry; detection limit; Dielectric Spectroscopy; electrochemistry; electrons; Enzyme Stability; Enzymes, Immobilized; Female; Glycerol Kinase; Glycerol kinase nanoparticles (GKNPs); Glycerol phosphate oxidase nanoparticles (GPONPs); Glycerolphosphate Dehydrogenase; graphene; Graphite; Humans; hydrogen peroxide; hypertriglyceridemia; Hypertriglyceridemia - blood; Lipase; Lipase nanoparticles; Male; Microscopy, Electron; nanoparticles; Nanoparticles - ultrastructure; Pencil graphite electrode; people; scanning electron microscopy; silver; silver chloride; temperature; Triglyceride; Triglyceride biosensor; Triglycerides - analysis; Triglycerides - blood; triolein; Lipase nanoparticles; Glycerol kinase nanoparticles (GKNPs); Triglyceride biosensor; Triglyceride; Pencil graphite electrode; Glycerol phosphate oxidase nanoparticles (GPONPs)
• ISSN: 0141-0229; 1879-0909; 1879-0909
• DOI: 10.1016/j.enzmictec.2017.01.009

•Constructed an improved amperometric triglyceride (TG) biosensor based on co- immobilization of lipase nanoparticles (NPs), glycerol-kinase NPs, glycerol-3-phosphate oxidase NPs onto pencil graphite (PG) electrode.•Biosensor showed optimum current within 2.5s, at 0.1V, pH 7.0 and 35°C.•The limit of detection and working range of biosensor were 0.1nM and 0.1–45mM respectively.•The enzyme electrode lost 20% of its initial activity following 240days of its normal uses, while being stored at 4°C.•Biosensor measured TG level in sera of apparently healthy adults and persons experiencing hypertryglyceridemia. Nanoparticles (NPs) of commercial lipase from Candida rugosa, of glycerol kinase (GK) from Cellulomonas species, of glycerol-3- phosphate oxidase (GPO) from Aerococcus viridans were prepared, characterized and co-immobilized onto a pencil graphite (PG) electrode. The morphological and electrochemical characterization of PG electrode was performed by scanning electron microscopy (SEM), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) before and after co-immobilization of enzyme nanoparticles (ENPs). An improved amperometric triglyceride (TG) biosensor was fabricated using Lipase NPs/GKNPs/GPONPs/PG electrode as the working electrode, Ag/AgCl as the standard electrode and Pt wire as auxiliary electrode. The biosensor showed optimum response within 2.5s at a pH 7.0 and temperature of 35°C. The biosensor measured current due to electrons generated at 0.1V against Ag/AgCl, from H2O2, which is produced from triolein by co-immobilized ENPs. A linear relationship was obtained over between a wide triolein concentration range (0.1mM–45mM) and current (mA) under optimal conditions. The Lipase NPs/GKNPs/GPONPs/PG electrode showed high sensitivity (1241±20mAcm−2mM−1); a lower detection limit (0.1nM) and good correlation coeficient (R2=0.99) with a standard enzymic colorimetric method. Analytical recovery of added triolein in serum was 98.01%, within and between batch coefficients of variation (CV) were 0.05% and 0.06% respectively. The biosensor was evaluated and employed for determination of TG in the serum of apparently healthy subject and persons suffering from hypertriglyceridemia. The biosensor lost 20% of its initial activity after its continued uses over a period of 240days, while being stored at 4°C.