Fabrication of glycerol biosensor based on co-immobilization of enzyme nanoparticles onto pencil graphite electrode

Glycerol kinase (GK) and glycerol-3- phosphate oxidase (GPO) nanoparticles (NPs) were prepared, characterized and immobilized onto pencil graphite (PG) electrode to fabricate an improved amperometric glycerol biosensor (GKNPs/GPONPs/PGE). GKNPs/GPONPs/PGE worked in optimum conditions of pH 7.0, temperature 30 °C, at an applied potential of -0.3 V. The biosensor exhibited wide linear response in a concentration range of glycerol (0.01-45 mM) with detection limit 0.0001 μM. The biosensor revealed high sensitivity (7.24 μAmM^-1cm^-2), low response time (2.5s) and a good agreement with the standard enzymic colorimetric method with a correlation coefficient (R² = 0.99). The evaluation study of biosensor offered a good analytical recovery of 98.73% when glycerol concentration was added to the sera sample. In addition, within and between batches study of working electrode showed coefficients of variation as 0.105% and 0.14%, respectively. The application of biosensor is performed in the serum of apparently healthy subject and patients affected by cardiogenic shock. There was a 20% loss in initial activity of biosensor after its regular use over a time period of 180 days, while being stored at 4 °C.