A Biosensor Utilizing l-Glutamate Dehydrogenase and Diaphorase Immobilized on Nanocomposite Electrode for Determination of l-Glutamate in Food Samples

摘要

Amperometric biosensor utilizing bienzymatic composition consisting of l-glutamate dehydrogenase and diaphorase for the determination of l-glutamate has been developed. Enzymes were immobilized between chitosan layers onto the surface of planar nanocomposite electrodes consisting of multi-walled carbon nanotubes (diameter=60–100 nm; length=5–15 mu m, 95+% purity). Linear response was obtained from 10 to 3,495 mu M in phosphate buffer solution of pH 9.0 and in the presence of enzyme cofactor NAD+ (2mM) and mediator ferricyanide (5 mM). The limit of detection was 5.4 mu M, and sensitivity was found to be 28 nA mu M-1 cm-2. The biosensor showed a short response time (within 60 s), good storage (no loss of activity for at least 3 months), and operational (response ability above 90 % after 7 days since its first use) stability. Finally, the results obtained from measurements of the food samples were compared with those obtained with an enzymatic–spectrophotometric method and correlated well. Analytical performance of the biosensor indicated that the bienzyme system utilizing diaphorase as a secondary enzyme could be a general basis for other biosensors based on NAD+-dependent dehydrogenases.