Development of a novel reagentless, screen-printed amperometric biosensor based on glutamate dehydrogenase and NAD~+, integrated with multi-walled carbon nanotubes for the determination of glutamate in food and clinical applications

摘要

A screen printed carbon electrode (SPCE) containing the electrocatalyst Meldola's Blue (MB) has been investigated as the base transducer for a reagentless glutamate biosensor. The biopolymer chitosan (CHIT) and multiwalled carbon nanotubes (MWCNTs) were used to encapsulate the enzyme glutamate dehydrogenase (GLDH) and the co-factor nicotinamide adenine dinucleotide (NAD~+). The biosensor was fabricated by sequentially depositing the components on the surface of the transducer (MB-SPCE) in a layer-by-layer process, details of which are included in the paper. Each layer was optimised to construct the reagentless device. The biosensor was used in conjunction with amperometry in stirred solution using an applied potential of +0.1 V (vs. Ag/AgCl). Optimum conditions for the analysis of glutamate were found to be: temperature, 35 ℃; phosphate buffer, pH 7 (0.75 mM, containing 0.05 M NaCl). The linear range of the reagentless biosensor was found to be 7.5-105 μM, and limit of detection was found to be 3 μM (based on n = 5, CV: 8.5% based on three times signal to noise) and the sensitivity was 0.39 nA/μM (±0.025, coefficient of variation (CV) of 6.37%, n = 5). The response time of the biosensor was 20-30 s. A food sample was analysed for monosodium glutamate (MSG). The endogenous content of MSG was 90.56 mg/g with a CV of 7.52%. The reagentless biosensor was also used to measure glutamate in serum. The endogenous concentration of glutamate was found to be 1.44 mM (n = 5), CV: 8.54%. The recovery of glutamate in fortified serum was 104%(n = 5),CV of 2.91%.