A new immobilization and sensing platform for nitrate quantification

Nitrate reductase (NR) from Aspergillus niger was covalently coupled to the epoxy affixed gold nanoparticles (epoxy/AuNPs) with a conjugation yield of 35.40±0.01 μg/cm2 and 93.90±0.85% retention of specific activity. The bare and NR bound epoxy/AuNPs support was characterized using scanning electron microscopy and Fourier Transform Infrared Spectroscopy. The immobilized enzyme system was optimized with respect to pH, temperature and substrate concentrations and successfully employed for determination of nitrate contents in ground water. The minimum detection limit of the method was 0.05 mM with linearity from 0.1 to 10.0 mM. The % recoveries of added nitrates (0.1 and 0.2 mM) were >95.0% and within-day and between-day coefficients of variations were 1.012% and 3.125% respectively. The method showed good correlation (R2=0.998) with the popular Griess reaction method. Epoxy/AuNPs bound NR showed good thermal and storage stabilities and retained 50% activity after 16 reuses.