A novel nitrite biosensor based on conductometric electrode modified with cytochrome c nitrite reductase composite membrane

A conductometric biosensor for nitrite detection was developed using cytochrome c nitrite reductase (ccNiR) extracted from Desulfovibrio desulfuricans ATCC 27774 cells immobilized on a planar interdigitated electrode by cross-linking with saturated glutaraldehyde (GA) vapour in the presence of bovine serum albumin, methyl viologen (MV), Nafion^®, and glycerol. The configuration parameters for this biosensor, including the enzyme concentration, ccNiR/BSA ratio, MV concentration, and Nafion^® concentration, were optimized. Various experimental parameters, such as sodium dithionite added, working buffer solution, and temperature, were investigated with regard to their effect on the conductance response of the biosensor to nitrite. Under the optimum conditions at room temperature (about 25 °C), the conductometric biosensor showed a fast response to nitrite (about 10 s) with a linear range of 0.2-120 μM, a sensitivity of 0.194 μS/μM [NO₂^-], and a detection limit of 0.05 μM. The biosensor also showed satisfactory reproducibility (relative standard deviation of 6%, n = 5). The apparent Michaelis-Menten constant (K_M,app) was 338 μM. When stored in potassium phosphate buffer (100 mM, pH 7.6) at 4 °C, the biosensor showed good stability over 1 month. No obvious interference from other ionic species familiar in natural waters was detected. The application experiments show that the biosensor is suitable for use in real water samples.