Development and characterization of a nitrite biosensor based on NirB nitrite reductase for environmental monitoring

Nitrite is prevalent both in the natural environment and the human body. However, high concentrations of nitrite can threaten the ecosystem, and sensitive detection of nitrite is of great significance for the environment and human health. Nitrite measurement in environmental samples can be challenging due to the interference of the coexisting ions. Enzyme-based biosensors with their substrate-specific bioagents have a high potential for environmental monitoring and speedy on-site detection. The discovery of novel enzymes specific to environmental pollutants is important for the development of biosensors with high specificity and low interference. The recombinant assimilatory nitrite reductase (NirB) enzyme of Escherichia coli was used in the present study to generate an electrochemical amperometric nitrite biosensor, and its characteristics were improved for nitrite measurement in environmental samples. The NirB/MWCNTs/CHIT/MV/GC electrode was prepared by enzyme immobilization in methyl viologen-modified carbon nanotube-chitosan layers. Increasing the amount of chitosan to 32 ng in the composite matrix improved the sensitivity (6965 mA/M.cm^-2) and LOD (0.22 ± 0.07 µM) of the electrode in PBS. The biosensor did not show any cathodic response to contaminants, including sulfite, ammonium, formate, acetate, chlorine, or sulfate, but to nitrate. A 17% decrease in the response to nitrite was observed when all the tested contaminants were included in the working buffer. Rainwater and seawater samples were used to test biosensor accuracy. The LOD of the biosensor for two different rainwater samples and a seawater sample was measured as 1.21 ± 0.24, 1.89 ± 0.28, and 1.54 ± 0.33 µM, respectively. The sensor provides accurate results when the nitrite concentration of the environmental sample is above the respective LOD levels. The NirB-based nitrite biosensor developed in this study may offer a wide range of potential applications in environmental monitoring and food safety.