Development of a bienzyme system for the electrochemical determination of nitrate in ambient air

This work reports the development of a bienzyme system consisting of salicylate hydroxylase (SHL) and nitrate reductase (NaR) for the electrochemical determination of nitrate. This method measures the concentration of nitrate directly under ambient air without suffering from oxygen interferences. The determination is based on the detection of NADH consumption, and the principle is as follows: NADH initiates the irreversible decarboxylation and hydroxylation of salicylate by SHL in the presence of oxygen to produce catechol, which results in a detectable signal due to its oxidation at the working electrode; the second enzyme, NaR, in the presence of nitrate, reduced the availability of NADH, and consequently, the current difference after the injection of nitrate is proportional to its concentration. This method shows high performance characteristics for nitrate determination with a broad detection range between 10 microM and 1,000 microM, a short measuring time of around 5 min, and a simple operation without sample pretreatment by inert gas purge or oxygen scavenger.