Simplified Nitrate-Reductase-Based Nitrate Detection by a Hybrid Thin-Layer Controlled Potential Coulometry/Spectroscopy Technique
By Wang, Tingting; Schlueter, Kevin T.; Riehl, Bill L.; Johnson, Jay M. & Heineman, William R.
Published in Analytical Chemistry
2013
Abstract
A novel method for the detection of nitrate was developed using simplified nitrate reductase (SNaR) that was produced by genetic recombination techniques. The SNaR consists of the fragments of the Mo -molybdopterin (MO -MPT) binding site and nitrate reduction active site and has high activity for nitrate reduction. The method is based on a unique combination of the enzyme-catalyzed reduction of nitrate to nitrite by thin-layer coulometry followed by spectroscopic measurement of the colored product generated from the reaction of nitrite with Griess reagents. Coulometric reduction of nitrate to nitrite used methyl viologen (MV2+) as the electron transfer mediator for SNaR and controlled potential coulometry in an indium tin oxide (ITO) thin-layer electrochemical cell. Absorbance at 540 nm was proportional to the concentration of nitrate in the sample with a linear range of 1 -160 μM and a sensitivity of 8000 AU M -1. The method required less than 60 μL of sample. Detection of nitrate could also be performed by measuring the charge associated with coulometry. However, the spectroscopic procedure gave superior performance because of interference from the large background charge associated with coulometry. Results for the determination of nitrate concentration in several natural water samples using this device with spectroscopic detection are in good agreement with analysis done with a standard method.