Optical biosensing of nitrite ions using cytochrome cd(1) nitrite reductase encapsulated in a sol-gel matrix

Nitrite is an important human health and environmental analyte. As such, th e European Union (EU) has imposed a limit for nitrite in potable water of 0 .1 mg l(-1) (2.18 muM). In order to develop an optical biosensing system fo r the determination of nitrite ions in environmental waters, cytochrome cd( 1) nitrite reductase has been extracted and purified from the bacterium Par acoccus pantotrophus. The protein has been spectroscopically characterised in solution and important kinetic parameters of nitrite reduction of the cy tochrome cd(1) enzyme, i.e., K-m, V-max and k(cat) have been determined. Th e influence of pH on the activity of the cytochrome cd(1) has been investig ated and the results suggest that this enzyme can be used for the determina tion of nitrite in the pH range 6-9. Biosensing experiments with the cytoch rome cd(1) in solution suggested that the decrease in intensity of the abso rption band associated with the d(1) haem (which is the nitrite binding sit e), at 460 nm, with increasing nitrite concentrations would enable the meas urement of this analyte with the optimum limit of detection. The cytochrome cd(1) has been encapsulated in a bulk sol-gel monolith with no structural changes observed and retention of enzymatic activity. The detection of nitr ite ions in the range 0.075-1.250 muM was achieved, with a limit of detecti on of 0.075 muM. In order to increase the speed of response, a sol-gel sand wich thin film structure was formulated with the cytochrome cd(1). This str ucture enabled the determination of nitrite concentrations within ca. 5 min . The sol-gel sandwich entrapped cytochrome cd(1) enzyme was found to be st able for several months when the films were stored at 4 degreesC.