Optical biosensing of nitrite ions using cytochrome cd1 nitrite reductase encapsulated in a sol–gel matrix

Abstract

Nitrite is an important human health and environmental analyte. As such, the European Union (EU) has imposed a limit for nitrite in potable water of 0.1 mg l⁻¹ (2.18 μM). In order to develop an optical biosensing system for the determination of nitrite ions in environmental waters, cytochrome cd₁ nitrite reductase has been extracted and purified from the bacterium Paracoccus pantotrophus. The protein has been spectroscopically characterised in solution and important kinetic parameters of nitrite reduction of the cytochrome cd₁ enzyme, i.e., K_m, V_max and k_cat have been determined. The influence of pH on the activity of the cytochrome cd₁ has been investigated and the results suggest that this enzyme can be used for the determination of nitrite in the pH range 6-9. Biosensing experiments with the cytochrome cd₁ in solution suggested that the decrease in intensity of the absorption band associated with the d₁ haem (which is the nitrite binding site), at 460 nm, with increasing nitrite concentrations would enable the measurement of this analyte with the optimum limit of detection. The cytochrome cd₁ has been encapsulated in a bulk sol–gel monolith with no structural changes observed and retention of enzymatic activity. The detection of nitrite ions in the range 0.075–1.250 μM was achieved, with a limit of detection of 0.075 μM. In order to increase the speed of response, a sol–gel sandwich thin film structure was formulated with the cytochrome cd₁. This structure enabled the determination of nitrite concentrations within ca. 5 min. The sol–gel sandwich entrapped cytochrome cd₁ enzyme was found to be stable for several months when the films were stored at 4 °C.