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 microM). 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., Km, Vmax and kcat 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 microM was achieved, with a limit of detection of 0.075 microM. 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 degrees C.

Nitrite Biosensing via Selective Enzymes—A Long but Promising Route

“…Similar works published by Ferreti et al [97] and Rosa et al [98] have suggested the employment of cd 1 NiR from Paracoccus pantotrophus ( P. pantotrophus ) in optical nitrite biosensors. In both cases, signal transduction was based on the intensity variation of the absorption band assigned to the catalytic d 1 -type heme (λ = 460 nm) following nitrite addition.…”

A Nitrite Biosensor Based on Co-immobilization of Nitrite Reductase and Viologen-modified Chitosan on a Glassy Carbon Electrode

“…The obtained linear detection range fully covers the nitrite concentration range encountered in water samples, for example, the acceptable nitrite maximum in drinking water is ∼2.2 μM for the EU standard [7–9,15]. Deviation from linearity at higher concentration of nitrite was observed, which is a typical phenomenon for an enzyme electrode, and can be ascribed partially to the saturation of the immobilized NiR, or inhibition of the immobilized NiR by the enzymatic nitric oxide product [26].…”

Biosensors for environmental pollutants and food contaminants

“…An unusual approach was described by Sarkar, in which proteases are used for the detection of dust particles [82]. Among other environmental monitoring analytes are heavy metals partly also focusing on their bioavailability [83,84,85,86]), and nitrite requiring a detection limit of 0.1 mg L -1 in potable water [23].…”

“…Aside from statistical significance of single-cell and single-molecule analysis, the pathogenic or toxic effect needs to be taken into account when deciding on detection limits required of our analytical tools. Prominently, this topic has been addressed with biosensors detecting the bioavailability of toxins and pollutants in environmental samples, rather than total concentrations present, and a few biosensors are cited addressing this point [23,24,25].…”