Optical biosensor based on nitrite reductase immobilised in controlled pore glass

Abstract

The increasing concentration of nitrite in groundwater, rivers and lakes brings serious risks to the public health and to the environment. The aim of this work was the development of an optical biosensor for quantifying nitrite based on the activity of cytochrome cd 1 nitrite reductase immobilised in controlled pore glass (CPG) beads. The developed biosensor operates by measuring the optical reflectance of nitrite reductase, which shows spectroscopic changes when nitrite reversibly binds to the reduced form and oxidises the enzyme. The optimisation of the immobilisation procedure showed that the immobilisation efficiency is highly dependent on the pH, being very low at basic pH, and that the maximum capacity of the CPG for the immobilisation of cd 1 was estimated in 57±10 mg cd 1 /g CPG. The CPG/ cd 1 specific activity remained stable at 4 °C, decreasing only 10% in 15 days. No observed effects of the immobilisation on the enzyme characteristics were detected, regarding both the red/ox absorbance spectra and the enzyme specific activity, since the red/ox spectra are in good agreement with similar ones obtained for cd 1 in solution, and the specific activity at time zero (0.6 μ moles of NO 2 − reduced min −1 mg of protein −1) is similar to that found for the soluble enzyme. The biosensor shows a sensitive response to increasing concentrations of nitrite in solution, especially at 460 nm, at which it showed higher sensitivity. The corresponding detection limit of 0.93 μM is well below the maximum admissible concentration imposed by European Community norms, of 2.2 μM.