No 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.

No abstract

Biosensor miniaturisation often requires the construction of micro volume cells using micro-machining techniques. In this work, a micro flow cell made of a transparent polymer [poly(methyl methacrylate)] developed for using with a fluorescence immunoglobulin G (IgG) immunosensor is described. The micro flow cell provides space to enclose a small amount of controlled pore glass (CPG), a transparent support that can host and covalently bind the biomolecules. The immobilisation of IgG on CPG permits the development of an optical immunosensor for the detection of fluorescein isothiocyanate (FITC)-labelled anti-IgG. In this immunosensor the excitation light is provided by an argon ion laser and guided by an optical fibre to the flow cell, where the fluorescence signal is filtered by a long-pass barrier filter (OG515) and then detected by a close positioned photodiode. This signal was found to be proportional to the amount of anti-IgG-FITC bound to the immobilised IgG during a direct immunochemical reaction. Characterisation of the CPG as an optical medium and immobilisation support was performed. CPG produces intense light scattering and good permeability to fluids, and also a typical immobilisation rate for IgG of about 90% of the initial amount of antibody. The described immunosensor shows a detection limit for anti-IgG-FITC of 6.3 nM and a sensitivity of 9.5 mV nM 21 . This immunoptode developed with a micro flow cell has been shown to be a suitable system for the detection of immunoglobulins.