RESUMEN
In this paper we propose the construction of a new non-mediated electrochemical biosensor for nitrite determination in complex samples. The device is based on the stable and selective cytochrome c nitrite reductase (ccNiR) from Desulfovibrio desulfuricans, which has both high turnover and heterogeneous electron transfer rates. In opposition to previous efforts making use of several redox mediators, in this work we exploited the capacity of ccNiR to display a direct electrochemical response when interacting with pyrolytic graphite (PG) surfaces. To enable the analytical application of such bioelectrode the protein was successfully incorporated within a porous silica glass made by the sol-gel process. In the presence of nitrite, the ccNiR/sol-gel/PG electrode promptly displays catalytic currents indicating that the entrapped ccNiR molecules are reduced via direct electron transfer. This result is noteworthy since the protein molecules are caged inside a non-conductive silica network, in the absence of any mediator species or electron relay. At optimal conditions, the minimum detectable concentration is 120 nM. The biosensor sensitivity is 430 mA M(-1) cm(-2) within a linear range of 0.25-50 microM, keeping a stable response up to two weeks. The analysis of nitrites in freshwaters using the method of standard addition was highly accurated.