Biosensors and Bioelectronics 22 (2007) 2485–2492 Biosensing nitrite using the system nitrite redutase/Nafion/methyl viologen—A voltammetric study M. Gabriela Almeida a,b,∗ , C´ elia M. Silveira a , Jos´ e J.G. Moura a a REQUIMTE, Departamento de Qu´ ımica, Centro de Qu´ ımica Fina e Biotecnologia, Faculdade de Ciˆ encias e Tecnologia, Universidade Nova de Lisboa, 2829-516 Monte de Caparica, Portugal b Escola Superior de Sa ´ ude Egas Moniz, Campus Universit´ ario Quinta da Granja, 2829-511 Caparica, Portugal Received 12 January 2006; received in revised form 15 September 2006; accepted 27 September 2006 Available online 13 November 2006 Abstract This work describes the construction and voltammetric characterization of a nitrite biosensor based on a cytochrome c-type nitrite reductase (ccNiR) and the Nafion ionomeric matrix loaded with methyl viologen as redox mediator. Despite the potential electrostatic repulsions between the anionic substrate and the Nafion sulfonate groups, the resulting bioelectrode exhibited electrocatalytic activity toward nitrite. This phenomenon must be due to the nonuniformity of the enzyme/Nafion membrane, which allows the direct interaction between the substrate and numerous enzyme molecules. Nevertheless, the anionic nature of Nafion exerted a certain diffusion barrier to nitrite, as revealed by the unusually elevated limits of the linear dynamic range and k app m . The irregularity of the composite membrane also contributed to slow down the rate of charge transfer throughout the Nafion polymer. The level of viologens incorporated within the Nafion membrane had a strong influence in the analytical parameters: as much mediator was present, lower was the sensitivity and wider was the linear range. For an optimized ratio enzyme/mediator the sensitivity was 445 ± 8 mA M -1 cm -2 , within the linear range 75–800 M; the lowest detected nitrite concentration was 60 M. The operational stability of the biosensor and the influence of some possible interferences were evaluated. © 2006 Elsevier B.V. All rights reserved. Keywords: Nitrite; Biosensor; Nitrite reductase; Nafion; Methyl viologen 1. Introduction In recent years, the recurrent anthropologic uses of nitrates and nitrites, combined with revelations of their potential toxi- city, have raised a number of public concerns. The most critical issue comes from agricultural practices. In fact, the surplus in the soil of nitrate fertilizers may have a broad ecological impact due to the unbalance of the biological nitrogen cycle, the eutrophication of aquatic ecosystems and the contamination of groundwater supplies (GEO-2000 Report, UNEP; Topic report 1/2003, EEA). The continuous ingestion of food containing nitrates and nitrites additives also brings potential hazards for human health. Nitrite is the main toxic agent (promotes the irre- ∗ Corresponding author at: Departamento de Qu´ ımica, Faculdade de Ciˆ encias e Tecnologia, Universidade Nova de Lisboa, Quinta da Torre, 2829-516 Monte de Caparica, Portugal. Tel.: +351 21 2948381; fax: +351 21 2948550. E-mail address: mga@dq.fct.unl.pt (M.G. Almeida). versible oxidization of hemoglobin to methemoglobin and the formation of carcinogenic nitrosoamines). Nevertheless, nitrate is easily converted to nitrite by bacterial action. Consequently, there is a growing demand to detect and quantify both anions in food, drinking water and environmental samples. The European Community, for instance, has established the maximum admis- sible levels of nitrates and nitrites in drinking water at 50 and 0.1 ppm, respectively. The majority of strategies for nitrate deter- mination rely upon nitrite detection. However, due to the huge variety of interferences usually present, few techniques enable the reliable quantification of nitrite in complex matrices (Ellis et al., 1998; Moorcroft et al., 2001). A promising alternative is the development of analytical probes based on the recognition properties of highly selective biological macromolecules, such as nitrite reducing enzymes, which remain relatively unex- ploited to date. In this work we propose the use of cytochrome c nitrite reductase (ccNiR) from the sulfate reducing bacterium Desulfovibrio desulfuricans (Dd) ATCC 27774, which catalyses the direct conversion of nitrite to ammonia in a six-electron 0956-5663/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2006.09.027