T1 Cu is located 7 ? beneath a hydrophobic surface patch which is surrounded by many negatively charged residues [30,33] and T2 Cu is located at the bottom of a 12 ?  or 16 ? [34,35] deep cavity formed at the interface between two adjacent monomers, and the inter-atomic distance between the two Cu sites is 12.6 ? . The NiR used in this study belongs to the copper-containing type from Rhodopseudomonas sphaeroides forma sp. denitrificans, which catalyzes 1-electron/2-proton reduction of nitrite to nitric oxide. This enzyme has a homodimeric structure (2 �� 39 kDa) and contains both T1 and T2 Cu centers per monomeric unit . The stability of this type NiR was reported to be good [36,37], which is essential to the preparation of a biosensor.
The redox potentials of T1 Cu of some Cu-NiRs are in the range of 0.
04�C0.1 V vs. Ag/AgCl at pH 7.0 , and that of Cu-NiR from Rhodobacter sphaeroides strain 2.4.3 is 0.05 V vs. Ag/AgCl at pH 7.0 . Several ET mediators have been tested to electrochemically reduce the T1 Cu of Cu-NiRs , and methyl viologen has been proven to be useful as mediator/Cu-NiR systems for catalytic reduction of nitrite . In this study, viologen was covalently linked to a chitosan backbone to effectively immobilize this mediator on the electrode surface by drop-coating of a HPU membrane. Chitosan is a natural biopolymer, and its unique properties such as biocompatibility, remarkable affinity for proteins, antibacterial property and environmentally friendly polyelectrolyte nature, etc.
render this material suitable for biofabrication .
Chitosan has been functionalized to be applied in catalysis , has been used to immobilizations of more than 60 enzymes [41,42]. In addition, it was reported that chitosan is able to increase the stability and activity of immobilized Brefeldin_A enzymes . Undoubtedly, these points are important Dacomitinib for a biosensor to be used practically.In this paper, we report the electrochemical characterization of a co-immobilized Cu-NiR and CHIT-V GCE as a reagentless biosensor for nitrite detection. The working principle of the proposed biosensor is shown in Figure 1. This kind of scheme is common in mediated catalytic reactions .Figure 1.Scheme of working principle of the co-immobilized Cu-NiR and CHIT-V GCE.2.?Experimental2.1. Chemicals and MaterialsSodium hydrogen phosphate (99.95%), sodium dihydrogen phosphate (99.999%), potassium chloride (99.999%), sodium chlorate (��99.0%), sodium sulfite (��98%), sodium sulfate (99.99+%), sodium nitrate (��99.0%), sodium nitrite (99.