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 ? [23] 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 ? [30]. 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 [36]. 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 [38], and that of Cu-NiR from Rhodobacter sphaeroides strain 2.4.3 is 0.05 V vs. Ag/AgCl at pH 7.0 [28]. Several ET mediators have been tested to electrochemically reduce the T1 Cu of Cu-NiRs [12], and methyl viologen has been proven to be useful as mediator/Cu-NiR systems for catalytic reduction of nitrite [17]. 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 [39].
Chitosan has been functionalized to be applied in catalysis [40], 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 [43]. 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 [44].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.