Abstract
An electrochemical nitrite biosensor based on co-immobilization of copper- containing nitrite reductase (Cu-NiR, from Rhodopseudomonas sphaeroides forma sp. denitrificans) and viologen-modified chitosan (CHIT-V) on a glassy carbon electrode (GCE) is presented. Electron transfer (ET) between a conventional GCE and immobilized Cu-NiR was mediated by the co-immobilized CHIT-V. Redox-active viologen was covalently linked to a chitosan backbone, and the thus produced CHIT-V was co-immobilized with Cu-NiR on the GCE surface by drop-coating of hydrophilic polyurethane (HPU). The electrode responded to nitrite with a limit of detection (LOD) of 40 nM (S/N = 3). The sensitivity, linear response range, and response time (t90%) were 14.9 nA/μM, 0.04−11 μM (r2 = 0.999) and 15 s, respectively. The corresponding Lineweaver-Burk plot showed that the apparent Michaelis-Menten constant (KMapp) was 65 μM. Storage stability of the biosensor (retaining 80% of initial activity) was 65 days under ambient air and room temperature storage conditions. Reproducibility of the sensor showed a relative standard deviation (RSD) of 2.8% (n = 5) for detection of 1 μM of nitrite. An interference study showed that anions commonlyfound in water samples such as chlorate, chloride, sulfate and sulfite did not interfere with the nitrite detection. However, nitrate interfered with a relative sensitivity of 64% and this interference effect was due to the intrinsic character of the NiR employed in this study.
Highlights
Nitrite is a typical inorganic pollutant in environmental, food, industrial and physiological systems [1,2,3], various methods for detection and determination of nitrite have been reported, involving spectroscopic, chromatographic, electrochemical detection methods, etc. [3,4,5]
Direct electron transfers (DET) between electrode and nitrate reductase (NiR) were seldom reported [11,20,21], most of the reported electrochemical nitrite biosensors were based on co-immobilization of NiR and respective mediators
We report the electrochemical characterization of a co-immobilized Cu-NiR and CHIT-V glassy carbon electrode (GCE) as a reagentless biosensor for nitrite detection
Summary
Nitrite is a typical inorganic pollutant in environmental, food, industrial and physiological systems [1,2,3], various methods for detection and determination of nitrite have been reported, involving spectroscopic, chromatographic, electrochemical detection methods, etc. [3,4,5]. 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. Viologen was covalently linked to a chitosan backbone to effectively immobilize this mediator on the electrode surface by drop-coating of a HPU membrane. It was reported that chitosan is able to increase the stability and activity of immobilized enzymes [43] These points are important for a biosensor to be used practically. This kind of scheme is common in mediated catalytic reactions [44]
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