Simplified nitrate-reductase-based nitrate detection by a hybrid thin-layer controlled potential coulometry/spectroscopy technique.
Simplified nitrate-reductase-based nitrate detection by a hybrid thin-layer controlled potential coulometry/spectroscopy technique.
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DOI：
10.1021/ac400987u
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发表时间：
2013-09
影响因子：
7.4
通讯作者：
Tingting Wang;K. Schlueter;B. L. Riehl;Jay M. Johnson;W. Heineman
中科院分区：
文献类型：
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作者：
Tingting Wang;K. Schlueter;B. L. Riehl;Jay M. Johnson;W. Heineman
利用基因重组技术产生的简化硝酸还原酶（SNaR）建立了一种检测硝酸盐的新方法。SNaR由钼-蝶呤（MO-MPT）结合位点和硝酸盐还原活性位点的片段组成，具有较高的硝酸盐还原活性。该方法是基于一个独特的组合的酶催化还原硝酸盐为亚硝酸盐的薄层库仑法，然后从亚硝酸盐与格里斯试剂反应产生的有色产物的光谱测量。硝酸盐库仑还原为亚硝酸盐使用甲基紫精（MV（2+））作为电子转移介体SNaR和控制电位库仑法在氧化铟锡（ITO）薄层电化学电池。在540 nm处的吸光度与样品中硝酸根的浓度成正比，线性范围为1-160 μM，灵敏度为8000 Au M（-1）。该方法所需样品量小于60 μL。硝酸盐的检测也可以通过测量库仑法相关的电荷来进行。然而，由于库仑分析法产生的背景电荷较大，因此光谱分析法具有上级性能。用该装置对几种天然水样中硝酸盐浓度的测定结果与标准方法的分析结果一致。
A novel method for the detection of nitrate was developed using simplified nitrate reductase (SNaR) that was produced by genetic recombination techniques. The SNaR consists of the fragments of the Mo-molybdopterin (MO-MPT) binding site and nitrate reduction active site and has high activity for nitrate reduction. The method is based on a unique combination of the enzyme-catalyzed reduction of nitrate to nitrite by thin-layer coulometry followed by spectroscopic measurement of the colored product generated from the reaction of nitrite with Griess reagents. Coulometric reduction of nitrate to nitrite used methyl viologen (MV(2+)) as the electron transfer mediator for SNaR and controlled potential coulometry in an indium tin oxide (ITO) thin-layer electrochemical cell. Absorbance at 540 nm was proportional to the concentration of nitrate in the sample with a linear range of 1-160 μM and a sensitivity of 8000 AU M(-1). The method required less than 60 μL of sample. Detection of nitrate could also be performed by measuring the charge associated with coulometry. However, the spectroscopic procedure gave superior performance because of interference from the large background charge associated with coulometry. Results for the determination of nitrate concentration in several natural water samples using this device with spectroscopic detection are in good agreement with analysis done with a standard method.