Use of a Phosphatase-Like DT-Diaphorase Label for the Detection of Outer Membrane Vesicles.,Analytical Chemistry

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Use of a Phosphatase-Like DT-Diaphorase Label for the Detection of Outer Membrane Vesicles.
Analytical Chemistry ( IF 6.7 ) Pub Date : 2019-03-18 00:00:00 , DOI: 10.1021/acs.analchem.9b00064
Andi Muhammad Ichzan ₁ , Sohee Lee ₁ , Chiew San Fang ₁ , Ponnusamy Nandhakumar ₁ , Hyeri Ha ₁ , Jung Min Joo ₁ , Kwang-Sun Kim ₁ , Haesik Yang ₁

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DT-diaphorase (DT-D) is known to mainly catalyze the two-electron reduction of quinones and nitro(so) compounds. Detection of Gram-negative bacterial outer membrane vesicles (OMVs) that contain pyrogenic lipopolysaccharides (LPSs, also called endotoxins) is required for evaluating the toxic effects of analytical samples. Here, we report that DT-D has a high dephosphorylation activity: DT-D catalyzes reductive dephosphorylation of a phosphate-containing substrate in the presence of NADH. We also report that sensitive and simple OMV detection is possible with a sandwich-type electrochemical immunosensor using DT-D and two identical LPS-binding antibodies as a catalytic label and two sandwich probes, respectively. The absorbance change in a solution containing 4-nitrophenyl phosphate indicates that dephosphorylation occurs in the presence of both DT-D and NADH. Among the three phosphate-containing substrates [4-aminophenyl phosphate, ascorbic acid phosphate, and 1-amino-2-naphthyl phosphate (ANP)] that can be converted into electrochemically active products after dephosphorylation, ANP shows the highest electrochemical signal-to-background ratio, because (i) the dephosphorylation of ANP by DT-D is fast, (ii) the electrochemical oxidation of the dephosphorylated product (1-amino-2-naphthol, AN) is rapid, even at a bare indium–tin oxide electrode, and (iii) two redox cycling processes significantly increase the electrochemical signal. The two redox cycling processes include an electrochemical–enzymatic redox cycling and an electrochemical–chemical redox cycling. The electrochemical signal in a neutral buffer (tris buffer, pH 7.5) is comparable to that in a basic buffer (tris buffer, pH 9.5). When the immunosensor is applied to the detection of OMV from Escherichia coli, the detection limit is found to be 8 ng/mL. This detection strategy is highly promising for the detection of biomaterials, including other extracellular vesicles.

中文翻译：

磷酸酶样DT-Diaphorase标记在检测外膜囊泡中的用途。

已知DT-黄递酶（DT-D）主要催化醌和硝基（so）化合物的双电子还原。需要检测含有热解脂多糖（LPS，也称为内毒素）的革兰氏阴性细菌外膜囊泡（OMV），以评估分析样品的毒性作用。在这里，我们报告DT-D具有很高的去磷酸化活性：DT-D在NADH的存在下催化含磷酸盐的底物的还原性去磷酸化。我们还报告说，使用DT-D和两个相同的LPS结合抗体分别作为催化标记和两个夹心探针的夹心式电化学免疫传感器，可以实现灵敏且简单的OMV检测。含有4-硝基苯基磷酸酯的溶液中的吸光度变化表明，在同时存在DT-D和NADH的情况下会发生去磷酸化。可以在脱磷酸后转化为电化学活性产物的三种含磷酸盐的底物[4-氨基苯基磷酸盐，抗坏血酸磷酸盐和1-氨基-2-萘基磷酸盐（ANP）]中，ANP显示出最高的电化学信噪比。本底比率，因为（i）DT-D对ANP的脱磷酸作用很快，（ii）即使在裸露的铟锡氧化物下，脱磷酸化产物（1-氨基-2-萘酚，AN）的电化学氧化作用也很快电极，以及（iii）两种氧化还原循环过程会显着增加电化学信号。这两个氧化还原循环过程包括电化学-酶氧化还原循环和电化学-化学氧化还原循环。中性缓冲液（tris缓冲液，pH 7.5）中的电化学信号与碱性缓冲液（tris缓冲液，pH 9.5）中的电化学信号相当。当免疫传感器用于检测OMV时大肠杆菌的检出限为8 ng / mL。这种检测策略对于检测包括其他细胞外囊泡在内的生物材料非常有前途。

更新日期：2019-03-18

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