A novel cholinesterase assay for the evaluation of neurotoxin poisoning based on the electron-transfer promotion effect of thiocholine on an Au electrode
A novel cholinesterase assay for the evaluation of neurotoxin poisoning based on the electron-transfer promotion effect of thiocholine on an Au electrode
复制标题
DOI：
10.1016/j.snb.2019.126893
复制
发表时间：
2019-11
期刊：
影响因子：
--
通讯作者：
H. Shimada;Y. Kiyozumi;Y. Koga;Y. Ogata;Y. Katsuda;Yusuke Kitamura;M. Iwatsuki;Katsuhiko Nishiyama;Hideo Baba;T. Ihara
中科院分区：
文献类型：
--
作者：
H. Shimada;Y. Kiyozumi;Y. Koga;Y. Ogata;Y. Katsuda;Yusuke Kitamura;M. Iwatsuki;Katsuhiko Nishiyama;Hideo Baba;T. Ihara
提出了一种简便、经济的胆碱酯酶测定方法。硫代胆碱（TCh）通过胆碱酯酶的作用从底物中产生，促进[Fe（CN）6]3-/4-与Au电极之间的电子转移。电流随着TCh浓度的增加而增加;因此，当胆碱酯酶被神经毒性化学物质（如有机磷和氨基甲酸酯化合物）抑制时，电流会降低。首先，我们监测了乙酰胆碱酯酶对胆碱酯酶抑制剂的反应。在Tris−HCl缓冲液中用乙酰胆碱酯酶消化底物乙酰硫代胆碱，然后将反应混合物与乙腈合并。将抛光的Au电极浸入混合溶液中30 s，以将酶水解产物TCh附着在电极表面上。然后在浓度低至1 mM的KCl作为支持电解质的存在下，在0.5mM [Fe（CN）6]3-中对电极进行差分脉冲伏安法。酶处理的最佳条件是在30 °C、1.25 mU/mL乙酰胆碱酯酶和100 μM乙酰硫代胆碱终浓度下处理10 min。敌敌畏（有机磷）和灭多威（氨基甲酸酯）对乙酰胆碱酯酶活性的抑制作用可以监测。该系统适用于人血清样品含有丁酰胆碱酯酶。敌敌畏和灭多威对丁酰胆碱酯酶的抑制作用甚至在人血清中也被成功地监测到。最后，10名志愿者捐献的真实的人血进行了胆碱酯酶测定使用该系统。结果表明，该方法与传统的5，5 ′-二硫代双（2-硝基苯甲酸）法测定结果具有良好的相关性。本方法被认为是有效的作为一种新的人血胆碱酯酶测定。
A simple and cost-effective cholinesterase assay is presented. Thiocholine (TCh), which is generated from substrates by the action of cholinesterase, promotes the electron transfer between [Fe(CN)6]3–/4–and Au electrodes. The electric current increases with the TCh concentration; therefore, the current decreases when cholinesterase is inhibited by neurotoxic chemicals, such as organophosphorus and carbamate compounds. First, we monitored the responses of acetylcholinesterase to cholinesterase inhibitors. The substrate acetylthiocholine was digested with acetylcholinesterase in Tris−HCl buffer, then the reaction mixture was combined with acetonitrile. A polished Au electrode was immersed in the mixed solution for 30 s to immobilize the enzymatic hydrolysis product, TCh, on the electrode surface. The electrode was then subjected to differential pulse voltammetry in 0.5 mM [Fe(CN)6]3–in the presence of KCl as a supporting electrolyte, with a concentration as low as 1 mM. The optimum conditions for the enzymatic treatment were for 10 min at 30 °C with 1.25 mU/mL acetylcholinesterase and 100 μM acetylthiocholine as final concentrations. The inhibition of acetylcholinesterase activities from DDVP (organophosphorus) and methomyl (carbamates) could be monitored. This system was applied to human serum samples containing butyrylcholinesterase. The inhibition of butyrylcholinesterase by DDVP and methomyl were successfully monitored even in human serum. Finally, real human blood donated by 10 volunteers were subjected to a cholinesterase assay using this system. The results from the present method showed good correlation with those obtained from the conventional method using 5,5′-dithiobis-(2-nitrobenzoicacid). The present method was considered valid as a novel ChE assay for human blood.