Microfluidic assays of acetylcholinesterase inhibitors
Microfluidic assays of acetylcholinesterase inhibitors
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DOI：
10.1021/ac990591f
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发表时间：
1999-11-15
影响因子：
7.4
通讯作者：
Ramsey, JM
中科院分区：
文献类型：
--
作者：
Hadd, AG;Jacobson, SC;Ramsey, JM
描述了一种用于乙酰胆碱酯酶（AChE）抑制剂的流动注射分析和电泳分离的微型装置。抑制剂，酶，底物和衍生剂的溶液混合在微芯片的通道内使用计算机控制的电动运输。乙酰胆碱酯酶催化水解乙酰硫代胆碱的硫代胆碱与香豆素苯基马来酰亚胺的芯片上的反应中测量，并通过激光诱导荧光检测所得的硫醚。抑制剂降低了荧光信号，并产生了诊断抑制类型的负峰。对于竞争性抑制剂观察到高斯峰，而对于不可逆抑制剂观察到宽的负峰。从他克林的微芯片测定中，推导出抑制常数Ki为1.5 +/- 0.2 nM，与标准比色皿测定法相当。对两种不可逆抑制剂克百威和毒扁豆碱进行了立即注射测定。采用Ei-min停流反应时间，获得了10 nM克百威的检测限。作为一种潜在的多重筛选装置，四种阳离子抑制剂，他克林，依洛芬铵，四甲基和四乙基氯化铵的混合物，分离和检测在70秒内。
A microfabricated device for flow injection analysis and electrophoretic separation of acetylcholinesterase (AChE) inhibitors is described. Solutions of inhibitor, enzyme, substrate, and derivitizing agent were mixed within the channels of the microchip using computer-controlled electrokinetic transport. AChE-catalyzed hydrolysis of acetylthiocholine to thiocholine was measured in an onchip reaction of thiocholine with coumarinylphenylmaleimide, and the resulting thioether was detected by laser-induced fluorescence. Inhibitors reduced the fluorescence signal and produced a negative peak diagnostic for the type of inhibition. A Gaussian peak was observed for competitive inhibitors, whereas a broad negative peak was observed for irreversible inhibitors. From a microchip assay for tacrine, an inhibition constant, K-i, of 1.5 +/- 0.2 nM was derived, which compared well with a standard cuvette assay, A now injection assay of two irreversible inhibitors, carbofuran and eserine, was performed. With a Ei-min stopped-flow reaction time, a detection limit of 10 nM carbofuran was obtained. As a potential multiplex screening device, a mixture of four cationic inhibitors, tacrine, edrophonium, and tetramethyl- and tetraethylammonium chloride, was separated and detected within 70 s.