Kohli Neeraj, Srivastava Devesh, Sun Jun, Richardson Rudy J, Lee Ilsoon, Worden Robert M
Department of Chemical Engineering and Materials Science, Michigan State University, East Lansing, Michigan 48824, USA.
Anal Chem. 2007 Jul 15;79(14):5196-203. doi: 10.1021/ac0701684. Epub 2007 Jun 8.
Neuropathy target esterase (NTE) is a membrane protein found in human neurons and other cells, including lymphocytes. Binding of certain organophosphorus (OP) compounds to NTE is believed to cause OP-induced delayed neuropathy (OPIDN), a type of paralysis for which there is no effective treatment. Mutations in NTE have also been linked with serious neurological diseases, such as motor neuron disease. This paper describes development of the first nanostructured biosensor interface containing a catalytically active fragment of NTE known as NEST. The biosensor was fabricated using the layer-by-layer assembly approach, by immobilizing a layer of NEST on top of multilayers consisting of a polyelectrolyte (poly-L-lysine) and an enzyme (tyrosinase). The biosensor has a response time on the order of seconds and gives a concentration-dependent decrease in sensor output in response to a known NEST (and NTE) inhibitor. Potential applications of the biosensor include screening OP compounds for NTE inhibition and investigating the enzymology of wild-type and mutant forms of NTE. Although the development of a NEST biosensor was the primary purpose of this study, we found that the approach developed for NEST could also be extended to measure the activity of other esterases involved in neural processes, such as acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). On the basis of measured sensitivities, phenyl valerate was the preferred substrate for NEST and BChE, whereas phenyl acetate was better for AChE.
神经病靶酯酶（NTE）是一种存在于人类神经元和其他细胞（包括淋巴细胞）中的膜蛋白。某些有机磷（OP）化合物与NTE的结合被认为会导致OP诱导的迟发性神经病（OPIDN），这是一种尚无有效治疗方法的瘫痪类型。NTE的突变也与严重的神经疾病有关，如运动神经元病。本文描述了首个包含NTE催化活性片段（称为NEST）的纳米结构生物传感器界面的开发。该生物传感器采用逐层组装方法制造，通过将一层NEST固定在由聚电解质（聚-L-赖氨酸）和酶（酪氨酸酶）组成的多层膜之上。该生物传感器的响应时间在秒级，并且响应已知的NEST（和NTE）抑制剂时，传感器输出会呈现浓度依赖性下降。该生物传感器的潜在应用包括筛选抑制NTE的OP化合物以及研究野生型和突变型NTE的酶学。尽管开发NEST生物传感器是本研究的主要目的，但我们发现为NEST开发的方法也可以扩展到测量参与神经过程的其他酯酶的活性，如乙酰胆碱酯酶（AChE）和丁酰胆碱酯酶（BChE）。根据测量的灵敏度，戊酸苯酯是NEST和BChE的首选底物，而乙酸苯酯对AChE更合适。
