SENSORS: Nanoparticles-based Biosensor for Direct Detection of Organophosphate Chemical Warfare Agents and Neurotoxic Pesticides

神经毒性有机磷酸盐（OP）具有广谱性，广泛分布于环境中，用于控制昆虫和生物病原体。此外，还有生物恐怖主义威胁和涉及化学战剂沙林和VX（也是神经毒性OPs）的实际袭击。这些神经毒素作为大规模杀伤性武器的潜在威胁需要开发强大而敏感的OP检测方法，这些方法可以区分正常使用下对社会几乎没有威胁的普通花园杀虫剂和可能摧毁军事和民用目标的大规模杀伤性武器。该项目探索基于酶的生物传感器，它与金纳米颗粒支架相连，可以直接检测地下水、废水、食物和土壤等多组分环境中超低浓度（10- 10m）的OP神经毒素。主要的生物传感器元件由金属纳米表面、一个或多个广谱有机磷酸酶-酶生物识别元件、与感兴趣的神经毒素特异性结合的荧光诱饵和用于荧光检测的光学系统组成。纳米粒子-分子界面被设计成在与生物识别元件结合时改变荧光诱饵的光学特性，产生一个独特的信号，当它被释放时发生变化。这项技术的发展成为一个强大的、敏感的、有鉴别能力的化学传感器家族，能够识别和量化有机磷（OP）神经毒剂和农药，包括：(i)开发适当的诱饵，可以与不同的药物特异性竞争，（ii）通过合理的、位点导向的诱变来选择或修饰酶，以微调催化酶的特性（亲和力和底物特异性）；（iii）开发最佳传感器平台，包括纳米颗粒特性、附着化学和光收集光学系统；（iv）鲁棒传感器性能检测算法设计。拟议的生物传感器的预期应用包括监测土壤、空气和/或水质，这将允许对环境污染进行及时、准确的报告，从而在部署解毒程序和污染场地的补救措施中对有毒物质作出适当的反应。众所周知，许多有机磷，无论是以杀虫剂（三磷酸酯和硫代膦酸酯）的形式，还是以化学战（CW）剂（氟化膦酸酯和硫代膦酸酯）的形式，作为乙酰胆碱和丁胆碱酯酶的抑制剂，都具有神经毒性。现有的有机磷检测方法鉴别能力差，技术复杂。这种能力不太适合野外条件，对第一响应者、军事行动、小公司、农民和社区都不起作用。因此，为了保护公众健康和确保国土安全，需要像本项目开发的那样坚固、易于使用、敏感和选择性的有机磷传感器。这个跨学科的项目为研究生和本科生提供了一个极好的培训机会，以确保国土安全。这个项目也构成了一个容易理解的例子，为高中和初中的科学课应用生物技术和技术来解决重要的社会问题。

Award 0330189, SimonianThere is a broad spectrum of neurotoxic organophosphates (OP) that are subject to widespread distribution in the environment for insect and biopathogen control. In addition, there have been bioterrorism threats and actual attacks involving the chemical warfare agents Sarin and VX (also neurotoxic OPs). The potential threats of these neuroxins as weapons of mass destruction necessitate the development of robust and sensitive methods for OP detection that can discriminate between common garden pesticides that pose little threat to society under normal usage and the weapons of mass destruction that could decimate military and civilian targets. This project explores enzyme-based biosensors, which are linked to gold nanoparticle scaffolds, that permit the direct detection of ultra low concentrations (10-10 M) of OP neurotoxins in multi-component environments such as ground water, waste water, food, and soil. The primary biosensor element consists of a metal nanosurface, one or more broad-spectrum organophosphate hydrolase-enzyme biorecognition elements, fluorescent decoys that compete specifically for binding with neurotoxins of interest, and an optical system for fluorescence detection. The nanoparticle-molecular interface is designed to alter the optical properties of the fluorescent decoy when bound by the biorecognition element, giving rise to a unique signal that changes when it is released. The development of this technology into a family of robust, sensitive, and discriminating chemical sensors capable of identifying and quantifying organophosphorus (OP) nerve agents and pesticides involves: (i) the development of appropriate decoys that can compete specifically with different agents, (ii) the selection or modification of enzymes via rational, site-directed mutagenesis to finely tune catalytic enzyme properties (both affinity and substrate specificities); (iii) the development of the optimum sensor platform, both in terms of nanoparticle properties and attachment chemistries and optical systems for light collection; and (iv) the design of detection algorithms for robust sensor performance. Intended applications of the proposed biosensor include the monitoring of soil, air, and/or water quality, which will allow prompt, accurate reporting on environmental contamination, thus initiating the appropriate response to toxic agents in deployment of detoxification procedures and remediation of contaminated sites. Many organophosphates, either in the form of pesticides (phosphotriesters and phosphonthioates) or chemical warfare (CW) agents (phosphonofluoridates and phosphono-thioates), are known to be neurotoxic as inhibitors of acetyl-choline and butryl-choline esterases. Existing methods for organophosphate detection are poorly discriminating and technologically complex. Such capabilities are poorly suited to field conditions and are not functionally available to first responders, military operations, nor small companies, farmers, and communities. Thus, robust, easy to use, sensitive, and selective organophosphate sensors, such as those developed in this project are needed, both to protect public health and to ensure homeland security. This inherently interdisciplinary project provides an excellent opportunity for training of graduate and undergraduate students in technologies appropriate for ensuring homeland security. This project also constitutes an easily understood example for high school and middle school science classes of the application of biotechnology and technology to solve important societal problems.