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

0330189，Simonian有广泛的神经毒性有机磷(OP)，它们在环境中广泛分布，用于昆虫和生物病原体控制。此外，还有生物恐怖主义威胁和涉及化学战剂Sarin和VX(也是神经毒性OP)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要求开发强大而灵敏的OP检测方法，可以区分正常使用情况下对社会几乎不构成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。该项目探索连接到金纳米支架上的基于酶的生物传感器，允许直接检测地下水、废水、食品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水解酶生物识别元件、与感兴趣的神经毒素竞争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-分子界面的设计目的是在与生物识别元件结合时改变荧光诱饵的光学性质，产生一种独特的信号，当它被释放时会发生变化。将这项技术发展成为能够识别和定量有机磷(OP)神经毒剂和农药的强大、灵敏和区分的化学传感器家族涉及：(I)开发能够与不同试剂竞争的适当诱饵；(Ii)通过合理的定点突变选择或修饰酶，以微调催化酶的性质(亲和力和底物特异性)；(Iii)开发最佳传感器平台，包括纳米粒子性质和附着化学以及用于光收集的光学系统；以及(Iv)设计检测算法，以实现稳健的传感器性能。建议的生物传感器的预期应用包括监测土壤、空气和/或水质，这将允许及时、准确地报告环境污染，从而在部署解毒程序和修复受污染地点时启动对有毒物质的适当反应。作为乙酰胆碱和丁基胆碱酯酶的抑制剂，许多有机磷酸盐，无论是杀虫剂(磷酸三酯和硫代膦)还是化学战剂(氟磷酸盐和硫代膦)，都具有神经毒性。现有的有机磷检测方法识别性差，技术复杂。这种能力不太适合现场条件，在功能上不适用于急救人员、军事行动，也不适用于小公司、农民和社区。因此，需要坚固、易用、灵敏和选择性强的有机磷传感器，如本项目中开发的传感器，以保护公众健康和确保国土安全。这一本质上是跨学科的项目为研究生和本科生提供了一个极好的机会，培训他们掌握适用于确保国土安全的技术。该项目也是高中和中学理科班应用生物技术和技术解决重要社会问题的一个容易理解的例子。

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.