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

Simonian获得的奖项0330189神经毒性有机磷酸酯（OP）在环境中广泛分布，用于昆虫和生物病原体控制。此外，还发生了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需要开发用于OP检测的稳健和灵敏的方法，该方法可以区分在正常使用下对社会几乎不构成威胁的普通花园农药和可能毁灭军事和民用目标的大规模杀伤性武器。该项目探索了基于酶的生物传感器，该传感器与金纳米颗粒支架相连，允许直接检测多组分环境中的超低浓度（10-10 M）OP神经毒素，如地下水，废水，食物和土壤。主要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水解酶-酶生物识别元件、特异性竞争与感兴趣的神经毒素结合的荧光诱饵和用于荧光检测的光学系统组成。纳米颗粒-分子界面被设计成在被生物识别元件结合时改变荧光诱饵的光学性质，从而产生在释放时改变的独特信号。将该技术开发成一系列能够识别和定量有机磷（OP）神经毒剂和农药的稳健、灵敏和有鉴别力的化学传感器，包括：（i）开发可以与不同试剂特异性竞争的适当诱饵，（ii）通过合理的方法选择或修饰酶，定点诱变以精细调节催化酶性质（亲和力和底物特异性）;（iii）开发最佳传感器平台，包括纳米颗粒特性和附着化学以及用于光收集的光学系统;以及（iv）用于鲁棒传感器性能的检测算法的设计。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，这将允许及时，准确地报告环境污染，从而启动适当的反应，以部署解毒程序和污染场地的补救有毒试剂。 许多有机磷酸酯，无论是以农药（磷酸三酯和硫代膦酸酯）或化学战剂（磷酰氟和磷酰硫代酸酯）的形式，已知作为乙酰胆碱和丁酰胆碱酯酶的抑制剂具有神经毒性。 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，并且在功能上不适用于第一响应者，军事行动，也不适用于小公司，农民和社区。 因此，强大的，易于使用的，敏感的，和选择性的有机磷酸盐传感器，如在这个项目中开发的需要，既保护公众健康，以确保国土安全。 这一固有的跨学科项目为研究生和本科生提供了一个培训适合确保国土安全的技术的绝佳机会。该项目还为高中和初中科学课提供了一个易于理解的例子，说明如何应用生物技术和技术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.