A NEW BIOSENSOR BASED ON CATASTROPHIC PROTEIN CHANGES

描述（由申请人提供）：该第一阶段项目将测试机械应力敏感的荧光聚合物可以检测蛋白质与小分子抑制剂反应后诱导的结构变化的想法。这一概念将使用重组乙酰胆碱酯酶（AChE）进行测试和证明，已知这种酶在与有机磷酸盐（OP）抑制剂反应时会发生灾难性变性。这一想法的科学和技术价值将在第一阶段进行测试，并进行优化，以确定第二阶段项目的行动方针。长期目标是开发和商业化一种全新的基于灾难性变性的聚合物传感器。在第一阶段，我们将附加或放置rAChE到结构反应性聚合物膜传感器元件的方式，保留酶的活性。rAChE膜将暴露于OP抑制剂，引起蛋白质结构响应。这种酶抑制剂变性过程导致附着的反应性薄膜中的相应结构变化，这导致聚合物中的荧光/颜色变化，其可以通过视觉或仪器测量来检测。该I期项目的主要挑战是确定：（a）rAChE是否可以附着或插入到含有结构反应元件的生物基质或聚合物表面上，以及（B）rAChE是否在附着到膜上时保留功能，并且在暴露于OP化合物时受到抑制。项目优势在于ATERIS Technologies开发了聚合反应元件，在AChE和OP-AChE相互作用方面拥有超过三十年的经验，包括重组蛋白的生产。ATERIS将利用这些专业领域的组合来实现以下里程碑：AIM 1。将rAChE（供试品）和天然AChE（对照品）连接到模型表面膜（脂质体）和ATERIS的聚丁二炔（PDA）薄膜上，并优化连接以保留酶功能。AIM 2.观察PDA薄膜包被的rAChE或脂质体包埋的rAChE与OP杀虫剂oxon反应产生的颜色或荧光变化。 公共卫生相关性：在美国，每年有150，000和300，000例暴露于有机磷（OP）杀虫剂的毒性事件报告，全世界有数百万人接受治疗。结构相似的OP化学神经毒剂继续对平民和军事人员构成威胁，并可能危害公共健康，伤害或致命伤害人类。实现拟议的传感器设备将允许快速评估环境或军事OP暴露。

DESCRIPTION (provided by applicant): This Phase I project will test the idea that mechanostress-sensitive, fluorescent polymers can detect structural changes induced in proteins following reaction of the protein with small molecule inhibitors. This concept will be tested and proven using recombinant acetylcholinesterase (AChE) an enzyme that is known to undergo catastrophic denaturation when it reacts with organophosphate (OP) inhibitors. The scientific and technical merits of the idea will be tested in this Phase I period, and optimized to define a course of action for a Phase II project. The long-term goal is to develop and commercialize an entirely new class of polymeric sensors based on catastrophic denaturation. In Phase I, we will attach or place rAChE onto structurally reactive polymer film sensor elements in a manner that retains the enzyme's activity. The rAChE-film will be exposed to OP inhibitors causing the protein to respond structurally. This enzyme-inhibitor denaturation process causes a corresponding structural change in the attached reactive thin film, which results in a fluorescent/color change in the polymer that can be detected visually or though instrumental measurements. The principle challenges for this Phase I project are to determine: (a) if rAChE can be attached or inserted onto a biological matrix or polymeric surface containing a structural response element, and (b) if rAChE retains function when attached to the film and is inhibited when exposed to OP compounds. Project advantages are that ATERIS Technologies has developed polymeric response elements, has over three decades of experience with AChE and OP-AChE interactions including the production of recombinant protein. ATERIS will use these combined areas of expertise to accomplish the following milestones: AIM 1. Attach rAChE (test species) and natural AChE (control) to a model surface membrane (liposome) and ATERIS' polydiacetylene (PDA) thin film and optimize the attachment to retain enzyme function. AIM 2. Visualize a color or fluorescence change resulting from the reaction of PDA thin film coated- rAChE or liposome embedded rAChE with an OP insecticide oxon. PUBLIC HEALTH RELEVANCE: There are 150,000 and 300,000 toxicity incidences reported yearly in the US for exposure to organophosphate (OP) insecticides and millions treated worldwide. Structurally similar OP chemical nerve agents continue as a threat to civilian and military personnel and can compromise public health, injure or fatally harm humans. Realization of the proposed sensor device will allow for rapid assessment of environmental or military OP exposure.