A NEW BIOSENSOR BASED ON CATASTROPHIC PROTEIN CHANGES

基于灾难性蛋白质变化的新型生物传感器

• 批准号: 7907049
• 负责人: charles mark thompson
• 金额: $ 10.6万
• 依托单位: ATERIS TECHNOLOGIES, LLC
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-17 至 2010-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7907049
• 关键词: Acetylcholinesterase; Antidotes; Area; Binding; Biochemical; Biological; Biosensor; Chemical Warfare Agents; Chemicals; Color; Detection; Development; Devices; Elements; Enzyme Inhibitor Drugs; Enzyme Inhibitors; Enzymes; Exposure to; Film; Fluorescence; Fracture; Goals; Human; Incidence; Indium; Insecticides; Lead; Life; Liposomes; Lysine; Measurement; Membrane; Military Personnel; Modeling; Mus; Organophosphates; Oximes; Phase; Polymers; Process; Production; Proteins; Public Health; Reaction; Recombinant Proteins; Recombinants; Reporting; Response Elements; Structure; Surface; System; Technology; Testing; Toxic effect; base; enzyme activity; experience; falls; inhibitor/antagonist; injured; nerve agent; neurotransmission; phase 1 study; polydiacetylene; protein structure; public health relevance; research study; response; sensor; small molecule; sound; toxic organophosphate insecticide exposure

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.

描述(申请人提供)：这个第一阶段的项目将测试机械应力敏感的荧光聚合物可以检测到蛋白质与小分子抑制剂反应后引起的结构变化的想法。这一概念将使用重组乙酰胆碱酯酶(AChE)进行测试和验证，AChE是一种已知在与有机磷(OP)抑制剂反应时会发生灾难性变性的酶。这一想法的科学和技术优点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。我们的长期目标是开发一种全新的基于灾难性变性的聚合物传感器并将其商业化。在第一阶段，我们将以保持酶活性的方式将RAChE附着或放置在具有结构反应性的聚合物薄膜传感器元件上。Rach膜将暴露在OP抑制剂中，使蛋白质在结构上做出反应。这种酶抑制剂的变性过程会导致附着的反应性薄膜发生相应的结构变化，从而导致聚合物中的荧光/颜色变化，这种变化可以用肉眼或通过仪器测量来检测。这个第一阶段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响应元件的生物基质或聚合物表面，以及(B)RACHE在附着到膜上时是否保留功能，而当暴露在OP化合物中时是否被抑制。该项目的优势在于，ATERIS技术公司开发了聚合物反应元件，在AChE和OP-AChE相互作用方面拥有30多年的经验，包括生产重组蛋白质。ATERIS将利用这些专业知识的结合领域来实现以下里程碑：目标1.将RACH(试验物种)和天然AChE(对照)连接到模型表面膜(脂质体)和ATERIS的聚二乙炔(PDA)薄膜，并对附着进行优化，以保留酶功能。目的2.观察PDA薄膜包被的RACH或脂质体包埋的RACH与OP杀虫剂Oxon反应后的颜色或荧光变化。 公共卫生相关性：据报道，美国每年有15万和30万起因接触有机磷(OP)杀虫剂而中毒的事件，全球有数百万人接受了治疗。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，并可能危及公共健康，伤害或致命伤害人类。建议的传感器装置的实现将允许对环境或军事OP暴露的快速评估。