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（由申请人提供）：该阶段项目将测试以下想法：与小分子抑制剂反应后，蛋白质反应后，具有机械敏感的荧光聚合物可以检测蛋白质诱导的结构变化。该概念将使用重组乙酰胆碱酯酶（ACHE）进行测试和证明，当它与有机磷酸盐（OP）抑制剂反应时，已知会经历灾难性变性的酶。该想法的科学和技术优点将在此I阶段时期进行测试，并进行了优化，以定义II期项目的行动方案。长期目标是基于灾难性变性开发和商业化一类全新的聚合传感器。在第一阶段，我们将以保留酶活性的方式附着或将Rache固定在结构反应性的聚合物膜传感器元件上。 Rache-FILM将暴露于OP抑制剂，导致蛋白质在结构上做出反应。该酶抑制剂变性过程会导致附着的反应性薄膜发生相应的结构变化，从而导致聚合物的荧光/颜色变化，可以在视觉上检测到或仪器测量。该阶段I项目的主要挑战是确定：（a）如果可以将Rache连接或插入包含结构响应元件的生物基质或聚合物表面上，并且（b）如果Rache在附着在膜上时会保留功能并在暴露于OP化合物时被抑制。项目优势是ATERIS Technologies开发了聚合物响应元素，在酸痛和OP-ACHES相互作用（包括重组蛋白的产生）方面具有超过三十年的经验。 ATERI将使用这些组合的专业知识领域来完成以下里程碑：AIM1。将Rache（测试物种）和自然ACHE（对照）附加到模型表面膜（脂质体）和Ateris的聚乙烯二乙烯（PDA）薄膜，并优化固定膜以保留酶的功能。 AIM 2。可视化由PDA薄膜涂层或脂质体嵌入的Rache与OP杀虫剂氧子的反应产生的颜色或荧光变化。 公共卫生相关性：美国每年在美国每年报告有15万和300,000毒素发生率，以暴露于有机磷酸盐（OP）杀虫剂，并且在全球范围内接受了数百万的治疗。在结构上相似的化学神经毒剂一直是对平民和军事人员的威胁，并可能损害公共卫生，伤害或致命的人类。拟议的传感器设备的实现将允许快速评估环境或军事运输。