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）：这个第一阶段的项目将测试机械应力敏感，荧光聚合物可以检测蛋白质与小分子抑制剂反应后引起的蛋白质结构变化的想法。这一概念将使用重组乙酰胆碱酯酶（AChE）进行测试和验证，AChE是一种已知的酶，当它与有机磷（OP）抑制剂反应时，会发生灾难性的变性。在第一阶段，我们将测试这个想法的科学和技术优点，并对其进行优化，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，我们将以保持酶活性的方式将rAChE附着或放置在结构反应性聚合物薄膜传感器元件上。rache膜将暴露于OP抑制剂，导致蛋白质在结构上作出反应。这种酶抑制剂变性过程在附着的反应性薄膜中引起相应的结构变化，从而导致聚合物的荧光/颜色变化，这种变化可以通过视觉或仪器测量来检测。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上，以及(b) rAChE是否在附着在膜上时保持功能，并在暴露于OP化合物时受到抑制。该项目的优势在于，ATERIS技术公司开发了聚合物响应元件，在AChE和OP-AChE相互作用方面拥有超过30年的经验，包括重组蛋白的生产。ATERIS将利用这些综合领域的专业知识来完成以下里程碑：将rAChE（被试种）和天然AChE（对照）附着在模型表面膜（脂质体）和ATERIS的聚二乙炔（PDA）薄膜上，并优化附着以保留酶的功能。目标2。将包被rach的PDA薄膜或包被rach的脂质体与OP杀虫剂oxon反应所产生的颜色或荧光变化可视化。