A NEW BIOSENSOR BASED ON CATASTROPHIC PROTEIN CHANGES

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.