Detection and Remediation of Organophosphate Contamination

NON-TECHNICAL SUMMARY: OP compounds represent the single largest class of urban and rural pesticides used commercially in various locals throughout the world. The acute toxicity of these broad-range neurotoxins is of great environmental concern. The main goal of this proposal is the development of new chemical and biochemical technologies that permit on-site detection and remediation of OP chemical contamination.
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APPROACH: 1 Develop New Enzyme Biocatalysts for Detection and Destruction of Pesticides and other Organophosphates.OP-hydrolyzing enzymes will be modified by site-specific and combinatorial methods to create a library of novel enzymes that have the ability to detect and ultimately detoxify the various neurotoxic pesticides. Additional native enzymes will be identified and collected by genomic screening and cloning of a broad array of OPH family members. The catalytic activities of the enzyme library will be assessed with OP-pesticides and pesticide analogs. The most promising candidates will be further characterized by kinetic assays with the CW-agents in collaboration with scientists from the U.S. Army Edgewood Chemical and Biological Center. <P>
2 Develop Biosensing Systems for Discriminating Between Different Classes of Neurotoxins Based on Coupled AChE-OPH Enzyme Monitors. The development of discriminative, enzyme-based field biosensor will be based on coupling the neurotoxic responsive activity of Acetyl Cholineesterase (AchE) in a chemically discriminating array utilizing variants of OPH and carbamate recognition element. These biological sensitive elements will be combined with modified physical transducers to enhance the chemical sensitivity of the system, and additional technological packaging will be designed to provide a field-ready system. Specific tasks include the develop a new biosensor strategy that involves a combination of biorecognition elements based on both neurotoxic esterases and OP hydrolases. The discriminative biosensor will be expressed on modern carbon-past printed electrodes with three enzymes (AChE, Choline oxidase and OPH) simultaneously immobilized on the surface of electrode. Our experimental plan for the next five year period is to continue with both target and saturation mutagenesis of the active site of PTE. Mutants will be rapidly identified with catalytic properties that are fine-tuned for use in both remediation and detection. New enzymes will be added to the collection using gene homologies to identify families of proteins predicted to share catalytic characteristics. Once a sufficient number of mutants have been characterized with varied catalytic properties for organophosphate hydrolysis, then catalytic assemblies can be constructed as the basis for highly sensitive chemical sensors. Detection of enzyme activity within a single cell or a small group of cells can be quantitatively measured using fluorophores specific to the products of the desired catalytic reaction. For example, the hydrolysis of organophosphates by hydrolases expressed in the cells will produce local pH changes and the release of anionic hydrolysis products. The type of anion is dependent on the substrate used to screen the mutants. Local pH changes will be detected using pH-sensitive fluorophores such as SNARF and anions detected by measuring the anion-induced quenching of fluorophores such as SPQ (a quinolinium salt) and SPA (an acridinium salt).