The long-term objective of this project is the development of a rapid and low cost detection tool, based on novel biosensor receptor elements, to test human urine for organophosphate pesticide exposure - a global public health issue. Chronic low dose exposure can result in a variety of neuropathies, increased incidence of leukemia, birth defects and learning disabilities in children. Organophosphates are metabolized in the body and dialkylphosphate metabolites are concentrated in the urine. Current methods of detection are too complex for broad population screening.
The specific aims of this project are 1) to identify 2-4 insect chemosensory proteins which bind dialkylphosphates with high affinity and then 2) to identify lower affinity competitor ligands which will facilitate development of a competitive, lateral flow-based assay and then 3) to demonstrate a multiplex lateral flow assay for dialkylphosphates based on insect chemosensory protein receptor elements. To achieve these aims a library of over 100 insect chemosensory proteins will be screened with the 4 most common dialkylphosphates detected in urine using a proprietary plate-based fluorescent assay. Proteins displaying significant binding affinity will then be screened against a combinatorial chemical library to identify lower affinity competitor ligands. At least two of the insect chemosensory proteins and competitor ligands will then be incorporated in a multiplex lateral flow assay in which the presence of dialkylphosphates in the sample will prevent binding of the insect chemosensory protein to the competitor ligand, producing a visible readout within 15 minutes. Insect chemosensory proteins are a large, diverse population which has evolved to be ideal receptors for small organic compounds, especially those that are poorly immunogenic and difficult to detect with specificity using current biosensor technology. They are small and readily expressed in high concentration. Both termini are distant from the binding pocket and available for modification, facilitating attachment to solid phases. They display high thermal stability allowing operation of biosensors under harsh physical conditions. Collectively, the structural features, diversity, and versatility of insect chemosensory proteins will facilitate the development of novel biosensors.
Pesticide exposure is a global public health issue. Chronic low dose exposure can result in a variety of neuropathies, increased incidence of leukemia, birth defects and learning disabilities in children. Current monitoring methods are inappropriate for broad population screening so a simple, low cost device for monitoring exposure through urine tests would facilitate more widespread testing and more rapid detection and treatment of exposed individuals.
