Handheld FRET Aptamer Sensor to Satisy the Beaches Act

Operational Technologies Corporation (OpTech) proposes to couple the ultrasensitivity of fluorescence-based detection with the high affinity and selectivity of DNA aptamers as improved replacements for antibodies to detect fecal indicator organisms in fresh and marine recreational waters. Fluorescence Resonance Energy Transfer (FRET) aptamer assays for fecal indicator organisms will be demonstrated in Phase I. Most other detection technologies are incapable of ultrasensitive detection to the level o f 100 or fewer microbes per 100 mL of water. The aptamer FRET assays will be developed to detect less than the EPA recommended 126 E. coli and 33 enterococci per 100 mL in fresh water and 35 enterococci per 100 mL in salt water samples, which will enable rapid compliance with the Beaches Act. The stoichiometry of aptamer FRET also makes such reactions highly accurate and reliable for quantitative analyses. OpTech has successfully developed preliminary FRET aptamer assays for HSARPA and the U.S. Army in the area of chemical and biological defense. Under this Phase I project, OpTech will develop FRET aptamers by it paten pending process against common surface antigens of all fecal coliforms such as core lipopolysaccharide (LPS) antigens (ethanolamine, specific saccharides, etc.) and glycerol teichoic acids of E. faecalis or E. faecium,thereby enabling detection across broad ranges of coliform and Enterococcus species. OpTech will then develop a 100 mL sample filtration protocol using a syringe filter to concentrate the sample prior to analysis. The entire procedure will require little or no experience and training, so that all laymen will be able to perform the assays almost immediately. In Phase II, OpTech will clone and sequence the individual FRET aptamers and use those of greatest merit in the ultimate assay formulations. Finally, OpTech will lyophilize these assays in plastic curvettes and integrate the assays with commercially available, battery-operated, handheld fluorometer for rapid, facile, and inexpensive detection of fecal contamination on site at beaches. OpTech will provide clear proof-of-concept data with bacterial spikes samples in Phase I, followed by assay refinement, testing at real recreational water sites, packaging and delivery of the system to the EPA by the end of Phase II. Clearly, the one-step FRET assay approach coupled to a handheld florometer will have numerous other commercial applications in clinical point-of-care diagnostics, including highly accurate glucose detection, veterinary diagnostics, environmental and agricultural field portable detection as well as homeland defense and military chemical or biological warfare agent detection.