Handheld FRET Aptamer Sensor to Satisfy 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 of 100 or fewer microbes per 100mL of water. The aptamer FRET assays will be developed to detect less than the U.S. Environmental Protection Agency (EPA) recommended 126 Escherichia coli and 33 enterococci per 100 mL in fresh water and 35 enterococci per 100mL 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 Homeland Security Advanced Research Projects Agency and the U.S. Army in the area of chemical and biological defense. Under this Phase I project, OpTech will develop FRET aptamers by its patent-pending process against common surface antigens of all fecal coliforms such as core lipopolysaccharide 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 100mL 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 cuvettes and integrate the assays with a commercially available, battery-operated, handheld fluorometer for rapid, facile, and inexpensive detection of fecal indicator organisms.