Final Report: Handheld FRET Aptamer Sensor to Satisfy the Beaches ActEPA Contract Number: EPD07055
Title: Handheld FRET Aptamer Sensor to Satisfy the Beaches Act
Investigators: Bruno, John G.
Small Business: Operational Technologies Corporation
EPA Contact: Manager, SBIR Program
Project Period: March 1, 2007 through August 31, 2007
Project Amount: $69,993
RFA: Small Business Innovation Research (SBIR) - Phase I (2007) RFA Text | Recipients Lists
Research Category: SBIR - Water and Wastewater , Small Business Innovation Research (SBIR)
Operational Technologies Corporation (OpTech) is developing a series of patent-pending fluorescence resonance energy transfer (FRET)-DNA aptamers and assays to whole lipopolysaccharide (LPS) and its components as well as lipoteichoic acid to rapidly detect and quantify E. coli and Enterococcus bacteria at low levels in environmental water samples. The project is pushing the limits of sensitivity using high affinity and highly specific aptamers, which are labeled with fluorophores, quenched by quencher-labeled bacteria, and compete with unlabeled bacteria in concentrated samples reduced 100:1 by filtration from 100 ml environmental water samples. The goal is to meet the detection requirements of the federal Beaches Act, which mandates daily monitoring of recreational waters for very low levels of the fecal indicators E. coli and Enterococcus. The FRET-aptamer assays will be lyophilized and interface with a commercially available handheld, battery-operated fluorometer to achieve rapid detection (within 20–30 minutes) in the field.
At the end of Phase I, OpTech has amassed 58 candidate aptamer DNA sequences to whole LPS form E. coli O111 and its components (glucosamine, KDO, and rough Ra core antigen). In addition, OpTech sequenced 8 candidate aptamers that bind lipoteichoic acid from Enterococcus faecalis. The secondary stem-loop structures of these aptamers were determined by a free energy minimization computer program (Vienna RNA software) using DNA parameters, and they appear to cluster into several structural families.
The aptamers were fluorophore-labeled by PCR or asymmetric PCR using AlexaFluor™ (AF) 546-dUTP and complexed with Black Hole Quencher-2-labeled heat-killed E. coli. In competitive FRET experiments, the polyclonal (SELEX round 5) aptamer system appeared capable of detecting between 10 and 100 bacteria per mL. Using one of the KDO aptamer sequences that were AF 546-dUTP labeled by asymmetric PCR, OpTech achieved a detection limit of 500 E. coli per mL, suggesting that other sequences in the aptamer pool contributed to the exceptionally low detection limit seen with the polyclonal FRET experiments.
The FRET-aptamer assay reagents also have been dried using a Savant Speed Vac with no significant loss of assay performance upon reconstitution. These data suggest that the prototype system (reader and assays) resulting from a Phase II effort could be freeze-dried for long shelf life. The tests might require a cocktail of several known LPSs or lipoteichoic acid aptamer sequences to cover a broad dynamic range of bacterial concentrations, because, like monoclonal antibodies, monoclonal aptamers seem to exhibit a more narrow dynamic range of concentrations for analyte detection.
The results from Phase I are very encouraging and strongly suggest that OpTech can optimize and produce FRET-aptamer assays for E. coli and Enterococcus in Phase II, which will likely involve the use of several aptamer sequences in a freeze-dried cocktail. OpTech is forming alliances with an assay contract manufacturer and the manufacturer of the handheld fluorescence reader to bring this system to market. If successful, the OpTech system can either replace or act as an initial detection system to augment the currently slow centralized laboratory instruments (including PCR-based detectors) to ensure the safety of recreational waters. Other uses would include on-site monitoring of the effluents from sewage treatment facilities before the treated effluents are returned to the environment and rapid, on-site monitoring for fecal pathogens in farm irrigation water.
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||Ikanovic M, Rudzinski WE, Bruno JG, Allman A, Carrillo MP, Dwarakanath S, Bhahdigadi S, Rao P, Kiel JL, Andrews CJ. Fluorescence assay based on aptamer-quantum dot binding to Bacillus thuringiensis spores. Journal of Fluorescence 2007;17:193-199.||