In-situ Detection of Indicator Organisms by Digitization and Concentration in Microfluidic Picoliter Droplets

R/CONT-219
Start/End: February, 2013 to January, 2014

The researcher has designed but not fully tested an intensely high-tech approach for monitoring low concentrations of pathogenic bacteria in water samples. In the method, water samples are mixed with probes that enzymes in bacteria convert to a fluorescent-colored product. Picoliter (trillionth-of-a-liter) droplets are formed from this mixture and the brightly colored drops are then counted to estimate bacterial concentrations. The key step in the approach is the ability to form the droplets, within which a single cell will have a very high effective concentration, on the order of 10^9 cfu/mL. Besides amplifying the pathogenic signal, the approach also reduces the assay time for detecting bacteria, which is critical for protecting public health. The main objective of this proof-of-concept project is to demonstrate the ability to form the droplets and count cells for the fecal indicator bacteria Escherichia coli and Enterococcus sp. The method’s accuracy will be verified for samples with known cell counts. The scientist will also characterize the rate at which color intensity builds in “incubating” droplets, as a function of droplet size, to identify an optimal drop size and assay time for the bacteria. The enzyme-substrate probe technology to be employed in this project has been approved and is expected to become adopted by the EPA. Outcomes from this project will further efforts to quantitatively measure low concentrations of water-borne pathogens through a technique that “packages” the EPA method in picoliter containers.