A microfluidic device and instrument prototypes for the detection of Escherichia coli in water samples using a phage-based bioluminescence assay

Abstract

Current quantification methods of Escherichia coli (E. coli) contamination in water samples involve long incubation, laboratory equipment and facilities, or complex processes that require specialized training for accurate operation and...

Rapid, sensitive, and low-cost detection of Escherichia coli bacteria in contaminated water samples using a phage-based assay

“…Note that, with strain ATCC 25922, there does not seem to be an advantage nor a disadvantage in incubating the bacteria with the phages for more than 2 h as we observe the signal plateauing after 2 h. Thus, for subsequent experiments with field samples that contain uncharacterized bacterial populations and within the microfluidic device 22 , we will use a phage incubation time of 3 h to ensure that all bacteria have time to produce the enzymatic reporter.…”

“…A vacuum source connected to outlet ports was used to control the fluidic flow within the microfluidic device. In this experiment, the processed volume of water was 10 mL, which we have shown in a previous study to yield equivalent results to the same number of bacteria in a 100 mL sample volume 22 . The assay was run for a total of 5 h, divided into a 2-h bacteria recovery period followed by a 3-h phage infection to ensure enough time for the cells to produce the NanoLuc reporter enzyme.…”

“…Thus, at the time of this field evaluation, our cocktail was at an early stage of development and contained four phages, namely Phi3, Phi7, RB69, and T7. The performance of the preliminary phage cocktail was tested in combination with an early microfluidic chip prototype designed to replace the manual steps of the assay, as described in our previous work 22 (Fig. 4 a).…”

“…As for our field evaluation of the partial cocktail, the assay was carried out using an in-house designed microfluidic chip, this time with an updated version shown in Fig. 5 a and further described in previous work 22 . The new version of the microfluidic device contains all the same functional units of the previous version; however, small modifications were made to allow for an injection-molded manufacturing process, which considerably reduced the overall cost of the test.…”

“…The phage cocktail has a broad E. coli host range that allows using the assay on any water source. Finally, the assay is performed in a microfluidic chip designed to integrate with an automated instrument prototype (a filtration unit, a liquid handling unit, and a detection unit) 22 for use in the field. The overall cost of the disposable microfluidic device is less than $1, and the associated instrumentation meets the cost targets set forth by the UNICEF TPP 8 , making this platform a suitable alternative to current E. coli field test kits.…”