Phage-Based Microfluidic Assay for Detecting Viable E. coli in Water Samples

Begin with a contaminated water sample likely to harbor fecal coliforms, including E. coli.

Perform a serial dilution in distilled water to reduce turbidity and microbial load.

Mix the desired diluted sample with the nutrient medium and load it into a microfluidic chip.

Use vacuum filtration to capture bacteria on a membrane within the serpentine channel of the chip.

Add nutrient medium to the upper chamber and incubate to enhance bacterial recovery.

Replace the medium with a solution of E. coli-specific phages carrying the luciferase gene.

Incubate to allow phage infection, where the phage DNA replicates, degrades host DNA, and forms new phages along with producing luciferase. Then, the infected cells lyse to release the luciferase.

Apply vacuum filtration to transfer the luciferase to a capture membrane.

Add a substrate that reacts with luciferase to generate a luminescent signal.

Using a photomultiplier tube, measure the luminescence, which is proportional to the number of viable E. coli cells in the water sample.