| Summary: | Biological water contamination detection-based assays are essential to test water quality; however, these assays are prone to false-positive results and inaccuracies, are time-consuming, and use complicated procedures to test large water samples. Herein, we show a simple detection and counting method for <i>E. coli</i> in the water samples involving a combination of DNAzyme sensor, microfluidics, and computer vision strategies. We first isolated <i>E. coli</i> into individual droplets containing a DNAzyme mixture using droplet microfluidics. Upon bacterial cell lysis by heating, the DNAzyme mixture reacted with a particular substrate present in the crude intracellular material (CIM) of <i>E. coli</i>. This event triggers the dissociation of the fluorophore-quencher pair present in the DNAzyme mixture leading to a fluorescence signal, indicating the presence of <i>E. coli</i> in the droplets. We developed an algorithm using computer vision to analyze the fluorescent droplets containing <i>E. coli</i> in the presence of non-fluorescent droplets. The algorithm can detect and count fluorescent droplets representing the number of <i>E. coli</i> present in the sample. Finally, we show that the developed method is highly specific to detect and count <i>E. coli</i> in the presence of other bacteria present in the water sample.
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