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Digital microfluidics is rapidly becoming the microfluidic technology of choice for a large variety of applications. The idea of using discrete droplets to maintain a high concentration of sub-cellular contents for digital detection at single molecule or cell level is highly promising as it can greatly enhance the detection sensitivity and quantification accuracy. However, realization of this idea has been hampered by the need for complicated target amplification and off-chip sample processing. Here we extend the repertoire of digital microfluidic applications to include a versatile “sample-to-answer” platform for pathogen detection. The proposed platform can perform integrated sample preparation and amplification-free pathogen detection on a microfluidic chip. The integration enables digital detection in a continuous flow format and thereby high-throughput measurement of single cells.