An off-the-shelf digital microfluidic platform for integrated extracellular vesicle isolation and detection

Extracellular vesicles (EVs) are membrane-bound nanoparticles that serve as minimally invasive biomarkers, yet their clinical translation is hampered by limitations in existing isolation and detection strategies. Here, we systematically benchmarked EVs secreted from HeLa cell cultures using ultracentrifugation, microfluidic ultrafiltration, and magnetic bead–based capture, the latter implemented both conventionally and on an off-the-shelf digital microfluidic (DMF) platform. This comparative evaluation encompassed key performance metrics, including yield, purity, workflow complexity, and operational efficiency. Nanoparticle tracking analysis and scanning electron microscopy revealed that EVs isolated via DMF exhibited recovery rates and vesicle morphology on par with those obtained through conventional bead-based workflows, while requiring markedly reduced reagent volumes. Western blotting and flow cytometry confirmed robust expression of tetraspanin EV markers (CD63 and CD81), across a dynamic concentration range. On-chip ELISA performed directly on the DMF device demonstrated strong linearity across EV concentrations ranging from 5×10 6 to 1×10 9 particles/mL (R 2 =0.8935), in close agreement with conventional benchtop measurements (R 2 =0.8388). Together, these results establish the DMF platform used in this study as a versatile, and cost-efficient solution capable of integrating EV isolation and detection within a single, unmodified device. By uniting conventional laboratory workflows with miniaturized, digitally controlled operations, this work underscores the potential of accessible DMF technologies to advance portable, point-of-care EV diagnostics in the future.