All-in-one multiple extracellular vesicle miRNA detection on a miniaturized digital microfluidic workstation

Extracellular vesicles (EVs) and EV-derived microRNAs (EV-miRNAs) are emerging as promising circulating biomarkers for early detection of malignant tumors such as non-small cell lung cancer (NSCLC). However, utilization of the gold standard method of RNA detection, the reverse transcription- quantitative polymerase chain reaction (RT-qPCR), on EV-miRNAs is hindered by laborious sample purification requirements and timeconsuming multi-step procedures. Herein, we propose and demonstrate a miniaturized digital microfluidic (DMF) workstation for all-in-one EV-miRNA detection based on RT-qPCR. In comparison with the previously reported DMF platform for EV isolation, the system further integrates parallel on-chip real-time PCR capability with a comparable detection sensitivity with in-vitro RT-qPCR (limit of detection = 2 copies/mu L), realizing automated, miniaturized, and facile EV-miRNA detection. Meanwhile, major methodological improvements were made, including one-step stem-looped RT-qPCR for miRNAs with both high sensitivity and specificity, and a simplified DMF substrate rework strategy for cost-effectiveness. As a demonstration, the detection of NSCLCrelated EV-miRNAs within 20 mu L of plasma samples was implemented, indicating the potential applicability of the DMF workstation and its automated protocol on point-of-care diagnosis of a wide range of diseases.