Microfluidic Extraction of Extracellular Vesicle-Associated Nucleic Acids for Downstream Molecular Analysis

Aim: To develop a microfluidic workflow for extracting extracellular vesicle (EV)-associated nucleic acids (NAs) from biofluids, such as blood plasma and serum, for downstream molecular analysis. Methods: Healthy serum and plasma samples were spiked with EVs obtained from Hec-1-A cells carrying defined mutations and characterized in accordance with MISEV guidelines. The spiked samples were then processed using either the standard centrifugation-based ExoGAG protocol or a microfluidic workflow. The latter combines ExoGAG mediated aggregation of EVs with mechanical entrapment of the resulting EV aggregates in a microfluidic filtration device (EVNA-chip). The retained material was washed and on-chip lysed to release its NA cargo. NAs were subsequently purified using a commercial silica column, producing an output suitable for molecular analysis through droplet digital PCR (ddPCR). Results: The EVNA-chip recovery yield was 42.0 +/- 4.28% and 38.1 +/- 1.46% for double-stranded DNA (dsDNA) and microRNA respectively. The recovered dsDNA remained compatible with ddPCR detection of the KRAS p.G12D variant (c.35G>A) and with the assessment of MSI-associated alterations using BAT25, BAT26, NR21, NR24, and MONO27. The method was demonstrated for pilot cohort of patients with endometrial cancer. The RNA workflow supported reverse transcription and was demonstrated for the quantification of miR-X, a potential marker of Fabry disease, using patient samples and healthy controls. Conclusion: Although recovery was lower than with the standard protocol (~45% for DNA and for RNA), the centrifugation-free EVNA-chip workflow demonstrates the compatibility of the method with downstream NA molecular analyses and is advantageous for the development of an integrated, automated platform for EV-based liquid biopsy applications.