Improved-Purity Extracellular Vesicle Isolation Using Antibody−Aptamer Conjugates by Double-Droplet Microfluidics

Extracellular vesicles (EVs) are promising non-invasive biomarkers, but their isolation from complex biofluids remains a major analytical challenge due to low abundance, heterogeneity, and co-isolation of contaminants. Immunoaffinity capture using magnetic beads offers high selectivity; however, conventional workflows require multiple manual steps and often rely on harsh elution conditions that compromise the integrity and purity of the vesicles. Here, we report a droplet microfluidic platform that integrates reversible antibody−aptamer immunocapture, allowing magnetic bead handling for the isolation and gentle release of EVs directly from human plasma. The approach exploits double-droplet emulsions and magnetically assisted bead extraction to minimize residual supernatant and reduce contamination. The system is first validated using model contaminants, demonstrating up to a tenfold reduction in contaminant carryover compared with the corresponding bench-scale protocol. Then, the droplet microfluidic platform is applied for EV isolation from the human plasma without pre-enrichment, showing a recovery comparable to that of the conventional bench-scale method and a significant reduction in non-specific binding, indicating improved sample purity. Overall, this strategy combines reversible affinity chemistry with droplet microfluidics to enable EV isolation with improved purity, preserved vesicle morphology, and detectable EV-associated markers.