Digital droplet microfluidics integrating DNA walkers and CRISPR-Cas13a for simultaneous surface protein and miRNA profiling in single exosomes

Tumor-derived exosomes carry multi-scale molecular signatures (e.g., surface proteins and nucleic acids) that reflect tumor heterogeneity, yet simultaneously profiling these biomarkers in single intact vesicles remains technically challenging. Herein, we developed a digital droplet microfluidic platform that integrates a DNA walker and a CRISPR/Cas13a system for the simultaneous detection of surface proteins (EpCAM, HER2) and miRNA (miR-21) at the single exosome level. This platform employed engineered liposome nanoprobes (eLipo-NPs) with EpCAM aptamers and hairpin probes (HPs) functionalized on their outer membranes, and encapsulated a CRISPR/Cas13a system within their lumen. Upon co-encapsulation with single exosomes into droplets, EpCAM-mediated membrane fusion redistributed HPs across the hybrid membrane and delivered CRISPR/Cas13a into the exosomes. The membrane-anchored DNA walker then bound HER2 and drove cyclic DNAzyme cleavage of HPs to restore red fluorescence. At the same time, crRNA-guided Cas13a recognized miR-21 and triggered trans-cleavage of reporters to generate green fluorescence. Digital counting of dual-color droplets enabled quantitative single-exosome analysis with a limit of detection (LOD) of 10 particles/μL, an assay time of 60 min, and throughput exceeding 8 × 10 3 droplets/s. Clinical validation using plasma-derived exosomes from 24 breast cancer patients and 14 healthy donors demonstrated distinct distributions among HER2-positive, HER2-negative, and healthy control groups, with dual-positive droplet fractions significantly correlated with clinical HER2 status, highlighting the platform's potential for liquid biopsy and precision oncology.