Reference-free single-vesicle profiling of small extracellular vesicles from liquid biopsies with the PICO assay

Small extracellular vesicles (sEV) are membrane-enclosed nanoparticles found in body fluids that carry molecular cargo from their cells of origin. Their stability and disease-associated molecular signatures make them promising targets for the development of non-, or minimally invasive liquid biopsies, yet scalable approaches enabling single-vesicle quantification of sEV while resolving their heterogeneity remain limited. Here, we present PICO (Protein Interaction Coupling), a reference-free quantitative assay adapted for sensitive multiplex profiling of individual intact vesicles. PICO detects vesicle markers by requiring colocalization of two or more copies of the same protein or of distinct proteins (e.g., CD9 or CD9/CD63) on individual vesicles, using DNA-barcoded antibodies and digital PCR (dPCR) for quantitative readout. We demonstrate that this unique architecture of the assay provides high specificity by distinguishing EV-bound proteins from soluble counterparts, and can be adapted to target either surface-exposed or intravesicular biomarkers. PICO requires minimal sample input (1 {micro}l) and no specialized instrumentation beyond standard digital PCR. In a head-to-head comparison with nano-flow cytometry, PICO achieved a comparable limit of detection for sEV subpopulations. Profiling sEV isolates from blood for canonical markers (CD9, CD63 and CD81) and HER2 demonstrates precise, high-resolution quantification of sEV subpopulations in complex clinical samples and supports integration of scalable single-EV analysis into research and diagnostic workflows.