Nanoscale detection of EpCAM-positive extracellular vesicles using magnetic beads and interferometric scattering microscopy

Extracellular vesicles (EVs) are nanoscale biomarkers that reflect the molecular state of their cell of origin. Epithelial cell adhesion molecule (EpCAM) is frequently enriched in tumor-derived EVs, offering diagnostic and monitoring potential for epithelial cancers. We developed a label-free detection method combining magnetic bead–based molecular targeting with interferometric scattering microscopy (iSCAT) for single-particle visualization of EpCAM-positive EVs. Streptavidin-coated magnetic nanoparticles (50 nm) were conjugated with biotinylated anti-EpCAM antibodies and incubated with EVs from breast cancer (MCF-7, high EpCAM) and murine colon adenocarcinoma (MC38, low EpCAM) cell lines. Nanoparticle tracking analysis confirmed bead functionalization, and Western blotting verified differential EpCAM expression. iSCAT imaging on thin-film substrates revealed distinct scattering patterns: bare beads showed uniform profiles, high EpCAM EV–bead complexes exhibited elongated morphologies, and low EpCAM samples showed minimal change. The method requires <1 μL of sample, avoids labeling artifacts, and enables real-time, high-contrast imaging. This adaptable platform can be tailored to other EV subpopulations, providing a sensitive, label-free approach for targeted EV detection in research and clinical diagnostics.