Single-Exosome EpCAM Heterogeneity Profiling for Breast Cancer Diagnosis and Progression Monitoring.

Exosomes are nanoscale vesicles that are attractive for liquid biopsy, but most methods deliver ensemble averages and obscure how surface biomarkers vary across individual vesicles. We develop a single-particle impact electrochemistry (SPIE) assay to profile epithelial cell adhesion molecule (EpCAM) on single exosomes. EpCAM-positive vesicles are labeled with silver nanoparticle-aptamer conjugates. Each labeled exosome yields an silver oxidation spike upon collision with a polarized ultramicroelectrode. Impact frequency reports the abundance of EpCAM-positive exosomes, while the integrated spike charge provides a semiquantitative proxy for per-vesicle EpCAM. Using MCF-7 (EpCAM-high) and HeLa (EpCAM-low) exosomes, we confirm EpCAM-dependent signals and resolve intrapopulation heterogeneity. For serum samples from healthy donors and breast cancer patients at different clinical stages, SPIE reveals stage-dependent increases in both EpCAM-positive exosome counts and per-vesicle EpCAM load, consistent with nanoflow cytometry. This nonoptical platform enables semiquantitative single-exosome protein phenotyping in complex matrices, offering potential for disease-state-associated exosome analysis, clinical research, and future multiplexed profiling.