김민섭 (한양대학교)

윤태현 (한양대학교)

김민섭 (한양대학교)

하승민 (한양대학교)

하은용 (한양대학교)

한예원 (한양대학교)

Zayakhuu Gerelkhuu (한양대학교)

윤태현 (한양대학교)

Extracellular vesicles (EVs) play a vital role in cell-to-cell communication and hold great promises for diagnostics and therapy. However, their small size, heterogeneity, and interference with other nanoparticles make them difficult to detect and characterize using traditional methods. In this study, we introduce a single-EV inductively coupled plasma time-of-flight mass spectrometry (sEV-ICP-TOFMS) technique that integrates mass cytometry with nanoparticle-conjugated antibodies and isotope ratio filtering to improve EV detection and quantification. The application of nanoparticle-conjugated antibodies significantly increased detection sensitivity compared to traditional metal isotope-labeled antibodies, allowing for reliable identification of EVs with low-abundance surface markers. The implementation of isotope ratio filtering enhanced signal specificity by distinguishing EV signals from background noise based on the distinct natural isotope abundance patterns of the nanoparticles. Quantitative analysis showed that applying isotope ratio filtering reduced background noise, resulting in an improved limit of detection (LOD) over standard intensity-based thresholding methods. Multiparametric profiling of surface proteins uncovered a bimodal distribution of CD9/CD63 coexpression, indicating EV heterogeneity. Further quantification revealed that CD9+ EVs carried an average of 15.9 ± 20.8 CD9 proteins per vesicle, whereas CD9+CD63+ EVs carried 22.8 ± 4.5 CD9 proteins per vesicle. This novel sEV-ICP-TOF-MS platform enhances both the sensitivity and specificity of single EV detection and enables high-resolution, multiparametric EV profiling. It presents a scalable approach with strong potential for biomarker discovery, disease diagnostics, and advancing our understanding of EV biology.