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They can interact with target cells to perform various functions, offering potential for therapeutic applications like drug delivery and diagnosis. The growing interest in EVs drives the need for robust methods for EV characterisation. One of the prevalent EV characterisation methods is scatter\u2010based nanoparticle tracking analysis (Sc\u2010NTA). This method measures the size and concentration of particles by tracking the scattered light from individual particles. However, Sc\u2010NTA has limitations in selectivity, as it detects all scattered light and fails to distinguish EVs from other nanoparticles, such as protein aggregates. To overcome this limitation, fluorescence\u2010based NTA (Fl\u2010NTA) is being utilised, where fluorescence tagging is used to selectively detect EVs. In previous studies, lipophilic dyes were employed for membrane labelling, but this resulted in false\u2010positive signals due to the staining of even non\u2010vesicular extracellular particles (NVEPs). Immunolabelling methods using antibodies that specifically bind to EV\u2010specific protein were also introduced; yet challenges with sensitivity and photostability of the organic dyes remained. To address the challenges, we conjugated quantum dots (QDs) to antibodies that specifically bind to EV\u2010specific markers, CD9, CD63 and then immunolabelled the EVs. Labelling conditions were optimised to develop a robust protocol for QD\u2010based immunolabelling. Detection sensitivity was evaluated by comparing QD\u2010based immunolabelling with Alexa dye\u2010based methods. Furthermore, size distribution analysis demonstrated the ability of QDs to detect smaller EV populations. Finally, subpopulations of EVs from various cell lines were profiled. This approach enhances the accurate characterisation of EVs, providing a reliable and reproducible method for EV quality control and improved insights into their heterogeneity.<\/jats:p>","DOI":"10.1002\/jex2.70072","type":"journal-article","created":{"date-parts":[[2025,7,22]],"date-time":"2025-07-22T11:09:45Z","timestamp":1753182585000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":5,"title":["Quantum Dot\u2010Based Immunolabelling of Extracellular Vesicles and Detection Using Fluorescence\u2010Based Nanoparticle Tracking Analysis"],"prefix":"10.1002","volume":"4","author":[{"ORCID":"https:\/\/orcid.org\/0009-0002-1249-9745","authenticated-orcid":false,"given":"Eunyong","family":"Ha","sequence":"first","affiliation":[{"name":"Department of Chemistry, College of Natural Sciences Hanyang University Seoul Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yewon","family":"Han","sequence":"additional","affiliation":[{"name":"Department of Chemistry, College of Natural Sciences Hanyang University Seoul Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Minseop","family":"Kim","sequence":"additional","affiliation":[{"name":"Department of Chemistry, College of Natural Sciences Hanyang University Seoul Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zayakhuu","family":"Gerelkhuu","sequence":"additional","affiliation":[{"name":"Research Institute for Convergence of Basic Science Hanyang University Seoul Republic of Korea"},{"name":"Institute of Next Generation Material Design Hanyang University Seoul Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sook Jin","family":"Kwon","sequence":"additional","affiliation":[{"name":"Institute of Next Generation Material Design Hanyang University Seoul Republic of Korea"},{"name":"Yoon Idea Lab. 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