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This study systematically deconstructs the sources and consequences of this limitation using high\u2010sensitivity nano\u2010flow cytometry (nFCM), revealing how the spatial footprint of antibody\u2010fluorophore conjugates fundamentally compromises co\u2010detection fidelity. To overcome this, we developed a sterically optimized framework through the sequential minimization of the spatial footprint of detection elements: first, by replacing large fluorophores with compact organic dyes and, most effectively, by employing nanobodies. This approach significantly enhanced the detection of dual\u2010protein\u2010positive EV subpopulations by mitigating mutual steric interference. Notably, the persistent labeling limitation observed for closely apposed protein pairs, such as CD9\/CD63, was repurposed as a sensitive probe for inferring nanoscale membrane topography. Clinically, simultaneous dual\u2010nanobody detection of PD\u2010L1 and CD47 on plasma\u2010derived EVs achieved an overall accuracy of 90.0% for breast cancer diagnosis. This work establishes a generalizable framework for accurate, multiplexed EV profiling in clinical diagnostics.<\/jats:p>","DOI":"10.1002\/smtd.71053","type":"journal-article","created":{"date-parts":[[2026,9,22]],"date-time":"2026-09-22T11:39:42Z","timestamp":1790077182000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Overcoming Steric Hindrance for Precise Multiplexed Protein Profiling of Single Extracellular Vesicles"],"prefix":"10.1002","author":[{"given":"Yunyun","family":"Hu","sequence":"first","affiliation":[{"name":"Department of Chemical Biology MOE Key Laboratory of Spectrochemical Analysis &amp; Instrumentation, Fujian Key Laboratory for Chemical Biology (Xiamen University) State Key Laboratory of Physical Chemistry of Solid Surfaces Collaborative Innovation Center of Chemistry for Energy Materials College of Chemistry and Chemical Engineering Xiamen University  Xiamen 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