Overcoming Steric Hindrance for Precise Multiplexed Protein Profiling of Single Extracellular Vesicles.

Multiplexed protein profiling of single extracellular vesicles (EVs) is crucial for precise liquid biopsy but is severely limited by steric hindrance arising from bulky detection probes. This study systematically deconstructs the sources and consequences of this limitation using high-sensitivity nano-flow cytometry (nFCM), revealing how the spatial footprint of antibody-fluorophore conjugates fundamentally compromises co-detection 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-protein-positive 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-nanobody detection of PD-L1 and CD47 on plasma-derived 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.