Advances in single extracellular vesicle characterization and multiplexed profiling

Extracellular vesicles (EVs) are nanoscale lipid bilayer particles that carry diverse molecular cargos and play essential roles in intercellular communication. However, their inherent heterogeneity and diversity present major challenges for bulk assays and conventional single EV profiling, as these approaches fail to identify rare subpopulations and the full molecular diversity, respectively. Advances in single EV characterization and multiplexed EV profiling technologies have begun to address these limitations, enabling precise, high-resolution analysis of individual vesicles. This review highlights recent developments in single EV characterization methods, including optical and mechanical approaches, and discusses multiplexed profiling platforms categorized into substrate-based, droplet-based, and solution-based strategies. These platforms integrate single EV partitioning with fluorescence and molecular coding schemes to achieve high-throughput and multidimensional biomarker analysis. We also outline four future perspectives for advancing multiplexed single EV profiling and facilitating its clinical application: 1) Integration of multi-omics in single EV profiling, 2) Multi-functional single EV profiling, 3) Rare and clinically relevant EV subpopulation analysis, 4) AI-driven data interpretation in multiplexed single EV analysis. • A comprehensive review of recent advances in the characterization of single extracellular vesicle (EV) and multiplexed single-EV analysis • Emphasis on the significance of multiplexed single-EV profiling in addressing EV heterogeneity • Focusing on the advanced multiplexed single-EV analysis, especially the integration of partitioning methods with multiplexing strategies • Discussion of current challenges and future perspectives for advancing multiplexed single-EV profiling toward clinical application