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Updated: Jun 5, 2025
Single Extracellular Vesicle Transmembrane Protein Characterization by Nano-Flow Cytometry
Published on: July 26, 2022
Size-based separation of extracellular vesicles investigating the relationship between Tetraspanins and RNA
Johan Yi1, Suyeon Kim2, Minyeob Lim2
1Department of Mechanical Engineering, Pohang University of Science and Technology, 77 Cheongam-Ro, Nam-Gu, Pohang, Gyeong-buk, 37673, Republic of Korea.
Background:
Extracellular vesicles (EVs), nano-sized particles released by cells, exhibit inherent heterogeneity, posing challenges for precise classification. This study introduces a method utilizing the coffee ring effect for size-based separation analysis, driven by the outward flow during droplet evaporation with Marangoni flow resulting from a surface tension gradient.
Results:
The controlled separation of nanoparticles based on size was applied to characterize EV and virus-like particle (VLP) samples. Tetraspanin markers exhibited distinct distribution patterns in dried droplets, with CD63-single-positive particles being smaller than those positive for CD9, CD81, or gag. Additionally, using previously developed fluorescence nanoparticle tracking analysis and transmission electron microscopy, single vesicle analysis validated the size disparities in CD-positive particles. RNA analysis through the coffee ring effect and fluorescent NTA revealed differential patterns in EVs and VLPs, providing different insights into RNA packaging.
Significance:
This multifaceted approach enhances understanding EV heterogeneity, emphasizing the potential influence of cellular origin and biogenesis pathways on particle characteristics.
