Small Methods | A Novel High-Sensitivity Technology for Extracellular Vesicle Detection

This study develops a VISTA technology based on single-molecule confocal microscopy, enabling high-sensitivity, high-specificity detection of extracellular vesicles (EVs) without purification steps, directly in biofluids. The method surpasses traditional techniques in detection limit and specificity, demonstrating significant translational potential.

 

Literature Overview
This article, "Fluorescence Characterization of Extracellular Vesicles Using Single-Molecule Confocal Microscopy", published in Small Methods, reviews and summarizes research on high-sensitivity, high-specificity EV detection using VISTA technology. The article demonstrates the technique's ability to accurately identify and quantify EVs in complex biological samples, avoiding measurement inaccuracies caused by interference from other particles in traditional methods. The writing is coherent and logically structured, ending with a full stop in English format.

Background Knowledge
Extracellular vesicles (EVs) are small membrane-enclosed particles secreted by cells that play critical roles in intercellular communication. They have recently gained significant attention as disease biomarkers. However, challenges remain in their detection due to low abundance and high heterogeneity. Current commonly used nanoparticle tracking analysis (NTA) relies on light scattering with low specificity, failing to distinguish EVs from similar particles like lipoproteins. Although methods like nano-flow cytometry offer specificity, they require expensive equipment and complex operations. VISTA technology combines dual-fluorescent antibodies with high-speed microfluidic chips, achieving high-sensitivity EV detection through single-molecule confocal microscopy. This overcomes limitations of existing methods and holds significant value for biomarker research and disease diagnostics. The technology enables direct analysis of serum and plasma samples without additional purification steps, reducing sample loss and improving detection efficiency. The study further compares recovery efficiency of different EV isolation methods, providing optimized strategies for EV analysis.

 

 

Research Methods and Experiments
The research team employed two fluorescent antibodies (AF488 and AF647) targeting the EV-specific surface marker CD9, combined with high-speed microfluidic chips and single-molecule confocal microscopy, to achieve dual-color colocalization detection of EVs. This method leverages multiple CD9 molecules on EV surfaces binding multiple antibodies, whereas single antibodies or non-EV particles only produce single-color fluorescence signals. By detecting colocalized fluorescence signals, researchers achieved highly specific EV identification.

Key Conclusions and Perspectives

• VISTA technology can detect EVs at sub-femtomolar concentrations (≈10⁷ EVs/mL), significantly surpassing traditional NTA methods (detection limit ≈10⁵ particles/mL)
• The method effectively distinguishes EVs from interfering particles like lipoproteins and protein aggregates in serum and plasma samples, demonstrating high specificity
• VISTA eliminates EV purification requirements, enabling direct biofluid analysis and reducing sample loss and contamination risks
• Comparison of different EV isolation methods revealed that SmartSEC (SEC) achieves higher recovery rates than ultracentrifugation (UC) in most samples, particularly in plasma

Research Significance and Prospects
VISTA provides a high-sensitivity, high-specificity solution for EV research, applicable to both fundamental studies and clinical diagnostics. Future applications could expand to detecting other EV surface markers (e.g., CD63, CD81, PSMA), enhancing identification capabilities for EV subpopulations. Additionally, VISTA's potential for automation and standardization offers broad application prospects in liquid biopsy, disease biomarker screening, and drug delivery research.

 

 

Conclusion
VISTA technology offers an innovative and efficient strategy for EV detection and quantification. By combining dual-color colocalization detection with high-speed microfluidic chips, it achieves high-sensitivity, high-specificity identification of EVs in complex biological samples. Compared to traditional methods, VISTA enables direct analysis of serum and plasma samples without purification steps, reducing sample loss while improving detection accuracy. The study demonstrates its wide applicability across various EV surface markers, showing excellent scalability and application flexibility. In the future, VISTA could become a standardized tool for EV research, advancing liquid biopsy and precision medicine by providing critical platform support for disease biomarker discovery and drug delivery system development.

 

Tianxiao Zhao, Noelia Pelegrina‐Hidalgo, Daniel C Edwards, Rebecca S Saleeb, and Mathew H Horrocks. Fluorescence Characterization of Extracellular Vesicles Using Single‐Molecule Confocal Microscopy. Small Methods.