A One-Step Workflow for Size-Based Separation of Extracellular Vesicles With Integrated Surface Marker Detection

Area of Science:

• Biotechnology
• Biomarker Discovery
• Nanotechnology

Background:

• Extracellular vesicles (EVs) are crucial biomarkers in liquid biopsies for disease diagnosis, particularly cancer.
• Current EV analysis requires extensive pre-purification from large biofluid volumes, causing delays and variability.
• There is a need for rapid, sensitive, and reproducible methods for EV biomarker characterization.

Purpose of the Study:

• To develop and validate a streamlined, one-step workflow for simultaneous size and surface marker analysis of EVs.
• To enable EV biomarker detection in minute volumes of complex biological fluids.
• To assess the workflow's potential for early cancer detection and patient stratification.

Main Methods:

• Coupling asymmetrical flow field-flow fractionation (AF4) with multi-angle light scattering (MALS) and a fluorescent light detector (FLD).
• Optimization of the AF4-MALS-FLD system for analyzing EV surface markers (CD9, CD63, CD81) and cancer biomarkers (PSMA, EpCAM, HER2).
• Application of the workflow to diverse sample types: purified EVs, cell culture supernatant, urine, and blood plasma.

Main Results:

• The AF4-MALS-FLD workflow successfully characterized EV size and surface markers in various biofluids.
• Detection of prostate cancer biomarkers (PSMA) in patient urine samples was achieved.
• Discrimination between breast cancer patients and healthy donors was demonstrated by quantifying specific EV biomarkers in plasma.

Conclusions:

• The one-step AF4-MALS-FLD workflow offers a rapid and robust method for extracellular vesicle biomarker detection.
• This approach significantly reduces sample volume requirements and technical variability in EV analysis.
• The workflow shows promise for advancing liquid biopsy applications in clinical diagnostics and personalized medicine.