Size-exclusion chromatography-based extracellular vesicle size subtyping and multiplex membrane protein profiling for differentiating gastrointestinal cancer prognosis.

Extracellular vesicles (EVs), as a type of subcellular structure, have been extensively researched for their potential for developing advanced diagnostic technologies for various diseases. However, the biomolecular and biophysical heterogeneity of EVs has restricted their application in clinical settings. In this article, we developed a size-exclusion chromatography-based technique for simultaneous EV size subtyping and protein profiling. By eluting fluorescent aptamer-treated patient plasma through a size-exclusion column, the mixture can be classified into 50 nm aptamer-bound EVs, 100 nm aptamer-bound EVs and free-floating aptamers, which could further enable multiplex EV membrane protein profiling by analyzing the fluorescence intensities of EV-bound aptamers. Using this technique, we successfully identified EV size subtypes for differentiating gastrointestinal cancer prognosis states. Overall, we developed a rapid, user-friendly and low-cost EV size subtyping and protein profiling technique, which holds great potential for identifying crucial EV size subtypes for disease diagnosis in the clinic.