ExtracellularVesicles Separation and Biomedical ApplicationBased on Affinity Recognition and Antifouling Coating BifunctionalMicrosphere

Extracellular vesicles (EVs) are crucial mediators in various physiological and pathological processes, facilitating intercellular communication and offering potential as diagnostic disease markers. However, existing EVs separation methods have limitations that hinder their clinical application. In this study, we present a novel approach using bifunctional silica microspheres (SiO₂-PTB-PS) for the specific, nondestructive isolation of EVs from complex biological media. The isolated EVs were subsequently used for direct cancer detection in clinical samples. The SiO₂-PTB-PS microspheres, functionalized with a phosphatidylserine (PS) recognition peptide (PSpep), specifically bound to PS on the EVs surface. Additionally, an anti-adhesion coating on the silica microspheres minimized protein contamination, enhancing purity. This affinity-based recognition and antifouling strategy ensured high-purity EVs separation. Furthermore, we developed a detection system combining SiO₂-PTB-PS microspheres with surface-enhanced Raman scattering (SERS) nanoprobes to identify protein tyrosine kinase 7 (PTK7) and epithelial cell adhesion (EpCAM) on the EVs membrane, achieving 80% precision in distinguishing cancer patients from healthy donors. The SiO₂-PTB-PS microsphere system shows significant promise as a biotechnology tool, advancing the clinical application of EVs-based diagnostics.