Spectroscopic approaches for the characterization of small extracellular vesicles: advances and applications in biomedical diagnostics

Exosomes, small extracellular vesicles (sEVs) of endosomal origin, have emerged as important mediators of intercellular communication and promising sources of noninvasive biomarkers owing to their diverse molecular cargo, including proteins, lipids, nucleic acids, and metabolites. However, their clinical translation remains limited by vesicle heterogeneity and the lack of standardized enrichment and characterization methods. While conventional techniques, such as western blotting, nanoparticle tracking analysis, and electron microscopy, provide essential information on exosome identity, morphology, and size distribution, they are often constrained by low sample throughput and limited molecular characterization. To address these limitations, spectroscopic approaches have advanced rapidly, offering high sensitivity and molecularly specific fingerprinting of exosomal phenotypes. This review summarizes current exosome enrichment strategies and highlights recent advances in spectroscopic approaches for exosome characterization, including fluorescence spectroscopy, surface-enhanced Raman scattering, Fourier-transform infrared spectroscopy, UV–visible spectroscopy, surface plasmon resonance, X-ray photoelectron spectroscopy, nuclear magnetic resonance spectroscopy, impedance spectroscopy, and mass spectrometry. Their potential for sensitive molecular profiling, biomarker discovery, and emerging applications in biomedical diagnostics is critically discussed. Finally, current challenges and future perspectives toward developing integrated, sensitive, and standardized platforms for exosome characterization and diagnostics are also presented.