Orthogonal analytical characterization of extracellular vesicles derived from healthy human saliva

Saliva-based diagnostics offer a compelling route toward accessible healthcare, particularly in low-resource settings where conventional laboratory infrastructure is limited. Extracellular vesicles (EVs) present in saliva carry molecular signatures reflective of physiological and pathological states, yet their clinical translation is hindered by reliance on complex instrumentation. Here, we report a label-free Raman spectroscopic approach for rapid biochemical profiling of salivary EVs, integrated with minimally invasive sampling and simplified processing workflows. EVs isolated from human saliva were characterized using cryogenic electron microscopy, nanoparticle tracking analysis, dynamic light scattering, and ExoView, confirming structural integrity and canonical marker expression. Raman spectroscopy enabled acquisition of distinct molecular fingerprints corresponding to proteins, lipids, and nucleic acids without labeling or extensive sample preparation. Importantly, we outline a pathway toward portable Raman-based detection systems, emphasizing low-cost optics, reduced power requirements, and compatibility with decentralized testing environments. This platform establishes an orthogonally validated structural and biochemical baseline for salivary EVs using label-free Raman spectroscopy and highlights that portable, point-of-care translation is a future opportunity enabled by this approach demonstrated in the present study.