Spectroscopic fingerprinting of extracellular vesicles from diverse cellular origins by ATR-FTIR for vibrational biomarkers of vector-host interactions.

Extracellular vesicles (EVs) are nanoscale lipid bilayer structures that facilitate intercellular communication across biological systems. Although extensively studied in mammals, their spectral and biochemical characteristics in invertebrate hosts relevant to viral transmission remain poorly understood. In this study, Attenuated Total Reflectance–Fourier Transform Infrared (ATR-FTIR) spectroscopy was used to characterize and discriminate EVs derived from human dermal fibroblasts (FibEVs), hepatocytes (HepEVs), and Aedes albopictus mosquito cells (MosqEVs), alongside synthetic EV-like vesicles (SynEVs). Spectral data were analyzed using Principal Component Analysis (PCA), Canonical Analysis of Principal Coordinates (CAP), and sparse Partial Least Squares–Discriminant Analysis (sPLS-DA). PCA revealed clear clustering according to EV origin, while CAP showed strong group separation (R² ≈ 0.99), with the first two canonical axes explaining 89% of total variance. sPLS-DA identified discriminant wavenumbers associated with lipids, proteins, and nucleic acids, achieving 93% classification accuracy and an average AUC of 0.99. MosqEVs displayed lipid-enriched spectral profiles consistent with insect membrane composition, whereas mammalian EVs were protein-dominant. These findings demonstrate that ATR-FTIR is a rapid, label-free approach for EV discrimination and provide the first vibrational characterization of mosquito-derived EVs, supporting future applications in vector biology, infection surveillance, and exosome quality assessment.