Positively charged gold–silver nanostar enabled molecular characterization of cancer associated extracellular vesicles

Direct surface-enhanced Raman scattering (SERS) has contributed to characterizing extracellular vesicles (EVs) by providing molecular signatures. However, little work has been carried out to understand the heterogeneity of EVs created by different methods or from different biological sources. Herein, we pioneered the use of positively charged gold-silver nanostars to explore the SERS profiles of different EVs. The physical features of EVs from cancer cells including the size, concentration, morphology and surface potential have been characterized via nanoparticle tracking analysis, transmission electron microscopy and zeta potential analysis. The results show that negatively charged EVs are attracted to positively charged gold-silver nanostar surfaces via electrostatic forces resulting in SERS spectra showing characteristic vibrational modes of the different components of EVs (i.e. proteins, lipids and nucleic acids). SERS data were complemented by other spectroscopic techniques including atomic force microscopy-infrared spectroscopy, UV-visible absorbance spectroscopy and fluorescence spectroscopy providing a more complete molecular picture of EVs. SERS signatures of EVs from different origins, batches, and isolation approaches were compared and analyzed. A statistical method (principal component analysis-linear discriminant analysis) was utilized to differentiate EV subtypes. Consequently, a desirable discrimination outcome for blind samples was obtained. This study provides novel insights to deepen our understanding of EV heterogeneity.