Volatolomic analysis of extracellular vesicles extracted from cultured cells

Extracellular vesicles (EVs) are pivotal mediators of intercellular communication and have emerged as key biomarkers in biomedical research. Their characterisation is conventionally based on physical and molecular approaches, including imaging, proteomics, genomics and lipidomics. Here, we introduce a complementary analytical strategy based on volatolomics, targeting volatile organic compounds (VOCs) released by EVs. EVs isolated from three human cell lines (EPI, Hep-G2, and HEK-293) were analysed using three different volatolomic techniques: gas chromatography-mass spectrometry (GC-MS), gas chromatography-ion mobility spectrometry (GC-IMS), and an electronic nose (e-nose) system. GC-MS enabled the identification of eight EV-associated VOCs, among which nonanal and 2,4-dimethylbenzaldehyde were absent in phosphate-buffered saline (PBS), the EV storage medium. GC-IMS provided distinctive signal patterns that allowed clear discrimination between the EVs originating from the three cell lines. Furthermore, e-nose analysis employing an array of ten porphyrinoid-functionalised quartz crystal microbalance sensors successfully differentiated the EVs from the control medium and generated well-defined, non-overlapping clusters for each cell type EV. Collectively, these results demonstrate that VOC profiling offers a powerful and previously unexplored approach for EV characterisation. This study represents the first application of GC-IMS and electronic-nose technologies to extracellular vesicle analysis, opening new avenues for EV profiling.