Simple SummaryIn this study, X-ray Photoelectron Spectroscopy (XPS) is used as a fast technique to characterize the composition of nanosized small extracellular vesicles (EVs), with the aim of evaluating its possible application in cancer screening and diagnosis. This spectroscopic technique is an ideal approach to characterize membranes due to its sensitivity and its penetration depth, in the nanometric range, meaning that the results will mainly correspond to the membrane composition, with little interference from the biomolecules in the EV´s lumen. We propose the use of XPS to analyze the membranes of small extracellular vesicles isolated from bodily fluids of cancer patients and compared them to those of healthy cells and donors. Notably, we have focused our analysis on the N chemical environment of EV membranes, as this can be related to previous complex chemical analyses of lipid and protein contents in EVs. We provide preliminary results on a fast and non-invasive novel strategy, as a method or tool for research-based EV investigations with potential application in cancer screening and monitoring.AbstractSmall extracellular vesicle (EV) membranes display characteristic protein-lipidic composition features that are related to their cell of origin, providing valuable clues regarding their parental cell composition and real-time state. This could be especially interesting in the case of cancer cell-derived EVs, as their membranes could serve as valuable tools in liquid biopsy applications and to detect changes in the tumor malignancy. X-Ray Photoelectron Spectroscopy (XPS) is a powerful surface analysis technique able to detect every chemical element present, being also sensitive to their chemical environment. Here we explore the use of XPS as a fast technique to characterize EV membrane composition, with possible application in cancer research. Notably, we have focused on the nitrogen environment as an indicator of the relative abundance of pyridine-type bonding, primary, secondary and tertiary amines. Specifically, we have analyzed how tumoral and healthy cells have different nitrogen chemical environments that can indicate the presence or absence of malignancy. In addition, a collection of human serum samples from cancer patients and healthy donors was also analyzed. The differential XPS analysis of EVs collected from patients confirmed that the patterns of amine evolution could be related to markers of cancer disease, opening the possibility of their use as a non-invasive blood biomarker.