Counting of peripheral extracellular vesicles in Multiple Sclerosis patients by an improved nanoplasmonic assay and dynamic light scattering

Extracellular vesicles (EVs) are vesicles naturally secreted by the majority of human cells. Being composed by a closed phospholipid bilayer secluding proteins and RNAs they are used to transfer molecular information to other cells, thereby influencing the recipient cell functions. Despite the increasingly recognized relevance of EVs, the clarification of their physiological role is hampered by the lack of suitable analytical tools for their quantification and characterization. In this study, we have implemented a nanoplasmonic assay, previously proposed for the purity of the EV fractions, to achieve a robust analytical protocol in order to quantify the total phospholipid concentration (CPL) and the EVs number. We show how the coupling of the nanoplasmonic assay with serial dilutions of the unknown sample allows, by simple visual inspection, to detect deviations from the physiological EVs content. The use of a response that depends on the absorbance values at three wavelengths permits to reduce the limit of detection of CPL to 5 ?M (total) and the limit of quantification to 35 ?M. We also propose a method that takes into account the spread in EV size when the concentration of phospholipids is turned into a concentration of vesicles. The proposed analytical protocol is successfully applied to a small cohort of Multiple Sclerosis patients examined in different stages of their clinical diseases.