Multiparametric orthogonal characterization of extracellular vesicles by liquid chromatography combined with in-line light scattering and fluorescence detection

Extracellular vesicles (EVs) are membrane-enclosed biological nanoparticles with potential as diagnostic markers and carriers for therapeutics. Characterization of EVs poses severe challenges due to their complex structure and composition, requiring the combination of orthogonal analytical techniques. ETH Zurich researchers demonstrate how liquid chromatography combined with multi-angle light scattering (MALS) and fluorescence detection in one single apparatus can provide multiparametric characterization of EV samples, including concentration of particles, average diameter of the particles, protein amount to particle number ratio, presence of EV surface markers and lipids, EV shape, and sample purity. The method requires a small amount of sample of approximately 107 EVs, limited handling of the sample and data analysis time in the order of minutes; it is fully automatable and can be applied to both crude and purified samples.

Schematic representation of the analytical system

Samples were injected into the chromatographic column (either size exclusion or ion exchange) to separate different species. The eluate was analyzed first by an in-line multi-angle light scattering (MALS) detector and then by a multiple wavelength fluorescence detector (FLD).

Normak K, Papp M, Ullmann M, Paganini C, Manno M, Bongiovanni A, Bergese P, Arosio P. (2023) Multiparametric Orthogonal Characterization of Extracellular Vesicles by Liquid Chromatography Combined with In-Line Light Scattering and Fluorescence Detection. Anal Chem. [Epub ahead of print]. [article]

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