Investigation of Extracellular Vesicle Corona Formation with Label-Free Optical Waveguide Lightmode Spectroscopy...

Extracellular vesicles (EVs) are lipid bilayer-delimited particles that are naturally released from all types of cells into the extracellular environment. During their release to the extracellular space, EVs adsorb biomolecules, collectively designated as EV corona. In this study, we developed a novel method for immobilizing EVs and assessing their capacity to bind biomolecules, using human serum albumin (HSA) as a model molecule for corona acquisition and optical waveguide lightmode spectroscopy as the analytical technique. Ten ng/mL poly-L-lysine (15-30 kDa), prepared in 42 mM TRIS buffer at pH 7.4, was used to achieve non-specific immobilization of EVs at a concentration of 4.5 × 10¹⁰ particles/mL, and also to provide surface passivation against proteins. The experiments were performed at 25 °C with a 100 µL/min flow rate. The optimal HSA detection range was between 1 and 100 ng/mL with EC₅₀ = 2.39 ng/mL. A mathematical model was also established to determine the estimated number of HSA molecules that can be bound on the surface of EVs. In saturation experiments it was demonstrated that the magnitude of HSA bound on the surface of EVs corresponded to the theoretical estimate. After its development, the method was further tested using human plasma and a chosen protein (DJ-1) as well as non-protein corona components, specifically double-stranded DNA and a microRNA (miR494).