Colorimetric Nanoplasmonic Assay To Determine Purity and Titrate Extracellular Vesicles

Abstract

Extracellular Vesicles (EVs) - cell secreted vesicles that carry rich molecular information of the parental cell and constitute an important mode of intercellular communication - are becoming a primary topic in translational medicine. EVs (that comprise exosomes and microvesicles/microparticles) have a size ranging from 40 nm to 1 μm and share several physicochemical proprieties, including size, density, surface charge, and light interaction, with other nano-objects present in body fluids, such as single and aggregated proteins. This makes separation, titration, and characterization of EVs challenging and time-consuming. Here we present a cost-effective and fast colorimetric assay for probing by eye protein contaminants and determine the concentration of EV preparations, which exploits the synergy between colloidal gold nanoplasmonics, nanoparticle-protein corona, and nanoparticle-membrane interaction. The assay hits a limit of detection of protein contaminants of 5 ng/μL and has a dynamic range of EV concentration ranging from 35 fM to 35 pM, which matches the typical range of EV concentration in body fluids. This work provides the first example of the exploitation of the nanoparticle-protein corona in analytical chemistry.

Interaction of Extracellular Vesicles with Si Surface Studied by Nanomechanical Microcantilever Sensors

“…The determined AI for the used preparation resulted below 25% of the AI of the dispersed AuNPs (Figure 2A), corresponding to a contaminant content <10 ng/µL (according to the calibration reported in Ref. [9]), that is a high purity preparation. Figure 2B shows the western blot analysis of the EV samples.…”

“…Before further analysis, EV pellets were resuspended in 50 µL PBS 10 mM supplemented with 1:1000 protease inhibitor cocktail. EV preparation purity was graded by a colorimetric nanoplasmonic (CONAN) assay [9], and related UV-vis spectroscopy measurements performed by a EnSight multimode plate reader (Perkin Elmer, Waltham, Massachusetts, United States. EV titration was performed as described in [23]; the calibration line was built with POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, Avanti Polar Lipids, Alabaster, AL, USA) liposomes of about 60 nm size.…”

“…For instance, the colloidal and interfacial properties of EVs have started to be tackled only very recently [9][10][11], and the mechanisms that govern direct interaction of EVs with synthetic surfaces is mostly unexplored.…”

“…The surface was selected as it is the most representative inorganic solid surface among the ones employed in biological applications, spanning from biosensors [14,15] to organoid-on-a-chip [16]. MC biosensors 2 of 11 were chosen for their ability to probe mass adsorption and interaction energetics at the solid-liquid interface of minute volumes of synthetic liposomes [17,18], comparable with standard EV formulations obtained from biological fluids or cell culture media, which are typically 100 µL solution volumes at few nM EV concentration (see for example [9]). Small EVs were isolated from an immortalized cell line of human vascular endothelial cell (HVEC) that is routinely used as model of EV-secreting cells, since it produces a good amount of highly pure exosomes compared to other cell types [19].…”

Therapeutic and diagnostic applications of extracellular vesicles

“…To increase both the vesicle yield as well as purity of the isolate, density gradient UC (iodixanol or sucrose) can be used [64,72]. To underscore the superior separation resolution, it was shown that viral particles could be physically separated from EVs by using an iodixanol-based density gradient [73].…”