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In particular, attribution of a purity grade and determination of the molar concentration (particle number) of EV preparations in a reproducible and scalable/cost-effective fashion requires further improvement ( Thery et al., 2018). However, EV biology investigation and clinical translation are not adequately supported by current manufacturing and characterization technologies ( Margolis and Sadovsky, 2019 Wiklander et al., 2019). EVs exhibit biomedical properties superior to even the most innovative synthetic nanomaterials, providing an array of possible applications in medical treatment and diagnostics, including regenerative medicine, immunology, neuroscience, microbiology, bio-nanotechnology, pharmacology, and others ( De Toro et al., 2015 Stremersch et al., 2016 Vader et al., 2016 Paolini et al., 2018). Such soft particles are composed of a lipid bilayer trimmed for safe and specific long-range transport and a hydrophilic core in which bio-macromolecules are stored. Among others, the secretion of membranous bio-nanoparticles called extracellular vesicles (EVs) has gained increasing attention. Over the past years, the conveyance of information by several “nanoscale” routes has been shown to be a key mechanism in numerous biological processes ( Baker, 2017 Maas et al., 2017). The assay is applicable to all classes of natural and artificial lipid microvesicles and nanovesicles.Ĭell communication is branched and complex. The CONAN assay proves to be robust and reliable and displays very interesting performances in terms of cost (inexpensive reagents, run by standard microplate readers), working volumes (1–2 μl of sample required), and time (full procedure takes <1 h). Instead, it automatically reports if the solution bears SAP contaminants, thus avoiding counting artifacts. For pure solutions, the assay also allows for determining the EV number, as the color shift is linearly dependent on the AuNP/EV molar ratio. The assay specifically targets SAPs, and not the EV-related proteins, with a detection limit <50 ng/μl (an order of magnitude higher resolution than that of the Bradford protein assay). The color change is visible by the naked eye or can be quantified by UV-Vis spectroscopy, providing an index of purity (a unique peculiarity to date). The solution turns blue if the EV preparation is pure, whereas it stays red if soluble exogenous single and aggregated proteins (SAPs often referred to as protein contaminants) are present. The CONAN assay consists of a solution of gold nanoparticles (AuNPs) into which the EV preparation is added.
#Purity fl studio 12 how to
This protocol paper describes how to assign a purity grade and to subsequently titrate extracellular vesicle (EV) solutions of a few microliters in volume by microplate COlorimetric NANoplasmonic (CONAN) assay.
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