Augmented COlorimetric NANoplasmonic (CONAN) Method for Grading Purity and Determine Concentration of EV Microliter Volume Solutions

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  • Andrea Zendrini, Food and Nutrition-Università Cattolica del Sacro Cuore, Consorzio Interuniversitario Nazionale per la Scienza e la Tecnologia dei Materiali, Florence, Italy.
  • ,
  • Lucia Paolini, University of Brescia, University of Florence
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  • Sara Busatto, University of Florence, Mayo Clinic
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  • Annalisa Radeghieri, University of Brescia, University of Florence
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  • Miriam Romano, University of Brescia, University of Florence
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  • Marca H M Wauben, Utrecht University
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  • Martijn J C van Herwijnen, Utrecht University
  • ,
  • Peter Nejsum
  • Anne Borup
  • Andrea Ridolfi, University of Florence, Istituto per lo Studio dei Materiali Nanostrutturati (CNR-ISMN)
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  • Costanza Montis, University of Florence
  • ,
  • Paolo Bergese, Consorzio Sistemi a Grande Interfase, Department of Chemistry, University of Florence, Sesto Fiorentino, Italy., University of Brescia

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. The CONAN assay consists of a solution of gold nanoparticles (AuNPs) into which the EV preparation is added. 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 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 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). 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. Instead, it automatically reports if the solution bears SAP contaminants, thus avoiding counting artifacts. 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). The assay is applicable to all classes of natural and artificial lipid microvesicles and nanovesicles.

TidsskriftFrontiers in Bioengineering and Biotechnology
Antal sider10
StatusUdgivet - feb. 2020

Bibliografisk note

Copyright © 2020 Zendrini, Paolini, Busatto, Radeghieri, Romano, Wauben, van Herwijnen, Nejsum, Borup, Ridolfi, Montis and Bergese.

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