TY - JOUR
T1 - Augmented COlorimetric NANoplasmonic (CONAN) Method for Grading Purity and Determine Concentration of EV Microliter Volume Solutions
AU - Zendrini, Andrea
AU - Paolini, Lucia
AU - Busatto, Sara
AU - Radeghieri, Annalisa
AU - Romano, Miriam
AU - Wauben, Marca H M
AU - van Herwijnen, Martijn J C
AU - Nejsum, Peter
AU - Borup, Anne
AU - Ridolfi, Andrea
AU - Montis, Costanza
AU - Bergese, Paolo
N1 - Copyright © 2020 Zendrini, Paolini, Busatto, Radeghieri, Romano, Wauben, van Herwijnen, Nejsum, Borup, Ridolfi, Montis and Bergese.
PY - 2020/2
Y1 - 2020/2
N2 - 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.
AB - 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.
KW - extracellular vesicles
KW - liposomes
KW - nanoparticles
KW - nanoplasmonics
KW - particle number
KW - purity
KW - synthetic vesicles
KW - titration
UR - http://www.scopus.com/inward/record.url?scp=85081054421&partnerID=8YFLogxK
U2 - 10.3389/fbioe.2019.00452
DO - 10.3389/fbioe.2019.00452
M3 - Journal article
C2 - 32117903
SN - 2296-4185
VL - 7
JO - Frontiers in Bioengineering and Biotechnology
JF - Frontiers in Bioengineering and Biotechnology
M1 - 452
ER -