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Multiplexed Affinity Measurements of Extracellular Vesicles Binding Kinetics

Extracellular vesicles (EVs) have attracted significant attention as impactful diagnostic biomarkers, since their properties are closely related to specific clinical conditions. However, designing experiments that involve EVs phenotyping is usually highly challenging and time-consuming, due to labor...

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Autores principales: Chiodi, Elisa, Daaboul, George G., Marn, Allison M., Ünlü, M. Selim
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8069658/
https://www.ncbi.nlm.nih.gov/pubmed/33918613
http://dx.doi.org/10.3390/s21082634
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author Chiodi, Elisa
Daaboul, George G.
Marn, Allison M.
Ünlü, M. Selim
author_facet Chiodi, Elisa
Daaboul, George G.
Marn, Allison M.
Ünlü, M. Selim
author_sort Chiodi, Elisa
collection PubMed
description Extracellular vesicles (EVs) have attracted significant attention as impactful diagnostic biomarkers, since their properties are closely related to specific clinical conditions. However, designing experiments that involve EVs phenotyping is usually highly challenging and time-consuming, due to laborious optimization steps that require very long or even overnight incubation durations. In this work, we demonstrate label-free, real-time detection, and phenotyping of extracellular vesicles binding to a multiplexed surface. With the ability for label-free kinetic binding measurements using the Interferometric Reflectance Imaging Sensor (IRIS) in a microfluidic chamber, we successfully optimize the capture reaction by tuning various assay conditions (incubation time, flow conditions, surface probe density, and specificity). A single (less than 1 h) experiment allows for characterization of binding affinities of the EVs to multiplexed probes. We demonstrate kinetic characterization of 18 different probe conditions, namely three different antibodies, each spotted at six different concentrations, simultaneously. The affinity characterization is then analyzed through a model that considers the complexity of multivalent binding of large structures to a carpet of probes and therefore introduces a combination of fast and slow association and dissociation parameters. Additionally, our results confirm higher affinity of EVs to aCD81 with respect to aCD9 and aCD63. Single-vesicle imaging measurements corroborate our findings, as well as confirming the EVs nature of the captured particles through fluorescence staining of the EVs membrane and cargo.
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spelling pubmed-80696582021-04-26 Multiplexed Affinity Measurements of Extracellular Vesicles Binding Kinetics Chiodi, Elisa Daaboul, George G. Marn, Allison M. Ünlü, M. Selim Sensors (Basel) Article Extracellular vesicles (EVs) have attracted significant attention as impactful diagnostic biomarkers, since their properties are closely related to specific clinical conditions. However, designing experiments that involve EVs phenotyping is usually highly challenging and time-consuming, due to laborious optimization steps that require very long or even overnight incubation durations. In this work, we demonstrate label-free, real-time detection, and phenotyping of extracellular vesicles binding to a multiplexed surface. With the ability for label-free kinetic binding measurements using the Interferometric Reflectance Imaging Sensor (IRIS) in a microfluidic chamber, we successfully optimize the capture reaction by tuning various assay conditions (incubation time, flow conditions, surface probe density, and specificity). A single (less than 1 h) experiment allows for characterization of binding affinities of the EVs to multiplexed probes. We demonstrate kinetic characterization of 18 different probe conditions, namely three different antibodies, each spotted at six different concentrations, simultaneously. The affinity characterization is then analyzed through a model that considers the complexity of multivalent binding of large structures to a carpet of probes and therefore introduces a combination of fast and slow association and dissociation parameters. Additionally, our results confirm higher affinity of EVs to aCD81 with respect to aCD9 and aCD63. Single-vesicle imaging measurements corroborate our findings, as well as confirming the EVs nature of the captured particles through fluorescence staining of the EVs membrane and cargo. MDPI 2021-04-09 /pmc/articles/PMC8069658/ /pubmed/33918613 http://dx.doi.org/10.3390/s21082634 Text en © 2021 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Chiodi, Elisa
Daaboul, George G.
Marn, Allison M.
Ünlü, M. Selim
Multiplexed Affinity Measurements of Extracellular Vesicles Binding Kinetics
title Multiplexed Affinity Measurements of Extracellular Vesicles Binding Kinetics
title_full Multiplexed Affinity Measurements of Extracellular Vesicles Binding Kinetics
title_fullStr Multiplexed Affinity Measurements of Extracellular Vesicles Binding Kinetics
title_full_unstemmed Multiplexed Affinity Measurements of Extracellular Vesicles Binding Kinetics
title_short Multiplexed Affinity Measurements of Extracellular Vesicles Binding Kinetics
title_sort multiplexed affinity measurements of extracellular vesicles binding kinetics
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8069658/
https://www.ncbi.nlm.nih.gov/pubmed/33918613
http://dx.doi.org/10.3390/s21082634
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