Multiplexed Affinity Measurements of Extracellular Vesicles Binding Kinetics.

Elisa Chiodi,Allison Marn,M Ünlü,George Daaboul

doi:10.3390/s21082634

Abstract

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.

Highlights

In the past few years, the interest in Extracellular Vesicles (EVs) as theranostic tools has significantly increased [1]
We developed a whole new application for kinetic measurements on Interferometric Reflectance Imaging Sensor by tailoring it to the multiplexed phenotyping of EVs
In order to satisfy the Minimal Information for Studies on Extracellular Vesicles (MISEV 2018 [19]) requirements, we characterized the studied EVs for size, protein content, and surface properties

Summary

Introduction

In the past few years, the interest in Extracellular Vesicles (EVs) as theranostic tools has significantly increased [1] These biological particles constitute a very heterogeneous population in the human body, in both origin and size. They range from vesicles of endosomic origin (small EVs, or exosomes, 50–150 nm) to microvesicles (50 nm–1 μm) released from the plasma membrane [2]. The heterogeneity of these biological nanoparticles can sometimes pose a challenge in terms of purification and phenotyping. By carrying pieces of information in the form of RNA fragments and biomarkers, EVs act as a “journal" of each individual’s health conditions and make a crucial contribution to the intracellular communication

Methods

Results

Discussion

Conclusion