scholarly journals Multiplexed Affinity Measurements of Extracellular Vesicles Binding Kinetics

Sensors ◽  
2021 ◽  
Vol 21 (8) ◽  
pp. 2634
Author(s):  
Elisa Chiodi ◽  
George G. Daaboul ◽  
Allison M. Marn ◽  
M. Selim Ünlü
Keyword(s):  
Extracellular Vesicles ◽  
Binding Affinities ◽  
Label Free ◽  
Surface Probe ◽  
Large Structures ◽  
Time Flow ◽  
Microfluidic Chamber ◽  
Clinical Conditions ◽  
Imaging Sensor

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.

scholarly journals Multiplexed Affinity Measurements of Extracellular Vesicles Binding Kinetics

2021 ◽  
Author(s):  
Elisa Chiodi ◽  
George G. Daaboul ◽  
Allison Marn ◽  
M. Selim Ünlü
Keyword(s):  
Extracellular Vesicles ◽  
Binding Affinities ◽  
Label Free ◽  
Surface Probe ◽  
Large Structures ◽  
Time Flow ◽  
Microfluidic Chamber ◽  
Clinical Conditions ◽  
Imaging Sensor

<p>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 of 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 hour) 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 antobodies, each spotted at six different concentrations, simultaneously. The affinity characterization is then analyzed through a model which 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. </p>

scholarly journals Multiplexed Affinity Measurements of Extracellular Vesicles Binding Kinetics

2021 ◽  
Author(s):  
Elisa Chiodi ◽  
George G. Daaboul ◽  
Allison Marn ◽  
M. Selim Ünlü
Keyword(s):  
Extracellular Vesicles ◽  
Binding Affinities ◽  
Label Free ◽  
Surface Probe ◽  
Large Structures ◽  
Time Flow ◽  
Microfluidic Chamber ◽  
Clinical Conditions ◽  
Imaging Sensor

<p>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 of 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 hour) 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 antobodies, each spotted at six different concentrations, simultaneously. The affinity characterization is then analyzed through a model which 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. </p>

scholarly journals Label-Free Prostate Cancer Detection by Characterization of Extracellular Vesicles Using Raman Spectroscopy

2018 ◽  
Vol 90 (19) ◽  
pp. 11290-11296 ◽  
Author(s):  
Wooje Lee ◽  
Afroditi Nanou ◽  
Linda Rikkert ◽  
Frank A. W. Coumans ◽  
Cees Otto ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Raman Spectroscopy ◽  
Extracellular Vesicles ◽  
Cancer Detection ◽  
Label Free ◽  
Prostate Cancer Detection

scholarly journals Comprehensive label-free characterization of extracellular vesicles and their surface proteins

2020 ◽  
Author(s):  
E. Priglinger ◽  
J. Strasser ◽  
B. Buchroithner ◽  
F. Weber ◽  
S. Wolbank ◽  
...  
Keyword(s):  
Quality Control ◽  
Extracellular Vesicles ◽  
Impedance Measurement ◽  
Surface Proteins ◽  
Label Free ◽  
Neuroprotective Effects ◽  
Characterization Methods ◽  
Minimal Sample ◽  
Flow Filtration

AbstractInterest in mesenchymal stem cell derived extracellular vesicles (MSC-EVs) as therapeutic agents has dramatically increased over the last decade. Preclinical studies show that MSC-EVs have anti-apoptotic and neuroprotective effects, boost wound healing, and improve the integration of allogeneic grafts through immunomodulation. Current approaches to the characterization and quality control of EV-based therapeutics include particle tracking techniques, Western blotting, and advanced cytometry, but standardized methods are lacking. In this study, we established and verified quartz crystal microbalance (QCM) as highly sensitive label-free immunosensing technique for characterizing clinically approved umbilical cord MSC-EVs enriched by tangential flow filtration and ultracentrifugation. Using QCM in conjunction with common characterization methods, we were able to specifically detect EVs via EV (CD9, CD63, CD81) and MSC (CD44, CD49e, CD73) markers and gauge their prevalence. Additionally, we characterized the topography and elasticity of these EVs by atomic force microscopy (AFM), enabling us to distinguish between EVs and non-vesicular particles (NVPs) in a therapeutic formulation. This measurement modality makes it possible to identify EV sub-fractions, discriminate between EVs and NVPs, and to characterize EV surface proteins, all with minimal sample preparation and using label-free measurement devices with low barriers of entry for labs looking to widen their spectrum of characterization techniques. Our combination of QCM with impedance measurement (QCM-I) and AFM measurements provides a robust multi-marker approach to the characterization of clinically approved EV formulations and opens the door to improved quality control.

scholarly journals Chemical characterization of extracellular vesicles of mesenchymal stromal cells: TOF-SIMS and BCARS approach

2021 ◽  
Vol 2086 (1) ◽  
pp. 012107
Author(s):  
A V Aybush ◽  
A A Gulin ◽  
A A Kuzoiatova ◽  
M V Gubina ◽  
F E Gostev ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Stromal Cells ◽  
Chemical Characterization ◽  
Time Frame ◽  
Signaling Proteins ◽  
Label Free ◽  
Reasonable Time Frame ◽  
Membrane Bound ◽  
Scale Data

Abstract Paracrine functions of mesenchymal stem (stromal) cells (MSCs) rely, at least partly, on membrane-bound extracellular vesicles (EVs) with rich composition of lipids, nucleic acids and signaling proteins. Elucidation the underlying chemistry could potentially lead to MSCs-free therapy. However, the secretome of MSCs (EVs’ composition) is non-static and depends on many other factors including surrounding cells and medium. Thus, the research techniques must be able to provide not only bulk but microscopy-scale data within a reasonable time frame. Two of these label-free techniques are subject of this work toward the question of chemical composition of the EVs.

scholarly journals Intraoperative visualization of the tumor microenvironment and quantification of extracellular vesicles by label-free nonlinear imaging

2018 ◽  
Vol 4 (12) ◽  
pp. eaau5603 ◽  
Author(s):  
Yi Sun ◽  
Sixian You ◽  
Haohua Tu ◽  
Darold R. Spillman ◽  
Eric J. Chaney ◽  
...  
Keyword(s):  
Tumor Microenvironment ◽  
Extracellular Vesicles ◽  
Cancer Progression ◽  
Spatial Information ◽  
Imaging System ◽  
Label Free ◽  
Breast Reduction Surgery ◽  
Diagnosis And Prognosis ◽  
Nonlinear Imaging

Characterization of the tumor microenvironment, including extracellular vesicles (EVs), is important for understanding cancer progression. EV studies have traditionally been performed on dissociated cells, lacking spatial information. Since the distribution of EVs in the tumor microenvironment is associated with cellular function, there is a strong need for visualizing EVs in freshly resected tissues. We intraoperatively imaged untreated human breast tissues using a custom nonlinear imaging system. Label-free optical contrasts of the tissue, correlated with histological findings, enabled point-of-procedure characterization of the tumor microenvironment. EV densities from 29 patients with breast cancer were found to increase with higher histologic grade and shorter tumor-to-margin distance and were significantly higher than those from 7 cancer-free patients undergoing breast reduction surgery. Acquisition and interpretation of these intraoperative images not only provide real-time visualization of the tumor microenvironment but also offer the potential to use EVs as a label-free biomarker for cancer diagnosis and prognosis.

Sign in / Sign up

Export Citation Format

Share Document