Multiplexed Affinity Measurements of Extracellular Vesicles Binding Kinetics

Area of Science:

• Biotechnology
• Nanotechnology
• Analytical Chemistry

Background:

• Extracellular vesicles (EVs) are crucial diagnostic biomarkers, but their phenotyping is hampered by time-consuming experimental protocols.
• Current methods often require lengthy incubation periods, delaying biomarker discovery and clinical application.

Purpose of the Study:

• To develop a rapid, label-free method for real-time extracellular vesicle (EV) detection and phenotyping.
• To optimize EV capture and binding affinity measurements on a multiplexed surface.
• To enable efficient characterization of EV-probe interactions within an hour.

Main Methods:

• Utilized Interferometric Reflectance Imaging Sensor (IRIS) in a microfluidic chamber for label-free kinetic binding measurements.
• Optimized assay conditions including incubation time, flow, surface probe density, and specificity.
• Performed kinetic characterization of 18 antibody-probe conditions simultaneously, analyzing multivalent binding kinetics.

Main Results:

• Achieved label-free, real-time detection and phenotyping of EVs in under one hour.
• Successfully optimized capture reactions and characterized binding affinities to multiplexed probes.
• Demonstrated higher affinity of EVs to aCD81 compared to aCD9 and aCD63, confirmed by single-vesicle imaging.

Conclusions:

• The developed IRIS-based method offers a significantly faster and more efficient approach for EV phenotyping and biomarker discovery.
• This technique streamlines assay development and provides valuable kinetic binding data for understanding EV interactions.
• The findings highlight the potential of rapid EV analysis for advancing diagnostic applications.