Elucidating Extracellular Vesicle Isolation Kinetics via an Integrated Off-Stoichiometry Thiol-Ene and Cyclic Olefin...

Extracellular vesicles (EVs) are promising biomarkers for diagnosing complex diseases such as cancer and neurodegenerative disorders. Yet, their clinical application is hindered by challenges in isolating cancer-derived EVs efficiently due to their broad size distribution in biological samples. This study introduces a microfluidic device fabricated using off-stoichiometry thiol-ene and cyclic olefin copolymer, addressing the absorption limitations of polydimethylsiloxane (PDMS). The device streamlines a standard laboratory assay into a semi-automated microfluidic chip, integrating sample mixing and magnetic particle separation. Using the microfluidic device, the binding kinetics between EVs and anti-CD9 nanobodies were measured for the first time. Based on the binding kinetics, already after 10 min the EV capture was saturated and comparable to standard laboratory assays, offering a faster alternative to antibody-based immunomagnetic protocols. Furthermore, this study reveals the binding kinetics of EVs to anti-CD9 nanobodies for the first time. Our findings demonstrate the potential of the microfluidic device to enhance clinical diagnostics by offering speed and reducing manual labor without compromising accuracy.