Microfluidic Size Exclusion Chromatography (μSEC) for Extracellular Vesicles and Plasma Protein Separation

Abstract

Extracellular vesicles (EVs) are recognized as next generation diagnostic biomarkers due to their disease‐specific biomolecular cargoes and importance in cell–cell communications. A major bottleneck in EV sample preparation is the inefficient and laborious isolation of nanoscale EVs (≈50–200 nm) from endogenous proteins in biological samples. Herein, a unique microfluidic platform is reported for EV‐protein fractionation based on the principle of size exclusion chromatography (SEC). Using a novel rapid (≈20 min) replica molding technique, a fritless microfluidic SEC device (μSEC) is fabricated using thiol‐ene polymer (UV glue NOA81, Young's modulus ≈1 GPa) for high pressure (up to 6 bar) sample processing. Controlled on‐chip nanoliter sample plug injection (600 nL) using a modified T‐junction injector is first demonstrated with rapid flow switching response time (<1.5 s). Device performance is validated using fluorescent nanoparticles (50 nm), albumin, and breast cancer cells (MCF‐7)‐derived EVs. As a proof‐of‐concept for clinical applications, EVs are directly isolated from undiluted human platelet‐poor plasma using μSEC and show distinct elution profiles between EVs and proteins based on nanoparticle particle analysis (NTA), Western blot and flow cytometry analysis. Overall, the optically transparent μSEC can be readily automated and integrated with EV detection assays for EVs manufacturing and clinical diagnostics.

Conducting Polymer-Coated Carbon Cloth Captures and Releases Extracellular Vesicles by a Rapid and Controlled Redox Process

“…They are also considered as a source of bioactive cargos, such as nucleic acids, proteins, growth factors, and lipids, and are indicative of the cell from which they are derived. , As such, EVs shed from cancerous cells are increasingly being studied as promising biological targets for therapeutics and detection of diseases . Numerous isolation techniques such as ultracentrifugation, size exclusion chromatography, immunoaffinity, precipitation, magnet, and antibody-based isolation methods have been studied by researchers for EV isolation . It is crucial that isolated EVs from biofluids preserve their functional activity for their use, for example, in disease diagnostics .…”

Recent Advances in Microfluidic Platform for Physical and Immunological Detection and Capture of Circulating Tumor Cells

“…S.C. Terry reported the first lab-on-a-chip (LOC) analysis system in 1979, which was investigated for gas chromatography applications [37]. Since then, microfluidic devices have been investigated for a variety of applications, including biosensors [38], separation [39], analysis [40], drug delivery [41,42], optoelectronics [43], cell manipulation [44], and chemical synthesis [45,46]. There has been much advancement in surface chemistry, which has enabled the development of smart surfaces and devices for various applications [47,48].…”

Microfluidic harvesting of breast cancer tumor spheroid-derived extracellular vesicles from immobilized microgels for single-vesicle analysis

“…30,31 Although these techniques produce three-dimensional cellular structures, the structures are still anisotropic as aggregation is induced by gravitational or hydrodynamic forces. Similarly, microfluidic devices have been utilized to enrich EVs from complex biofluids via immunoaffinity, 32 size-exclusion, 33 and inertial forces. 34 While these techniques offer insights into disease-associated molecular signatures, complex biofluids contain EVs from various cellular origins that convolute cell-specific signals.…”