Exosome Purification and Analysis Using a Facile Microfluidic Hydrodynamic Trapping Device

Abstract

Exosomes are nanosized (30–150 nm) extracellular vesicles (EVs) secreted by various cell types. They are easily accessible in biological fluids and contain specific disease biomarkers, making them attractive for diagnosis and prognosis applications. Accurate biological characterization of exosomes is an important step toward clinical applications that require effective and precise isolation of subpopulations of exosomes. It is therefore of particular importance to develop an efficient and reliable exosome purification technique to isolate exosomes from the heterogeneous extracellular fluids. In this work, we intend to isolate and visualize exosomes by combining an affinity-based method and passive microfluidic particle trapping. Microbeads with a diameter of 20 μm are first functionalized with streptavidin and biotinylated antibodies and then used to immobilize and enrich exosomes on their surfaces using antigen–antibody affinity binding. We have developed a microfluidic device with trapping arrays to efficiently trap a large number of individual microbeads with enriched exosomes at the single-particle level, i.e., one single bead per trapping site, on the basis of a passive hydrodynamic trapping principle. The large-scale microfluidic single-bead trapping permits massively multiplexed fluorescence detection and quantification of the individual beads, which prevents the optical interfering of background noise as well as allowing one to acquire an average fluorescence density of a single bead for an accurate fluorescence-based exosome quantification. In addition, on-chip elusion and lysis of the protein and RNA content of captured exosomes enable further molecular analysis of exosomes, including Western blot and quantitative polymerase chain reaction. This microfluidic device provides a rapid and straightforward capturing and quantification method to analyze EVs for a variety of biological studies and applications.

Exosomes for mRNA delivery: a novel biotherapeutic strategy with hurdles and hope

“…These methods include ultracentrifugation, ultrafiltration, size exclusion chromatography (SEC), precipitation with polymers, and separation by affinity-based methods [47,78,79]. Recently, the devices based on microfluidic technology indicate promising advances for isolation and analysis of exosomes [80][81][82]. Therefore, look at the pros and cons of each method to find the qualified method of exosome purification is essential.…”

The role of exosomes in liquid biopsy for cancer diagnosis and prognosis prediction

“…Immunoaffinity‐based capture is a common approach in the microfluidics‐based technique. Microbeads that were conjugated with anti‐CD63 antibody were designed as trapping arrays in a microfluidic hydrodynamic device, which could significantly reduce the interference of background noise to enhance the purity of exosomes 21 . Fang et al used the microfluidic chip in a similar principle to capture tumor‐derived exosomes to assist the clinical diagnosis of breast cancer 22 .…”

Zinc Sulfide-Based Hybrid Exosome-Coated Nanoplatform for Targeted Treatment of Glioblastoma in an Orthotopic Mouse Glioblastoma Model

“…[17] Culture supernatants were collected 2 days later and processed for exosome purification by differential centrifugation. [18]…”