Rapid Characterization and Quantification of Extracellular Vesicles by Fluorescence‐Based Microfluidic Diffusion Sizing

Abstract

Extracellular vesicles (EVs) are emerging as promising diagnostic and therapeutic tools for a variety of diseases. The characterization of EVs requires a series of orthogonal techniques that are overall time-and material-consuming. Here, a microfluidic device is presented that exploits the combination of diffusion sizing and multiwavelength fluorescence detection to simultaneously provide information on EV size, concentration, and composition. The latter is achieved with the nonspecific staining of lipids and proteins combined with the specific staining of EV markers such as EV-associated tetraspanins via antibodies. The device can be operated as a single-step immunoassay thanks to the integrated separation and quantification of free and EV-bound fluorophores. This microfluidic technique is capable of detecting and quantifying components associated to EV subtypes and impurities and thus to measure EV purity in a time scale of minutes, requiring less than 5 µL of sample and minimal sample handling before the analysis. Moreover, the analysis is performed directly in solution without immobilization steps. Therefore, this method can accelerate screening of EV samples and aid the evaluation of sample reproducibility, representing an important complementary tool to the current array of biophysical methods for EV characterization, particularly valuable for instance for bioprocess development.

High‐Yield Separation of Extracellular Vesicles Using Programmable Zwitterionic Coacervates

“…After demonstrating that the ZW+ coacervates can recruit and release intact liposomes in high yields and that the separation process is compatible with a wide range of product concentrations, we applied our polymeric coacervates on EVs produced from human HEK-293F cells (see characterization by microfluidic diffusion sizing, [26] NTA, and TEM in Figure S4, Supporting Information) and microalgae (see comprehensive characterization in refs. [ 18,27 ]).…”

Emerging micro-nanotechnologies for extracellular vesicles in immuno-oncology: from target specific isolations to immunomodulation

“…3E) Detection activity is measured utilizing nonspecific staining of lipids and proteins combined with the specific staining of EV markers such as EV-associated tetraspanins via antibodies. 5 2.2. EV characterization 2.2.1.…”

“…3 (D) Schematic diagram of digital detection of BAM-DNA-anchored EVs at the single-nanoparticle level, reliant on nucleic acid-based amplification 4. (E) Schematic of fluorescence-based microfluidic diffusion sizing for the multiparametric characterization and quantification of EVs and images of the microfluidic device used for EV analysis 5. (F) The hybrid macro-and nanomechanical oscillator-based exosome isolation system: EXO, photograph of the EXODUS station, on which an EXODUS device is installed.…”

Isolation of Extracellular Vesicles From Microalgae: A Renewable and Scalable Bioprocess

“…Here, we discuss and define a clear manufacturing practice for the implementation of nanoalgosome production, with optimized protocols for microalgal cultivation ( upstream processing ) and isolation of EVs by Tangential Flow Filtration (TFF), an isolation technique allowing to process large volumes of microalgae cultures, reaching concentrated EV samples ( downstream processing ). Our production pipeline is optimized thanks to quality controls, ensured by an extensive biophysical and biochemical characterization by different techniques, including dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), immunoblot analysis (IB analysis) of protein markers, atomic force microscopy (AFM), and BicinChoninic Acid assay (BCA assay) ( Romancino et al, 2018 ; Adamo et al, 2021 ; Paganini et al, 2021 ). Moreover, we demonstrate the possibility to recycle microalgal biomass after EVs harvesting to renew the cell culture and continue EVs production in a cyclic bioprocess ( renewable processing ).…”