Size Separation of Exosomes and Microvesicles Using Flow Field-Flow Fractionation/Multiangle Light Scattering and Lipidomic Comparison

Kim, Young Beom; Lee, Gwang Bin; Moon, Myeong Hee

doi:10.1021/acs.analchem.2c00806

Cited by 35 publications

(23 citation statements)

References 53 publications

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“…3,14 Flow rates in AF4 are quite large, causing significant dilution of the injected samples and requiring a postseparation enrichment step to enhance the concentration of the eluted EVs for downstream analysis. 14,20,22 Moreover, it is very difficult for AF4 to resolve EVs from low-density lipoproteins (LDLs) because they share similar sizes, 24 and resolution would get worse with samples containing high protein concentrations that may overload the column. Thus, AF4 methods for EV separation typically work with the relatively clean samples treated by UG or UF.…”

Section: ■ Introductionmentioning

confidence: 99%

“…The instrument also provides gentle separation to maintain sample integrity to a large extent because there is no concern about collision with column packing . These features make AF4 highly suitable for EV separations. ,,− AF4 is viewed to provide good resolution over a much wider particle size range and superior flexibility compared to SEC and can provide higher purity and integrity of the isolated EVs than UG and UF methods. , …”

Section: Introductionmentioning

confidence: 99%

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Offline Coupling of Asymmetrical Flow Field-Flow Fractionation and Capillary Electrophoresis for Separation of Extracellular Vesicles

Gao

Hutchins

et al. 2022

Anal. Chem.

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Extracellular vesicles (EVs) play important roles in cell-to-cell communications and carry high potential as markers targeted in disease diagnosis, prognosis, and therapeutic development. The main obstacles to EV study are their high heterogeneity; low amounts present in samples; and physical similarity to the abundant, interfering matrix components. Multiple rounds of separation and purification are often needed prior to EV characterization and function assessment. Herein, we report the offline coupling of asymmetrical flow field-flow fractionation (AF4) and capillary electrophoresis (CE) for EV analysis. While AF4 provides gentle and fast EV separation by size, CE resolves EVs from contaminants with similar sizes but different surface charges. Employing Western Blotting, ELISA, and SEM, we confirmed that intact EVs were eluted within a stable time window under the optimal AF4 and CE conditions. We also proved that EVs could be resolved from free proteins and high-density lipoproteins by AF4 and be further separated from the low-density lipoproteins co-eluted in AF4. The effectiveness of the coupled AF4-CE system in EV analysis was demonstrated by monitoring the changes in EV secretion from cells and by direct injection of human serum and detection of serum EVs. We believe that coupling AF4 and CE can provide rapid EV quantification in biological samples with much reduced matrix interference and be valuable for the study of total EVs and EV subpopulations produced by cells or present in clinical samples.

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Section: ■ Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Offline Coupling of Asymmetrical Flow Field-Flow Fractionation and Capillary Electrophoresis for Separation of Extracellular Vesicles

Gao

Hutchins

et al. 2022

Anal. Chem.

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“…While a couple of studies specified to have used both small and medium EVs, most studies discussed here primarily focus on the characterization of small EVs, still some of these works do not provide details on the specific range of sizes of the vesicles studied or their means for the isolation of the EVs. Currently, available isolation technologies have limitations regarding achieving a complete separation of small and medium EVs, [ 106 ] rendering it feasible that several other studies reviewed could also be applied for the characterization of the smaller sizes of vesicles classified as medium EVs.…”

Section: Techniques Comparison and Future Direction Of Extracellular ...mentioning

confidence: 99%

“…[11] Copyright 2020, American Chemical Society. a complete separation of small and medium EVs, [106] rendering it feasible that several other studies reviewed could also be applied for the characterization of the smaller sizes of vesicles classified as medium EVs.…”

Section: Techniques Comparison and Future Direction Of Extracellular ...mentioning

confidence: 99%

Nanostructured‐Based Optical Readouts Interfaced with Machine Learning for Identification of Extracellular Vesicles

Mata

Jeanne

Jalali

et al. 2022

Adv Healthcare Materials

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Extracellular vesicles (EVs) are shed from cancer cells into body fluids, enclosing molecular information about the underlying disease with the potential for being the target cancer biomarker in emerging diagnosis approaches such as liquid biopsy. Still, the study of EVs presents major challenges due to their heterogeneity, complexity, and scarcity. Recently, liquid biopsy platforms have allowed the study of tumor‐derived materials, holding great promise for early‐stage diagnosis and monitoring of cancer when interfaced with novel adaptations of optical readouts and advanced machine learning analysis. Here, recent advances in labeled and label‐free optical techniques such as fluorescence, plasmonic, and chromogenic‐based systems interfaced with nanostructured sensors like nanoparticles, nanoholes, and nanowires, and diverse machine learning analyses are reviewed. The adaptability of the different optical methods discussed is compared and insights are provided into prospective avenues for the translation of the technological approaches for cancer diagnosis. It is discussed that the inherent augmented properties of nanostructures enhance the sensitivity of the detection of EVs. It is concluded by reviewing recent integrations of nanostructured‐based optical readouts with diverse machine learning models as novel analysis ventures that can potentially increase the capability of the methods to the point of translation into diagnostic applications.

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“…The diameter of EVs largely depends on their origin and typically ranges from 30 to 200 nm . The quantification of EVs in a solution is mainly based on methods such as electron microscopy (EM), , nanoparticle tracking analysis (NTA), , dynamic light scattering (DLS), and ELISA tests . However, these methods work best with impurity-free solutions, and the presence of other types of membrane microbubbles in the analyzed solution in sizes similar to EVs significantly hinders NTA and DLS measurements .…”

Section: Introductionmentioning

confidence: 99%

Parallel SPR and QCM-D Quantitative Analysis of CD9, CD63, and CD81 Tetraspanins: A Simple and Sensitive Way to Determine the Concentration of Extracellular Vesicles Isolated from Human Lung Cancer Cells

et al. 2023

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Tetraspanins, including CD9, CD63, and CD81, are transmembrane biomarkers that play a crucial role in regulating cancer cell proliferation, invasion, and metastasis, as well as plasma membrane dynamics and protein trafficking. In this study, we developed simple, fast, and sensitive immunosensors to determine the concentration of extracellular vesicles (EVs) isolated from human lung cancer cells using tetraspanins as biomarkers. We employed surface plasmon resonance (SPR) and quartz crystal microbalance with dissipation (QCM-D) as detectors. The monoclonal antibodies targeting CD9, CD63, and CD81 were oriented vertically in the receptor layer using either a protein A sensor chip (SPR) or a cysteamine layer that modified the gold crystal (QCM-D) without the use of amplifiers. The SPR studies demonstrated that the interaction of EVs with antibodies could be described by the two-state reaction model. Furthermore, the EVs' affinity to monoclonal antibodies against tetraspanins decreased in the following order: CD9, CD63, and CD81, as confirmed by the QCM-D studies. The results indicated that the developed immunosensors were characterized by high stability, a wide analytical range from 6.1 × 10 4 particles•mL −1 to 6.1 × 10 7 particles•mL −1 , and a low detection limit (0.6−1.8) × 10 4 particles•mL −1 . A very good agreement between the results obtained using the SPR and QCM-D detectors and nanoparticle tracking analysis demonstrated that the developed immunosensors could be successfully applied to clinical samples.

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Size Separation of Exosomes and Microvesicles Using Flow Field-Flow Fractionation/Multiangle Light Scattering and Lipidomic Comparison

Cited by 35 publications

References 53 publications

Offline Coupling of Asymmetrical Flow Field-Flow Fractionation and Capillary Electrophoresis for Separation of Extracellular Vesicles

Offline Coupling of Asymmetrical Flow Field-Flow Fractionation and Capillary Electrophoresis for Separation of Extracellular Vesicles

Nanostructured‐Based Optical Readouts Interfaced with Machine Learning for Identification of Extracellular Vesicles

Parallel SPR and QCM-D Quantitative Analysis of CD9, CD63, and CD81 Tetraspanins: A Simple and Sensitive Way to Determine the Concentration of Extracellular Vesicles Isolated from Human Lung Cancer Cells

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