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

Rima, Xilal Y.; Zhang, Jingjing; Nguyen, Luong T. H.; Rajasuriyar, Aaron; Yoon, Min Jin; Chiang, Chi‐Ling; Walters, Nicole; Kwak, Kwang Joo; Lee, L James; Reátegui, Eduardo

doi:10.1039/d1lc01053k

Cited by 11 publications

(10 citation statements)

References 104 publications

(120 reference statements)

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“…Sung et al [ 101 ] described an automated and highly sensitive integrated microfluidic platform featuring a sample treatment to microRNA biomarker quantification from 20 µL plasma in ovarian cancer. Rima et al [ 102 ] developed a novel microfluidic system to collect EVs generated from breast cancer tumor spheroids, thereby enabling analysis at the single-vesicle level. Niu et al [ 103 ] designed a fluid nanoporous microinterface (FluidporeFace) in a microfluidic chip to enhance the isolation efficiency of EVs derived from the tumor.…”

Section: Methods Of Ev Isolationmentioning

confidence: 99%

Proteomic Research of Extracellular Vesicles in Clinical Biofluid

Fan

Poetsch

2023

Proteomes

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Extracellular vesicles (EVs), the lipid bilayer membranous structures of particles, are produced and released from almost all cells, including eukaryotes and prokaryotes. The versatility of EVs has been investigated in various pathologies, including development, coagulation, inflammation, immune response modulation, and cell–cell communication. Proteomics technologies have revolutionized EV studies by enabling high-throughput analysis of their biomolecules to deliver comprehensive identification and quantification with rich structural information (PTMs, proteoforms). Extensive research has highlighted variations in EV cargo depending on vesicle size, origin, disease, and other features. This fact has sparked activities to use EVs for diagnosis and treatment to ultimately achieve clinical translation with recent endeavors summarized and critically reviewed in this publication. Notably, successful application and translation require a constant improvement of methods for sample preparation and analysis and their standardization, both of which are areas of active research. This review summarizes the characteristics, isolation, and identification approaches for EVs and the recent advances in EVs for clinical biofluid analysis to gain novel knowledge by employing proteomics. In addition, the current and predicted future challenges and technical barriers are also reviewed and discussed.

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Section: Methods Of Ev Isolationmentioning

confidence: 99%

Proteomic Research of Extracellular Vesicles in Clinical Biofluid

Fan

Poetsch

2023

Proteomes

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“…Relative and total fluorescent intensities of the sample were obtained from custom-built algorithms that were previously reported. (31, 37)…”

Section: Methodsmentioning

confidence: 99%

“…Scatter plots were generated with the mean intensity and size of the single spots detected by TIRFM. Relative and total fluorescent intensities of the sample were obtained from custom-built algorithms that were previously reported (31, 37).…”

Section: Methodsmentioning

confidence: 99%

“…Virions are biogenetically and morphologically similar to extracellular vesicles (EVs) (25), which are cell-derived lipid nanoparticles containing bioactive molecules (26). The advent of research in EVs has sought to characterize biomolecular signatures to unravel their vast heterogeneity (27), which has promoted the engineering of in situ single-EV technologies (28)(29)(30)(31)(32)(33)(34)(35)(36). Coupling in situ labeling with high-resolution microscopy, such as total internal reflection fluorescence microscopy (TIRFM), has further enabled the colocalization of proteins and nucleic acids in single EVs (37).…”

Section: Introductionmentioning

confidence: 99%

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Integrated antigenic and nucleic acid detection in single virions and virion-infected host-derived extracellular vesicles

Nguyen,

Rima,

Nguyen

et al. 2023

Preprint

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The coronavirus disease of 2019 (COVID-19) led to the rapid development of novel assays to improve sensitivities for detecting the virion responsible for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Despite that, there have been over 767 million reported cases and over 6.9 million deaths worldwide. Therefore, tunable, sensitive, and high-throughput assays are warranted to control future outbreaks. Herein, we developed a tunablein situassay to selectively sort virions and infected host-derived extracellular vesicles (IHD-EVs) and simultaneously detect their antigens and nucleic acid cargo at a single-particle resolution. The Biochip Antigen and RNA Assay (BARATM) integrates positive immunoselection and infection dynamics to sort particles, immunofluorescence to detect antigens, and fluorescencein situhybridization to detect nucleic acids. BARATMenhanced sensitivities by detecting biomolecular signatures at the single-particle level, enabling the detection of virions in asymptomatic patients, and genetic mutations in single SARS-CoV-2 virions. Furthermore, BARATMrevealed the continued long-term expression of virion-RNA in the IHD-EVs of post-acute sequelae SARS-CoV-2 infection (PASC) patients. BARATMwas validated on saliva (30 healthy donors and 33 symptomatic and 20 asymptomatic patients) and nasopharyngeal swabs (19 healthy donors and 40 patients), revealing a highly accurate diagnosis by simultaneously detecting the spike glycoprotein and nucleocapsid-encoding RNA on single SARS-CoV-2 virions with sensitivities of 100 % and 95 %, respectively, and specificities of 100 % for both biofluids. Altogether, the single-particle detection of antigens and virion-RNA provides a tunable framework for the diagnosis, monitoring, and mutation screening of current and future outbreaks.Significance StatementViral outbreaks are imminent and rapidly translatable assays are necessary to keep contagion at bay. The Biochip Antigen and RNA Assay (BARATM) enables the simultaneous detection of antigens and nucleic acid cargo in virions and extracellular vesicles (EVs) from infected host cells at a single-particle resolution. Detecting single particles enhances sensitivities and specificities, enables long-term disease, and latent state monitoring, and provides a unique perspective into genetic mutations and virion lineages. Our work provides evidence for the utility of single-particle technologies as multifaceted diagnostic assays, providing more comprehensive information than traditional diagnostics.ClassificationBiological Sciences (Applied Biological Sciences)

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“…Due to the heterogeneity of EVs, the detection of single EVs is also significant. [ 87 ] Nguyen et al designed a highly sensitive immunogold‐based biochip, as shown in Figure 6 . First, a thin gold coating was deposited on glass coverslips by titanium, followed by coating with PEG, after which AuSERPs were assembled by attaching functionalized glass coverslips to silicone gaskets with 64 chambers, and then the treated glass surfaces with a mixture of streptavidin‐conjugated AuNP followed by biotinylated antibody mixture to capture specific EV subpopulations was functionalized.…”

Section: Membrane Fusion‐based In Situ Detectionmentioning

confidence: 99%

Emerging Sensing and In Situ Detection Technologies for the Analysis of Extracellular Vesicle miRNAs

Han,

Wang,

Zhu

et al. 2023

Advanced NanoBiomed Research

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Liquid biopsy has received increasing attention as a new disease detection modality because of its noninvasive, simple sampling, and reproducible assay advantages. Among the markers of liquid biopsy, extracellular vesicles (EVs) are considered as promising disease biomarkers because they contain a large amount of biological information and have a significant role in physiological activities. The emergence and progression of some of these diseases are associated with miRNAs carried by EVs (EV‐miRNAs). Therefore, high‐sensitive detection of EV‐miRNAs is essential in clinical applications. A growing number of strategies, including biosensors, in situ detection methods, and microfluidics have been developed for the detection of EV‐miRNA and have been applied in the diagnosis of diseases such as cancer. This review summarizes the probes, signal amplification, and detection methods for EV‐miRNA detection, as well as the application of membrane fusion‐based in situ detection and integrated microfluidic chips for EV‐miRNA detection. The challenges of these materials and techniques in clinical diagnostic applications are also discussed.

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

Abstract: Investigating cellular and vesicular heterogeneity in breast cancer remains a challenge, which encourages the development of controllable in vitro systems that mimic the tumor microenvironment. Although three-dimensional cell culture better...

Cited by 11 publications

References 104 publications

Proteomic Research of Extracellular Vesicles in Clinical Biofluid

Proteomic Research of Extracellular Vesicles in Clinical Biofluid

Integrated antigenic and nucleic acid detection in single virions and virion-infected host-derived extracellular vesicles

Emerging Sensing and In Situ Detection Technologies for the Analysis of Extracellular Vesicle miRNAs

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