Magnetic particle based liquid biopsy chip for isolation of extracellular vesicles and characterization by gene amplification

2021 ◽  
Vol 194 ◽  
pp. 113585
Author(s):  
Srinivas Bathini ◽  
Shanmugasundaram Pakkiriswami ◽  
Rodney J. Ouellette ◽  
Anirban Ghosh ◽  
Muthukumaran Packirisamy
Keyword(s):  
Gene Amplification ◽  
Extracellular Vesicles ◽  
Liquid Biopsy ◽  
Magnetic Particle

scholarly journals Single Step In Situ Detection of Surface Protein and MicroRNA in Clustered Extracellular Vesicles Using Flow Cytometry

2021 ◽  
Vol 10 (2) ◽  
pp. 319
Author(s):  
Hee Cheol Yang ◽  
Won Jong Rhee
Keyword(s):  
Extracellular Vesicles ◽  
Liquid Biopsy ◽  
Molecular Beacon ◽  
Disease Diagnosis ◽  
Single Step ◽  
Flow Cytometer ◽  
Surface Proteins ◽  
Biomarker Detection ◽  
In Situ Detection

Because cancers are heterogeneous, it is evident that multiplexed detection is required to achieve disease diagnosis with high accuracy and specificity. Extracellular vesicles (EVs) have been a subject of great interest as sources of novel biomarkers for cancer liquid biopsy. However, EVs are nano-sized particles that are difficult to handle; thus, it is necessary to develop a method that enables efficient and straightforward EV biomarker detection. In the present study, we developed a method for single step in situ detection of EV surface proteins and inner miRNAs simultaneously using a flow cytometer. CD63 antibody and molecular beacon-21 were investigated for multiplexed biomarker detection in normal and cancer EVs. A phospholipid-polymer-phospholipid conjugate was introduced to induce clustering of the EVs analyzed using nanoparticle tracking analysis, which enhanced the detection signals. As a result, the method could detect and distinguish cancer cell-derived EVs using a flow cytometer. Thus, single step in situ detection of multiple EV biomarkers using a flow cytometer can be applied as a simple, labor- and time-saving, non-invasive liquid biopsy for the diagnosis of various diseases, including cancer.

scholarly journals A multiparametric extraction method for Vn96-isolated plasma extracellular vesicles and cell-free DNA that enables multi-omic profiling

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Jeremy W. Roy ◽  
Catherine A. Taylor ◽  
Annie P. Beauregard ◽  
Surendar R. Dhadi ◽  
D. Craig Ayre ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Liquid Biopsy ◽  
Small Rna Sequencing ◽  
Isolation Method ◽  
Protein Biomarkers ◽  
Cell Free Dna ◽  
Extraction Protocol ◽  
Free Dna ◽  
Rich Material ◽  
Short Timeframe

AbstractExtracellular vesicles (EVs) have been recognized as a rich material for the analysis of DNA, RNA, and protein biomarkers. A remaining challenge for the deployment of EV-based diagnostic and prognostic assays in liquid biopsy testing is the development of an EV isolation method that is amenable to a clinical diagnostic lab setting and is compatible with multiple types of biomarker analyses. We have previously designed a synthetic peptide, known as Vn96 (ME kit), which efficiently isolates EVs from multiple biofluids in a short timeframe without the use of specialized lab equipment. Moreover, it has recently been shown that Vn96 also facilitates the co-isolation of cell-free DNA (cfDNA) along with EVs. Herein we describe an optimized method for Vn96 affinity-based EV and cfDNA isolation from plasma samples and have developed a multiparametric extraction protocol for the sequential isolation of DNA, RNA, and protein from the same plasma EV and cfDNA sample. We are able to isolate sufficient material by the multiparametric extraction protocol for use in downstream analyses, including ddPCR (DNA) and ‘omic profiling by both small RNA sequencing (RNA) and mass spectrometry (protein), from a minimum volume (4 mL) of plasma. This multiparametric extraction protocol should improve the ability to analyse multiple biomarker materials (DNA, RNA and protein) from the same limited starting material, which may improve the sensitivity and specificity of liquid biopsy tests that exploit EV-based and cfDNA biomarkers for disease detection and monitoring.

scholarly journals Peptide-Affinity Precipitation of Extracellular Vesicles and Cell-Free DNA Improves Sequencing Performance for the Detection of Pathogenic Mutations in Lung Cancer Patient Plasma

2020 ◽  
Vol 21 (23) ◽  
pp. 9083
Author(s):  
Catherine Taylor ◽  
Simi Chacko ◽  
Michelle Davey ◽  
Jacynthe Lacroix ◽  
Alexander MacPherson ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Extracellular Vesicles ◽  
Liquid Biopsy ◽  
Nsclc Patient ◽  
Egfr Mutations ◽  
Single Nucleotide Variants ◽  
Cell Free Dna ◽  
Free Dna ◽  
Nsclc Patients ◽  
Peptide Affinity

Liquid biopsy is a minimally-invasive diagnostic method that may improve access to molecular profiling for non-small cell lung cancer (NSCLC) patients. Although cell-free DNA (cf-DNA) isolation from plasma is the standard liquid biopsy method for detecting DNA mutations in cancer patients, the sensitivity can be highly variable. Vn96 is a peptide with an affinity for both extracellular vesicles (EVs) and circulating cf-DNA. In this study, we evaluated whether peptide-affinity (PA) precipitation of EVs and cf-DNA from NSCLC patient plasma improves the sensitivity of single nucleotide variants (SNVs) detection and compared observed SNVs with those reported in the matched tissue biopsy. NSCLC patient plasma was subjected to either PA precipitation or cell-free methods and total nucleic acid (TNA) was extracted; SNVs were then detected by next-generation sequencing (NGS). PA led to increased recovery of DNA as well as an improvement in NGS sequencing parameters when compared to cf-TNA. Reduced concordance with tissue was observed in PA-TNA (62%) compared to cf-TNA (81%), mainly due to identification of SNVs in PA-TNA that were not observed in tissue. EGFR mutations were detected in PA-TNA with 83% sensitivity and 100% specificity. In conclusion, PA-TNA may improve the detection limits of low-abundance alleles using NGS.

Separation of distinct exosome subpopulations: isolation and characterization approaches and their associated challenges

The Analyst ◽  
2021 ◽  
Author(s):  
Karishma Singh ◽  
Ruchika Nalabotala ◽  
Kevin M. Koo ◽  
Sudeep Bose ◽  
Ranu Nayak ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Liquid Biopsy ◽  
Therapeutic Use ◽  
Isolation And Characterization ◽  
Communications System

Exosomes are nano-sized extracellular vesicles that serve as a communications system between cells and has shown tremendous promise as liquid biopsy biomarkers in diagnostic, prognostic, and even therapeutic use in...

Protein biomarkers in breast cancer-derived extracellular vesicles for use in liquid biopsies

2021 ◽  
Author(s):  
Dan Li ◽  
Wenjia Lai ◽  
Di Fan ◽  
Qiaojun Fang
Keyword(s):  
Breast Cancer ◽  
Early Diagnosis ◽  
Extracellular Vesicles ◽  
Liquid Biopsy ◽  
Breast Cancer Diagnosis ◽  
Extracellular Vesicle ◽  
Detection Methods ◽  
Protein Markers ◽  
Liquid Biopsies ◽  
Diagnosis And Prognosis

Breast cancer is the most common malignant disease in women worldwide. Early diagnosis and treatment can greatly improve the management of breast cancer. Liquid biopsies are becoming convenient detection methods for diagnosing and monitoring breast cancer due to their non-invasiveness and ability to provide real-time feedback. A range of liquid biopsy markers, including circulating tumor proteins, circulating tumor cells, and circulating tumor nucleic acids, have been implemented for breast cancer diagnosis and prognosis, with each having its own advantages and limitations. Circulating extracellular vesicles are messengers of intercellular communication that are packed with information from mother cells and are found in a wide variety of bodily fluids; thus, they are emerging as ideal candidates for liquid biopsy biomarkers. In this review, we summarize extracellular vesicle protein markers that can be potentially used for the early diagnosis and prognosis of breast cancer or determining its specific subtypes.

scholarly journals Enhanced recovery of CD9-positive extracellular vesicles from human specimens by chelating reagent

2020 ◽  
Author(s):  
Ayako Kurimoto ◽  
Yuki Kawasaki ◽  
Toshiki Ueda ◽  
Tatsutoshi Inuzuka
Keyword(s):  
Extracellular Vesicles ◽  
Western Blotting ◽  
Biological Samples ◽  
Liquid Biopsy ◽  
Enhanced Recovery ◽  
Recovery Efficiency ◽  
Early Diagnostics ◽  
Isolation Methods ◽  
Chelating Reagent

AbstractExtracellular vesicles (EVs) have gained attention as potential targets of early diagnostics and prognosis in the field of liquid biopsy. Despite clinical potentials, the best method to isolate EVs from specimens remains controversial due to low purity, low specificity, and lack of reproducibility with current isolation methods. Here we show that a chelating reagent enhances the recovery efficiency of EVs from crude biological samples by immunoprecipitation using an anti-CD9 antibody. Proteomic and western blotting analyses show that the EVs isolated using the chelating reagent contain a wider variety of proteins than those isolated with PBS.

scholarly journals Analysis of cancer-related mutations in extracellular vesicles RNA by Droplet Digital™ PCR

BioTechniques ◽  
2020 ◽  
Vol 69 (2) ◽  
pp. 99-107
Author(s):  
Soo Ann Yap ◽  
Agnieszka Münster-Wandowski ◽  
Anika Nonnenmacher ◽  
Ulrich Keilholz ◽  
Sandra Liebs
Keyword(s):  
Colorectal Cancer ◽  
Tumor Progression ◽  
Extracellular Vesicles ◽  
Liquid Biopsy ◽  
Mek Inhibitor ◽  
Diagnostic Information ◽  
Braf V600e ◽  
Plasma Samples ◽  
Differential Centrifugation ◽  
Potential Biomarkers

Extracellular vesicles (EVs) are taking their place as potential biomarkers in the field of liquid biopsy. In this study, EVs were isolated from plasma samples of 31 patients with colorectal cancer and melanoma via differential centrifugation and Droplet Digital™ PCR (Bio-Rad, CA, USA) was used to profile BRAF V600E/K, KRAS G12A/C/D/V and KRAS G13D mutations from EV-derived cDNA. The concordance rates with corresponding tissue were 54% and 44% in the colorectal cancer and melanoma cohort, respectively. Two patients displayed mutations in EVs not previously detected in tissue as evidence for emerging molecular resistance to anti-EGFR and BRAF/MEK inhibitor therapy prior to radiological evidence of tumor progression. We concluded that EV-derived nucleic acids may provide clinically relevant diagnostic information and mirror evolution of the disease.

scholarly journals Bioactive DNA from extracellular vesicles and particles

2020 ◽  
Vol 11 (7) ◽  
Author(s):  
Ethan Z. Malkin ◽  
Scott V. Bratman
Keyword(s):  
Autoimmune Diseases ◽  
Extracellular Vesicles ◽  
Intercellular Communication ◽  
Liquid Biopsy ◽  
Molecular Biomarker ◽  
Comprehensive Overview ◽  
Cell Free Dna ◽  
Disease Biomarker ◽  
Functional Potential ◽  
Free Dna

Abstract Extracellular vesicles (EVs) and particles (EPs) have recently emerged as active carriers of molecular biomarkers and mediators of intercellular communication. While most investigations have focused exclusively on the protein, lipid and RNA constituents of these extracellular entities, EV/EP DNA remains poorly understood, despite DNA being found in association with virtually all EV/EP populations. The functional potential of EV/EP DNA has been proposed in a number of pathological states, including malignancies and autoimmune diseases. Moreover, the effectiveness of cell-free DNA as the biomarker of choice in emerging liquid biopsy applications highlights the role that EV/EP DNA may play as a novel disease biomarker. In this review, we provide a comprehensive overview of EV/EP DNA studies conducted to date, with a particular focus on the roles of EV/EP DNA as a functional mediator and molecular biomarker in various pathologic states. We also review what is currently known about the origins, structure, localisation and distribution of EV/EP DNA, highlighting current controversies as well as opportunities for future investigation.

scholarly journals Plasma extracellular vesicles detected by Single Molecule array technology as a liquid biopsy for colorectal cancer

2020 ◽  
Vol 9 (1) ◽  
pp. 1809765 ◽  
Author(s):  
Ping Wei ◽  
Fei Wu ◽  
Bin Kang ◽  
Xiaohua Sun ◽  
Fabienne Heskia ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Extracellular Vesicles ◽  
Single Molecule ◽  
Liquid Biopsy ◽  
Array Technology

scholarly journals Ultrasensitive quantification of tumor mRNAs in extracellular vesicles with an integrated microfluidic digital analysis chip

Lab on a Chip ◽  
2018 ◽  
Vol 18 (24) ◽  
pp. 3790-3801 ◽  
Author(s):  
Peng Zhang ◽  
Jennifer Crow ◽  
Divya Lella ◽  
Xin Zhou ◽  
Glenson Samuel ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Single Molecule ◽  
Ewing Sarcoma ◽  
Liquid Biopsy ◽  
Single Molecule Detection ◽  
Digital Analysis ◽  
Fusion Transcripts ◽  
Non Invasive ◽  
Mrna Analysis ◽  
Analysis Platform

A microwell-patterned microfluidic digital mRNA analysis platform enables PCR-free, single-molecule detection of EWS-FLI1 fusion transcripts in EVs towards liquid biopsy-based non-invasive diagnosis of Ewing Sarcoma.

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