Quantification Of Exosomes Research Articles

Advances in exosome analysis.

There is growing demand for novel biomarkers that detect early stage disease as well as monitor clinical management and therapeutic strategies. Exosome analysis could provide the next advance in attaining that goal. Exosomes are membrane encapsulated biologic nanometric-sized particles of endocytic origin which are released by all cell types. Unfortunately, exosomes are exceptionally challenging to characterize with current technologies. Exosomes are between 30 and 200nm in diameter, a size that makes them out of the sensitivity range to most cell-oriented sorting or analysis platforms, i.e., traditional flow cytometers. The most common methods for targeting exosomes to date typically involve purification followed by the characterization and the specific determination of their cargo. The whole procedure is time consuming, requiring thus skilled personnel as well as laboratory facilities and benchtop instrumentation. The most relevant methodology for exosome isolation, characterization and quantification is addressed in this chapter, including the most up-to-date approaches to explore the potential usefulness of exosomes as biomarkers in liquid biopsies and in advanced nanomedicine.

DNA-functionalized covalent organic framework capsules for analysis of exosomes

Exosomes have been deemed as hopeful liquid biopsy biomarkers for malignant tumor identification. However, proposing detection methods with high sensitivity still meets challenges. Herein, we have fabricated an electrochemical biosensor in this work via constructing nucleic acid functionalized covalent organic framework (COF) capsules for sensitive detection of exosomes. Firstly, nucleic acid functionalized COF capsules are prepared by modifying azide-labeled DNA onto alkynyl-containing COF capsules. Then, the capsules are used to fabricate an electrochemical biosensor for exosomes quantification. In this design of the sensor, the modification of DNA strands on COFs can greatly improve the binding efficiency, while the loading of plentiful horseradish peroxidase (HRP) facilitates signal amplification. So, the fabricated biosensor has effectively enhanced the sensitivity and lowered the detection limit to 87 particles/μL. Further studies show that the biosensor can be used for the identification of exosomes from non-small cell lung cancer. Moreover, by simply replacing the DNA sequences, the proposed strategy can generally be used to fabricate different DNA-COF capsules with diverse functions for broader biomedical applications.

An electrochemical biosensor for exosome detection based on covalent organic frameworks conjugated with DNA and horseradish peroxidase

Quantification of trace exosomes in biofluids is helpful for the early diagnosis of cancers. Electrochemical biosensors have attracted special attentions for exosome detection, while the employment of nanoconjugates in the construction of electrochemical biosensors can achieve better performance. So, in this work, we have designed and fabricated HRP-DNA-COF nanoconjugates (HDCs) that involve covalent organic frameworks (COFs) as the scaffold, cholesterol-tagged DNA to recognize exosomes and horseradish peroxidase (HRP) to amplify the signal readout. The successful preparation of HDCs have been verified experimentally, based on which an electrochemical biosensor has been constructed for exosome detection. Attributed to the specific recognition ability and excellent catalytic ability of HDCs, the biosensor achieves good performance to detect exosomes with simplicity and sensitivity in the linear range from 104 to 107 particles/μL. Besides, the strategy proposed in this work to construct COF-based nanoconjugates may promote the wider application of COFs in biomedical research.

Introduction of Nanomaterials to Biosensors for Exosome Detection: Case Study for Cancer Analysis.

Exosomes have been gaining attention for early cancer diagnosis owing to their biological functions in cells. Several studies have reported the relevance of exosomes in various diseases, including pancreatic cancer, retroperitoneal fibrosis, obesity, neurodegenerative diseases, and atherosclerosis. Particularly, exosomes are regarded as biomarkers for cancer diagnosis and can be detected in biofluids, such as saliva, urine, peritoneal fluid, and blood. Thus, exosomes are advantageous for cancer liquid biopsies as they overcome the current limitations of cancer tissue biopsies. Several studies have reported methods for exosome isolation, and analysis for cancer diagnosis. However, further clinical trials are still required to determine accurate exosome concentration quantification methods. Recently, various biosensors have been developed to detect exosomal biomarkers, including tumor-derived exosomes, nucleic acids, and proteins. Among these, the exact quantification of tumor-derived exosomes is a serious obstacle to the clinical use of liquid biopsies. Precise detection of exosome concentration is difficult because it requires clinical sample pretreatment. To solve this problem, the use of the nanobiohybrid material-based biosensor provides improved sensitivity and selectivity. The present review will discuss recent progress in exosome biosensors consisting of nanomaterials and biomaterial hybrids for electrochemical, electrical, and optical-based biosensors.

Nanomaterials assisted exosomes isolation and analysis towards liquid biopsy.

Exosomes has attracted tremendous research interests as they are emerging as a new paradigm of liquid biopsy. Although the concentration of exosomes in blood is relatively abundant, there still exists various vesicle-like nanoparticles, such as microvesicles, apoptotic bodies. It's an urgent need to isolate and enrich exosomes from the complex contaminants in biofluid samples. Moreover, the expressing level of exosomal biomarkers varies a lot, which make the sensitive molecular detection of exosomes in high demand. Unfortunately, the efficient isolation and sensitive molecular quantification of exosomes is still a major obstacle hindering the further development and clinical application of exosome-based liquid biopsy. Nanomaterials, with unique physiochemical properties, have been widely used in biosensing and analysis aspects, thus they are thought as powerful tools for effective purification and molecular analysis of exosomes. In this review, we summarized the most recent progresses in nanomaterials assisted exosome isolation and analysis towards liquid biopsy. On the one hand, nanomaterials can be used as capture substrates to afford large binding area and specific affinity to exosomes. Meanwhile, nanomaterials can also be served as promising signal transducers and amplifiers for molecular detection of exosomes. Furthermore, we also pointed out several potential and promising research directions in nanomaterials assisted exosome analysis. It's envisioned that this review will give the audience a complete outline of nanomaterials in exosome study, and further promote the intersection of nanotechnology and bio-analysis.

Ultrasensitive Detection of Exosomes Using an Optical Microfiber Decorated with Plasmonic MoSe2-Supported Gold Nanorod Nanointerfaces

Exosomes are potential and promising natural noninvasive biomarkers for liquid biopsies and can be involved in various biological and pathological processes in early-stage cancer. Thus, there is an urgent demand to develop low-cost, small-size, remarkable-specificity, and ultrasensitive exosome biosensors for early clinical point-of-care (POC) testing. Although various conventional tumor exosome detection methods have been generally proposed, the low detection sensitivity and specificity significantly hinder their use in cancer clinical diagnosis and prognosis. To address the above challenges, an optical microfiber integrated with MoSe2-supported gold nanorods is proposed. To tune the strong localized surface plasmon resonance (LSPR) of the nanointerfaces on the optical microfiber to be in accordance with the operating wavelength of the silica optical microfiber in the telecommunication band, gold nanorods with a high aspect ratio of approximately 10:1 are proposed. Due to the interaction between the excited LSPR effect and the evanescent field of the optical microfiber, the sensor can detect clear cell renal cancer exosomes within a wide concentration range from 100 particles/mL to 108 particles/mL, with an extremely low limit of detection (LOD) of 9.32 particles/mL, which is lower than that of current various state of the art methods. More importantly, the microfiber with high specificity can successfully differentiate pathological plasma and healthy controls, exhibiting very promising clinical applications in renal cancer diagnosis and prognosis. This work opens up a new approach for the in situ detection and quantification of exosomes with ultrahigh sensitivity in early clinical screening and diagnosis.

Fast and specific enrichment and quantification of cancer-related exosomes by DNA-nanoweight-assisted centrifugation.

Exosomes are nanoscale membrane vesicles actively released by cells and play an important role in the diagnosis of cancer-related diseases. However, it is challenging to efficiently enrich exosomes from extracellular fluids. In this work, we used DNA nanostructures as "nanoweights" during centrifugation to facilitate the enrichment of cancerous exosomes in human serum. Two different DNA tetrahedral nanostructures (DTNs), each carrying a specific aptamer for exosome biomarker recognition, were incubated with clinical samples simultaneously. One DTN triggered the cross-linking of multiple target exosomes and, therefore, enabled low-speed and fast centrifugation for enrichment. The other DTN further narrowed down the target exosome subtype and initiated a hybridization chain reaction (HCR) for sensitive signal amplification. The method enabled the detection of 1.8 × 102 MCF-7-derived exosomes per microliter and 5.6 × 102 HepG2-derived exosomes per microliter, with 1000-fold higher sensitivity than conventional ELISA and 10-fold higher sensitivity than some recently reported fluorescence assays. Besides, the dual-aptamer system simultaneously recognized multiple surface proteins, eliminating the interference risk from free proteins. Thus, this easy-to-operate method can enrich exosomes with excellent specificity and sensitivity and therefore will be appealing in biomedical research and clinical diagnosis.

Enhanced Extracellular Transfer of HLA-DQ Activates CD3+ Lymphocytes towards Compromised Treg Induction in Celiac Disease.

Celiac disease (CeD) manifests with autoimmune intestinal inflammation from gluten and genetic predisposition linked to human leukocyte antigen class-II (HLA-II) gene variants. Antigen-presenting cells facilitate gluten exposition through the interaction of their surface major histocompatibility complex (MHC) with the T cell receptor (TCR) on T lymphocytes. This fundamental mechanism of adaptive immunity has broadened upon recognition of extracellular exosomal MHC, raising awareness of an alternative means for antigen presentation. This study demonstrates that conditioned growth media (CGM) previously exposed to monocyte-derived dendritic cells from CeD significantly downregulates the CD3+ lineage marker of control T cells. Such increased activation was reflected in their elevated IL-2 secretion. Exosome localization motif identification and quantification within HLA-DQA1 and HLA-DQB1 transcripts highlighted their significant prevalence within HLA-DQB1 alleles associated with CeD susceptibility. Flow cytometry revealed the strong correlation between HLA-DQ and the CD63 exosomal marker in T cells exposed to CGM from MoDCs sourced from CeD patients. This resulted in lower concentrations of CD25+ CD127− T cells, suggestive of their compromised induction to T-regulatory cells associated with CeD homeostasis. This foremost comparative study deciphered the genomic basis and extracellular exosomal effects of HLA transfer on T lymphocytes in the context of CeD, offering greater insight into this auto-immune disease.

Dual Rolling Circle Amplification-Assisted Single-Particle Fluorescence Profiling of Exosome Heterogeneity for Discriminating Lung Adenocarcinoma from Pulmonary Nodules

Dual Rolling Circle Amplification-Assisted Single-Particle Fluorescence Profiling of Exosome Heterogeneity for Discriminating Lung Adenocarcinoma from Pulmonary Nodules

Elevated expression of RAB3B plays important roles in chemoresistance and metastatic potential of hepatoma cells

BackgroundCancer stem cells (CSCs) are thought to play important roles in carcinogenesis, recurrence, metastasis, and therapy-resistance. We have successfully induced cancer stem-like sphere cells (CSLCs) which possess enhanced chemoresistance and metastatic potential. To enable the development of targeted therapy against CSLCs, we identified a gene responsible for this phenotype in CSLC.MethodsHuman hepatoma cell line SK-HEP-1 was used for CSLC induction with a unique sphere inducing medium, and HuH-7 cells were used as non-sphere forming cells in the same condition. RNA-sequencing was performed followed by validation with quantitative RT-PCR and western blotting. Knockdown experiments were done by using CRISPR-Cas9 genome-editing, and the rescue experiments were performed using the expressing plasmid vector. Chemoresistance and liver metastasis of the cells, was studied following the splenic injection of cells to severely immune deficient mice and evaluated using the MTS assay. Quantification of exosomes in the medium was done using ELISA.ResultsRAB3B was identified as an up-regulated gene in both CSLCs and prognostically poor hepatocellular carcinoma (HCC) by RNA-sequencing. RAB3B-KD cells showed altered CSLC phenotypes such as sphere formation, chemoresistance, and metastatic potentials, and those were rescued by RAB3B complementation. Increased exosome secretion was observed in CSLCs, and it was not observed in the RAB3B-KD cells. In addition, the RAB3B expression correlated with the expression of ABCG2, APOE, LEPR, LXN, and TSPAN13.ConclusionThe up regulation of RAB3B may play an important role in the chemoresistance and metastatic potential of CSLCs.

Smartphone-Based Electrochemical Biosensors for Directly Detecting Serum-Derived Exosomes and Monitoring Their Secretion.

Exosomes are potential biomarkers, which play an important role in early diagnosis and prognosis prediction of cancer-related diseases. Nevertheless, direct quantification of exosomes in biological fluid, especially in point-of-care tests (POCTs), remains extremely challenging. Herein, we developed a sensitive and portable electrochemical biosensor in combination with smartphones for quantitative analysis of exosomes. The improved double-antibody sandwich method-based poly-enzyme signal amplification was adopted to detect exosomes. We could detect as low as 7.23 ng of CD63-positive exosomes in 5 μL of serum within 2 h. Importantly, we demonstrated that the biosensor worked well with microliter-level serum and cell culture supernatant. The biosensor holds great potential for the detection of CD-63-expressing exosomes in early diagnosis of prostate disease because CD63-positive exosomes were less detected from the prostate patient serum. Also, the biosensor was used to monitor the secretion of exosomes with the drug therapy, showing a close relationship between the secretion of exosomes and the concentration of cisplatin. The biosensing platform provides a novel way toward POCT for the diagnosis and prognosis prediction of prostate disease and other diseases via biomarker expression levels of exosomes.

Quantification and Imaging of Exosomes via Luciferase-Fused Exosome Marker Proteins: ExoLuc System.

Bioluminescence (BL) has been widely used to quantitatively monitor various biological phenomena. Here, we describe a protocol for preparing and using cells expressing exosomes labeled with luciferase. The BL of the culture medium of these cells is proportional to the number of secreted exosome particles obtained by well-established nanoparticle tracking analysis, allowing easy, rapid, and sensitive quantification of exosomes in vitro and in vivo. This method, designated the ExoLuc system, is a powerful tool for analyzing the molecular mechanisms of exosome biosynthesis, secretion, uptake, and biodistribution.

Isolation and Characterization of Salivary Exosomes for Cancer Biomarker Discovery.

Exosomes are small extracellular vesicles secreted by cells and are known to play a key role in intercellular communication. Several studies have associated exosomes with various roles in tumorigenesis and explored their potential as a source of biomarkers for diagnosis and prognosis in cancer research. Exosomes can be isolated from several body fluids, including those that are noninvasively accessible, such as human saliva. This book chapter provides a step-by-step detailed description of techniques that are used for the isolation, quantification, and characterization of exosomes from saliva. These include ultracentrifugation for the isolation, nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and western blot (WB) for characterization of exosomes. The NTA approach explores the Brownian motion and light scattering of particles to predict size and concentration. TEM enables visualization of the exosomes which often present a cup-shaped morphology. Western blot is used to detect commonly expressed exosome-associated proteins. Finally, salivary exosomes isolated using these protocols can further be characterized for downstream analysis according to their cargo (proteins, DNA, RNA, miRNA) and utilized for cancer biomarker discovery.

Ti3C2 MXene-anchored photoelectrochemical detection of exosomes by in situ fabrication of CdS nanoparticles with enzyme-assisted hybridization chain reaction.

Exosomes that carry large amounts of tumor-specific molecular information have been identified as a potential non-invasive biomarker for early warning of cancer. In this work, we reported an enzyme-assisted photoelectrochemical (PEC) biosensor for quantification of exosomes based on the in situ synthesis of Ti3C2 MXene/CdS composites with magnetic separation technology and hybridization chain reaction (HCR). First, exosomes were specifically bound between aptamer-labeled magnetic beads (CD63-MBs) and a cholesterol-labeled DNA anchor. The properly designed anchor ends acted as a trigger to enrich the alkaline phosphatase (ALP) through HCR. It catalyzed more sodium thiophosphate to generate the sulfideion (S2−), which combined with Cd2+ for in situ fabrication of CdS on Ti3C2 MXene resulting in elevated photocurrent. The Ti3C2 MXene-anchored PEC method was realized for the quantitative detection of exosomes, which exhibited the dynamic working range from 7.3 × 105 particles per mL to 3.285 × 108 particles per mL with a limit of detection of 7.875 × 104 particles per mL. The strategy showed acceptable stability, high sensitivity, rapid response and excellent selectivity. Furthermore, we believe that the PEC biosensor has huge potential as a routine bioassay method for the precise quantification of exosomes from breast cancer in the future.

Label-free imaging and biomarker analysis of exosomes with plasmonic scattering microscopy.

Exosome analysis is a promising tool for clinical and biological research applications. However, detection and biomarker quantification of exosomes is technically challenging because they are small and highly heterogeneous. Here, we report an optical approach for imaging exosomes and quantifying their protein markers without labels using plasmonic scattering microscopy (PSM). PSM can provide improved spatial resolution and distortion-free image compared to conventional surface plasmon resonance (SPR) microscopy, with the signal-to-noise ratio similar to objective coupled surface plasmon resonance (SPR) microscopy, and millimeter-scale field of view as a prism-coupled SPR system, thus allowing exosome size distribution analysis with high throughput. In addition, PSM retains the high specificity and surface sensitivity of the SPR sensors and thus allows selection of exosomes from extracellular vesicles with antibody-modified sensor surfaces and in situ analyzing binding kinetics between antibody and the surface protein biomarkers on the captured exosomes. Finally, the PSM can be easily constructed on a popular prism-coupled SPR system with commercially available components. Thus, it may provide an economical and powerful tool for clinical exosome analysis and exploration of fundamental issues such as exosome biomarker binding properties.

Correction: Rapid and sensitive leukemia-derived exosome quantification via nicking endonuclease-assisted target recycling

Correction for 'Rapid and sensitive leukemia-derived exosome quantification via nicking endonuclease-assisted target recycling' by Mengyang Zhou et al., Anal. Methods, 2021, 13, 4001-4007, https://doi.org/10.1039/D1AY00854D.

Determination of chemoresistance in ovarian cancer by simultaneous quantification of exosomes and exosomal cisplatin with surface enhanced Raman scattering

Ovarian cancer is one of the most prevalent and lethal cancers in the world. This disease is frequently associated with a resistance to conventional chemotherapy, often acquired during therapy. We report a new method of diagnosing chemoresistance in ovarian cancers by simultaneous quantification of tumor derived exosomes and the chemotherapy drug cisplatin excreted within them. This is accomplished by a surface enhanced Raman scattering modality using cysteine capped gold nanoparticles. Interaction between the nanoparticles and cisplatin causes the nanoparticles to become destabilized, and the rate of this aggregation is proportional to the concentration of the drug. The exosome spectra were subsequently used to develop regression and discriminant models using support vector machines (SVM), which were used to differentiate between histological subtypes of ovarian cancer and their chemoresponsiveness. This method is able to measure exosome-derived cisplatin down to a concentration of 0.17 µg/mL, and exosomes down to 65 nM. Combining these metrics with support vectors machine discriminant models is able to diagnose chemoresistance with greater than 90% accuracy.

Translating cancer exosomes detection into the color change of phenol red based on target-responsive DNA microcapsules

Emerging evidence indicates that exosomes can be used as a potential biomarker for monitoring diseases, including cancer. However, enhancing the sensing performance in terms of convenience and sensitivity remains an urgent demand for exosomes detection. In this study, a pH-sensitive colorimetric biosensing strategy was developed for exosomes detection by integrating stimuli-responsive DNA microcapsules and acetylcholinesterase to produce acetic acid. The constructed DNA microcapsules consisted of DNA shells crosslinked by anti-CD63 aptamers and loaded with acetylcholinesterase. With exosomes addition, an energetically stabilized aptamer-CD63 compound was produced and microcapsules dissociated due to the reaction of surface protein CD63 of exosomes and aptamer of CD63, resulting in the release of encapsulated AChE. Through a simple centrifugation separation, unreacted DNA microcapsules were removed and the supernatant containing released acetylcholinesterase collected, which was then used for colorimetric exosomes detection through the ability of acetylcholinesterase to hydrolyze acetylcholine to release acetic acid. The resulting decreased solution pH was detected with phenol red indicator, with the sharp color transition conveniently by naked eye. Exosomes quantification was also achieved using the solution's absorption intensity ratio of 558 vs. 432 nm. The linear range was from 2.0 × 103 to 5.0 × 105 particles/μL, and the limit of detection and limit of quantification were 1.2 × 103 particles/μL and 2.2 × 103 particles/μL, respectively. In addition, this proposed strategy for exosomes detection showed a relative standard deviation of 3.1% and high recovery efficiency (>94%), exhibiting a bright application future in exsomes analysis.

Tetrahedral DNA mediated direct quantification of exosomes by contact-electrification effect

Exosomes are membrane-enclosed extracellular vesicles carrying multiple biomolecules for intercellular communications. Accurate detection of exosomes could provide critical clinical information and show great significance for early diagnosis and personalized therapy of cancer. In this work, we propose a triboelectric sensing strategy for direct quantification of exosomes based on the contact-electrification effect. The target exosomes can be selectively captured on the three-dimensional tetrahedral DNA (TDNA) monolayer. The electrons transfer between abundant amino groups from exosomes and the tribo-materials contribute to the measured signal. Due to the specific output characteristic, it is able to directly discriminate 3 exosomes /μL with a linear range from 20 to 1000 exosomes/μL, even without any signal amplification. The challenges for distinguishing different cell line-derived exosomes and anti-interference in complicated biological serum systems show good performances. The presence of target exosomes can also be easily determined by visual observation of LED lighted by the generated electric energy. The proposed method can be used as a powerful tool for ultrasensitive analysis of exosomes, which is expected to have broad biological and analytical applications.

Advances in Biosensors Technology for Detection and Characterization of Extracellular Vesicles.

Exosomes are extracellular vehicles (EVs) that encapsulate genomic and proteomic material from the cell of origin that can be used as biomarkers for non-invasive disease diagnostics in point of care settings. The efficient and accurate detection, quantification, and molecular profiling of exosomes are crucial for the accurate identification of disease biomarkers. Conventional isolation methods, while well-established, provide the co-purification of proteins and other types of EVs. Exosome purification, characterization, and OMICS analysis are performed separately, which increases the complexity, duration, and cost of the process. Due to these constraints, the point-of-care and personalized analysis of exosomes are limited in clinical settings. Lab-on-a-chip biosensing has enabled the integration of isolation and characterization processes in a single platform. The presented review discusses recent advancements in biosensing technology for the separation and detection of exosomes. Fluorescent, colorimetric, electrochemical, magnetic, and surface plasmon resonance technologies have been developed for the quantification of exosomes in biological fluids. Size-exclusion filtration, immunoaffinity, electroactive, and acoustic-fluid-based technologies were successfully applied for the on-chip isolation of exosomes. The advancement of biosensing technology for the detection of exosomes provides better sensitivity and a reduced signal-to-noise ratio. The key challenge for the integration of clinical settings remains the lack of capabilities for on-chip genomic and proteomic analysis.