Sensitive Detection Of Exosomes Research Articles

Flexible Paper-Based Immunosensor for the Detection of Specific Cancer-Derived Exosomes

This study aims at an electrochemical paper-based device for the selective and sensitive detection of exosomes as an economic and flexible platform. Using a laser cutter, a three-electrode platform was patterned on a hydrophobic paper, and then synthetic carbon and silver ink were screen-printed on the paper. Anti-human CD9 antibody was covalently attached to the surface of a carbon ink electrode enriched with graphene oxide aerogel/CoFe2O4@chitosan nanocomposite. The immobilization, incubation and binding time of the capture antibody were investigated as effective parameters. Under optimum conditions, the differential pulse voltammetry (DPV) was used to establish a calibration plot that covered 500 to 5 × 106 exosoms μl−1 with a detection limit of 100 exosome μl−1. This method was implemented successfully for the detection of exosomes in a serum sample. As the results show, the fabricated biosensor is capable of detecting exosomes for the early diagnosis of cancers.

Sensitive detection of exosomes by gold nanoparticles labeling inductively coupled plasma mass spectrometry based on cholesterol recognition and rolling circle amplification

Exosomes, potential biomarkers for liquid biopsy, provide a promising opportunity for early cancer diagnosis in a noninvasive manner. However, direct analysis of exosomes in complicated biological samples is still challenging. Herein, a cholesterol recognition assay was proposed for detection of exosomes by gold nanoparticles (Au NPs) labeling inductively coupled plasma mass spectrometry. Specifically, exosomes were captured by anti-CD63, and then cholesterol-modified rolling circle amplification product was inserted into the membrane of exosomes for signal amplification. Simultaneously, Au NPs-labelled DNA was prepared for labeling and read-out signal. The proposed method achieved the quantification of the exosome concentration in a range of 10-106 particles/μL with a limit of detection of 1.5 particles/μL. The method was applied for the direct detection of exosomes in human serum in a purification-free way, and the analytical result coincided with that obtained by nanoparticle tracking analysis. Real sample analysis manifested that the exosome concentration in the serum of oral squamous cell carcinoma patients was higher than that in the healthy volunteers’ serum. The developed method is sensitive, selective, accurate and can be used for the direct detection of exosomes in human serum, providing a new opportunity for exosome-based liquid biopsy in early cancer diagnosis.

Single microbead-based fluorescent aptasensor (SMFA) for direct isolation and in situ quantification of exosomes from plasma.

Exosomes are cell-derived membrane-enclosed biological nanoparticles that carry lots of parental molecular information, and are recognized as an ideal biomarker for non-invasive diagnosis. However, due to the low abundance of exosomes in plasma samples and the interferences from complex biological matrices, the sensitive and direct detection of exosomes still remains a challenge. Here, by combining the direct magnetic isolation with in situ fluorescence imaging, we developed a Single Microbead-based Fluorescent Aptasensor (SMFA) for specific enrichment and sensitive quantification of exosomes from plasma. In the SMFA, a single aptamer-modified microbead (MB) served as the reaction carrier so that the specific exosomes inserted with a fluorescent anchor will be highly enriched on the single MB. By in situ fluorescence imaging to monitor the fluorescence signals on the single MB, sensitive detection of exosomes can be realized without the requirement of any signal amplification routes, and as low as 4.9 × 104 particles per μL of exosomes could be simply detected. More importantly, the SMFA could be applied for direct detection of the exosomes from small amounts of clinical plasma samples without prior purification procedures, indicating its great potential applications in clinical diagnostics.

Engineering of exosome-triggered enzyme-powered DNA motors for highly sensitive fluorescence detection of tumor-derived exosomes

Tumor-derived exosomes containing multiple proteins originating from parent cancer cells have emerged as biomarkers for cancer diagnosis. Herein, we propose a three-dimensional DNA motor-based exosome assay platform for the selective and sensitive detection of exosomes. The DNA motor used gold nanoparticle (GNP) tracks, consisting of fluorescein-labeled substrate strands and aptamer-locked motor strands. Recognition of the target protein on exosomes by its aptamer unlocked the motor strand and triggered the DNA motor process. Powered by restriction endonuclease, the motor strands autonomously walked along the GNP track. Each movement step cleaved one substrate strand and restored one fluorescein molecule. For exosome detection, the proposed method displayed a broad dynamic range acrossing 5 orders of magnitude with the detection limit as low as 8.2 particles/μL in PBS. The method also exhibited good selectivity among different tumor-derived exosomes and performed well in complex biological samples. The capability to profile exosomal surface proteins efficiently endowed our DNA motor great potential for developing a simple and cost-effective device for clinical diagnosis.

Ultrasensitive Detection of Exosomes by Target-Triggered Three-Dimensional DNA Walking Machine and Exonuclease III-Assisted Electrochemical Ratiometric Biosensing.

Exosomes are membrane-enclosed phospholipid extracellular vesicles. In spite of their great promise as noninvasive biomarkers for cancer diagnosis, sensitive detection of exosomes is still challenging. Herein, the detection of exosomes was changed to the detection of DNA after recognition of exosomes with its aptamers. CD63 aptamer and EpCAM aptamer were used for the detection of MCF-7 cell-secreted exosome. The recognition process was amplified through the movements of a three-dimensional DNA walker. And then, Exonuclease III- assisted electrochemical ratiometric sensor was applied for further signal amplification. Under optimal conditions, the detection limit of 1.3 × 104 particles/mL was obtained with excellent selectivity. Furthermore, clinical application test for the detection of exosomes in human serum was also verified.

An ultrasensitive electrochemical aptasensor for the determination of tumor exosomes based on click chemistry

Exosomes, lipid bilayer membrane vesicles, can guide various pathological and physiological processes. However, reliable, convenient and sensitive methods for exosome determination for early cancer diagnosis are still technically challenging. Herein, an electrochemical aptasensor based on click chemistry and the DNA hybridization chain reaction (HCR) for signal amplification has been developed for the ultrasensitive detection of tumor exosomes. CD63 aptamer was first immobilized on a glassy carbon electrode for capturing exosomes, and 4-oxo-2-nonenal alkyne (alkynyl-4-ONE) molecules, functionalized lipid electrophiles, were conjugated to the exosomes via the reaction of amino and aldehyde groups. Azide-labeled DNA probe as an anchor was then connected to the exosomes by copper (I)-catalyzed click chemistry. Signal amplification was achieved by HCR, and the numerous linked horseradish peroxidase (HRP) molecules could catalyze the reaction of o-phenylenediamine (OPD) and H2O2. The concentration of exosomes could be quantified by monitoring the electrochemical reduction current of 2,3-diaminophenazine (DAP). Under the optimal conditions, this method allowed the sensitive detection of exosomes in the range of 1.12 × 102 to 1.12 × 108 particles/μL with a limit of detection (LOD) of 96 particles/μL. Furthermore, the present assay enabled sensitive and accurate quantification of exosomes in human serum, and it has high potential for exosome analysis in clinical samples.

Avoiding Pre-Isolation Step in Exosome Analysis: Direct Isolation and Sensitive Detection of Exosomes Using Gold-Loaded Nanoporous Ferric Oxide Nanozymes.

Most of the current exosome-analysis strategies are time-consuming and largely dependent on commercial extraction kit-based preisolation step, which requires extensive sample manipulations, costly isolation kits, reagents, tedious procedures, and sophisticated equipment and is prone to bias/artifacts. Herein we introduce a simple method for direct isolation and subsequent detection of a specific population of exosomes using an engineered superparamagnetic material with multifunctional properties, namely, gold-loaded ferric oxide nanocubes (Au-NPFe2O3NC). In this method, the Au-NPFe2O3NC were initially functionalized with a generic tetraspanin (exosomes-associated) antibody (i.e., CD63) and dispersed in sample fluids where they work as "dispersible nanocarriers" to capture the bulk population of exosomes. After magnetic collection and purification, Au-NPFe2O3NC-bound exosomes were transferred to the tissue-specific, antibody-modified, screen-printed electrode. As a proof of principle, we used a specific placental marker, placenta alkaline phosphatase (PLAP), to detect exosomes secreted from placental cells. The peroxidase-like activity of Au-NPFe2O3NC was then used to accomplish an enzyme-linked immunosorbent assay (ELISA)-based sensing protocol for naked-eye observation along with UV-visible and electrochemical detection of PLAP-specific exosomes present in placental cell-conditioned media. We demonstrated excellent agreement in analytical performance for the detection of placental cell-derived exosomes (i.e., linear dynamic range, 103-107 exosomes/mL; limit of detection, 103 exosomes/mL; relative standard deviation (%RSD) of <5.5% for n = 3) using with and without commercial "total exosome isolation kit"-based preisolation step. We envisage that this highly sensitive, rapid, and inexpensive assay could be useful in quantifying specific populations of exosomes for various clinical applications, focusing on pregnancy complications.