Detection Of Exosomes Research Articles

Dual signal Amplification-Mediated Aptamer-Based electrochemical biosensor for sensitive detection of exosomes

Dual signal Amplification-Mediated Aptamer-Based electrochemical biosensor for sensitive detection of exosomes

Concentration of kidney markers and detection of exosomes in urine samples collected in cotton wool balls in preterm and term neonates

Collecting urine samples in neonates by catheterisation or suprapubic puncture causes trauma, whereas self-adhesive collection bags can damage fragile skin. An alternative method is the collection of samples from urine-soaked cotton wool balls placed in diapers. The aim of this study was to compare the concentration of albumin, creatinine, neutrophil gelatinase-associated lipocalin (NGAL), and uromodulin between clean-catch urine and samples collected in cotton balls in neonates and assess the efficiency of exosome extraction. Standard clean-catch urine samples (SSs) were assayed for albumin, creatinine, NGAL, and uromodulin using commercial ELISA kits. Concentrations were compared to the same urine samples extracted immediately from soaked cotton wool balls (sample 2, S2) or the urine extracted from cotton wool balls placed in diaper in a warm incubator for 2 h before extraction (sample 3, S3). Exosomes were extracted from all three samples of one patient for visualisation by electron microscopy. Twenty-six infants (17 males) of median gestational age at birth of 32+1 weeks had urine collected at a median age of 29 days at 37+6 weeks corrected age. Concentrations in S2 and S3 were within 10% of the concentration of SS in 46% and 35% of specimens for albumin, 69% and 58% for creatinine, 12% and 12% for NGAL, and 27% and 15% for uromodulin, respectively, without consistent positive or negative bias. Urine albumin/creatinine ratios (UACRs) were 4.3% less in S2 and 4.5% less in S3 than in SS. Exosomes were extracted and visualised from all three sample types. Neonatal urine samples extracted from cotton wool balls can be used to screen for relevant albuminuria ​but provide imprecise estimates of NGAL and uromodulin. The proof of exosome extraction from urine collection in cotton wool balls opens the potential to examine exosomal cargo.

Terminal deoxynucleotidyl transferase (TdT) based template-free signal amplification for the detection of exosomes in MUC1-positive cells

The Mucin1 (MUC1) protein, involved in cytoprotective and signaling pathways, is abnormally elevated in various cancers, making it a key cancer indicator. Exosomes, which reflect the status of their originating cells, offer potential for cancer diagnosis. Thus, developing a method to detect MUC1-positive exosomes is crucial for the early diagnosis of certain cancers. In this study, we developed a highly sensitive, specific, and simple UV–visible signal amplification method to detect MUC1-positive exosomes using terminal deoxynucleotidyl transferase (TdT). Initially, exosomes were captured on magnetic beads using a CD63 aptamer(apt). The Primer-AuNPs-MUC1 apt complex which we synthesized by low pH loading method was then attached MUC1 proteins on the surface of the exosomes to create a sandwich structure. TdT catalyzed the extension of Biotin-dATP at the 3′ end of the primer, introducing multiple biotin sites into the sandwich structure. These sites subsequently bound multiple streptavidin-horseradish peroxidase (streptavidin-HRP), which catalyzed the oxidative color change of the substrate, which can be detected by colorimetric method. This method can detect A549 exosomes in the range of 1.4E+6 to 4.2E+8 particles/mL and shows high specificity for cell lines with different MUC1 expression. Additionally, it successfully distinguished cholangiocarcinoma (CCA) patients (n=11) from healthy individuals (n=7) in clinical serum assays, demonstrating good performance in real sample detection.

Label-Free Exosome Analysis by Surface-Enhanced Raman Scattering Spectroscopy with Laser-Ablated Silver Nanoparticle Substrate.

Early diagnostics of breast cancer is crucial to reduce the risk of cancer metastasis and late relapse. Exosome, which contains distinct information of its origin, can be the target object as a liquid biopsy. However, its low sensitivity and inadequate diagnostic tools interfere with the point-of-care testing (POCT) of the exosome. Recently, Surface-enhanced Raman Scattering (SERS) spectroscopy, which amplifies the Raman scattering, has been proved as a promising tool for exosome detection. However, the fabrication process of SERS probe or substrate is still inefficient and far from large-scale production. This study proposes rapid and label-free detection of breast cancer-derived exosomes by statistical analysis of SERS spectra using silver-nanoparticle-based SERS substrate fabricated by selective laser ablation and melting (SLAM). Employing silver nanowires and optimizing laser process parameters enable rapid and low-energy fabrication of SERS substrate. The functionalities including sensitivity, reproducibility, stability, and renewability are evaluated using rhodamine 6G as a probe molecule. Then, the feasibility of POCT is examined by the statistical analysis of SERS spectra of exosomes from malignant breast cancer cells and non-tumorigenic breast epithelial cells. The presented framework is anticipated to be utilized in other biomedical applications, facilitating cost-effective and large-scale production performance.

Enhanced plasmonic scattering imaging via deep learning-based super-resolution reconstruction for exosome imaging.

Exosome analysis plays pivotal roles in various physiological and pathological processes. Plasmonic scattering microscopy (PSM) has proven to be an excellent label-free imaging platform for exosome detection. However, accurately detecting images scattered from exosomes remains a challenging task due to noise interference. Herein, we proposed an image processing strategy based on a new blind super-resolution deep learning neural network, named ESRGAN-SE, to improve the resolution of exosome PSI images. This model can obtain super-resolution reconstructed images without increasing experimental complexity. The trained model can directly generate high-resolution plasma scattering images from low-resolution images collected in experiments. The results of experiments involving the detection of light scattered by exosomes showed that the proposed super-resolution detection method has strong generalizability and robustness. Moreover, ESRGAN-SE achieved excellent results of 35.52036, 0.09081, and 8.13176 in terms of three reference-free image quality assessment metrics, respectively. These results show that the proposed network can effectively reduce image information loss, enhance mutual information between pixels, and decrease feature differentiation. And, the single-image SNR evaluation score of 3.93078 also showed that the distinction between the target and the background was significant. The suggested model lays the foundation for a potentially successful approach to imaging analysis. This approach has the potential to greatly improve the accuracy and efficiency of exosome analysis, leading to more accurate cancer diagnosis and potentially improving patient outcomes.

Surface protein analysis of breast cancer exosomes using visualized strategy on centrifugal disk chip

Breast cancer, the most common cancer among women worldwide, lacks specific tumor markers for accurate diagnosis. Recent advances have highlighted tumor-derived exosomes as a promising non-invasive biomarker for cancer detection. Continuous monitoring of surface protein markers on exosomes in the blood could offer valuable insights for breast cancer diagnosis. However, integrating the isolation and detection of exosomes from whole blood is bulky, time-consuming, and requires professional operations. To address this difficulty, we developed a method of integrated centrifugal disk chip (CD chip) exosome enrichment directly from whole blood followed by a colorimetric visualization strategy for multiplex analysis. The disc consists of multi-chambers and multi-microchannels with immediate smartphone-enabled processing of colorimetric results. The combination of CEA + CA125 + EGFR on-chip detection could significantly differentiate the different stages of cancer in tumor-bearing mice and successfully distinguish between breast cancer patients and healthy individuals. Crucially, small volumes (100 μL) of blood samples were adequate. In addition, the chip was simple and fast, with results within 10 min, which provides immediate exosomal information through consecutive blood sampling, which could potentially result in a more timely and well-informed clinical breast cancer diagnosis.

Harnessing Spatiotemporal-Specific Tumorous Exosome Dynamics: Ultra-Sensitive Lanthanide Luminescence Detection Strategy Enabled by Exosomal Membrane Engineering for Melanoma Immunotherapy Monitoring.

Focused on the newly secreted tumorous exosomes during melanoma immunotherapy, this work has pioneered an ultra-sensitive spatiotemporal-specific exosome detection strategy, leveraging advanced exosomal membrane engineering techniques. The proposed strategy harnesses the power of amplified lanthanide luminescence signals on these exosomes, enabling precise and real-time monitoring of the efficacy of melanoma immunotherapy. The methodology comprises two pivotal steps. Initially, Ac4ManNAz-associated metabolic labeling is employed to evolve azide groups onto the membranes of newly secreted exosomes with remarkable selectivity. These azide groups serve as versatile clickable artificial tags, enabling the precise identification of melanoma exosomes emerging during immunotherapy. Subsequently, lanthanide-nanoparticle-functionalized polymer chains are controllably grafted onto the exosome surfaces through click chemistry and in situ Fenton-RAFT polymerization, serving as robust signal amplifiers. When integrated with time-resolved fluorescence detection, this strategy yields detection signals with an exceptionally high signal-to-noise ratio, enabling ultra-sensitive detection of PD-L1 antigen expression levels on the spatiotemporal-specific exosomes. The detection strategy boasts a wide linear concentration range spanning from 1.7 × 104 to 1.7 × 109 particles/mL, with a remarkable theoretical detection limit of 1.28 × 103 particles/mL. The remarkable enhancements in detection sensitivity and accuracy facilitate the evaluation of the efficacy of immunotherapeutic interventions in the mouse B16 melanoma model, notably revealing a substantial disparity in PD-L1 levels between immunotherapy-treated and untreated groups (P < 0.01) and further emphasizing the cumulative therapeutic effect that intensifies with repeated treatments (P < 0.001).

Exosome in renal cell carcinoma progression and implications for targeted therapy.

Renal cell carcinoma is a urological malignancy with a high metastatic rate, while targeted therapy for renal cell carcinoma still has much room for improvement. Some cutting-edge researches have focused on exosome in cancer treatment and there are some breakthroughs in breast cancer, lung cancer, and pancreatic cancer. Up to now, exosome in renal cell carcinoma progression and implications for targeted therapy has been under research by scientists. In this review, we have summarized the structure, formation, uptake, functions, and detection of exosomes, classified the mechanisms of exosomes that cause renal cell carcinoma progression, and listed the promising utilization of exosomes in targeted therapy for renal cell carcinoma. In all, based on the mechanisms of exosomes causing renal cell carcinoma progression and borrowing the successful experience from renal cell carcinoma models and other cancers, exosomes will possibly be a promising target for therapy in renal cell carcinoma in the foreseeable future.

Advances in exosome plasmonic sensing: Device integration strategies and AI-aided diagnosis

Exosomes, as next-generation biomarkers, has great potential in tracking cancer progression. They face many detection limitations in cancer diagnosis. Plasmonic biosensors have attracted considerable attention at the forefront of exosome detection, due to their label-free, real-time, and high-sensitivity features. Their advantages in multiplex immunoassays of minimal liquid samples establish the leading position in various diagnostic studies. This review delineates the application principles of plasmonic sensing technologies, highlighting the importance of exosomes-based spectrum and image signals in disease diagnostics. It also introduces advancements in miniaturizing plasmonic biosensing platforms of exosomes, which can facilitate point-of-care testing for future healthcare. Nowadays, inspired by the surge of artificial intelligence (AI) for science and technology, more and more AI algorithms are being adopted to process the exosome spectrum and image data from plasmonic detection. Using representative algorithms of machine learning has become a mainstream trend in plasmonic biosensing research for exosome liquid biopsy. Typically, these algorithms process complex exosome datasets efficiently and establish powerful predictive models for precise diagnosis. This review further discusses critical strategies of AI algorithm selection in exosome-based diagnosis. Particularly, we categorize the AI algorithms into the interpretable and uninterpretable groups for exosome plasmonic detection applications. The interpretable AI enhances the transparency and reliability of diagnosis by elucidating the decision-making process, while the uninterpretable AI provides high diagnostic accuracy with robust data processing by a “black-box” working mode. We believe that AI will continue to promote significant progress of exosome plasmonic detection and mobile healthcare in the near future.

Ultrasensitive Electrochemiluminescence Biosensor Based on DNA-Bio-Bar-Code and Hybridization Chain Reaction Dual Signal Amplification for Exosomes Detection.

Exosomes have received considerable attention as potent reference markers for the diagnosis of various neoplasms due to their close and direct relationship with the proliferation, adhesion, and migration of tumor. The ultrasensitive detection of cancer-derived low-abundance exosomes is imperative, but still a great challenge. Herein, we report an electrochemiluminescence (ECL) biosensor based on the DNA-bio-bar-code and hybridization chain reaction (HCR)-mediated dual signal amplification for the ultrasensitive detection of cancer-derived exosomes. In this system, two types of aptamers were modified on the magnetic nanoprobe (MNPs) and gold nanoparticles (AuNPs) with numerous bio-bar-code DNA, respectively, which formed "sandwich" structures in the presence of specific target exosomes. The "sandwich" structures were separated under magnetic field, and the numerous bio-bar-code DNA were released by dissolving AuNPs. The released bio-bar-code DNA triggered the HCR procedure to produce a good deal of long DNA duplex structure for embedding in hemin, which generated strong ECL signal in the presence of coreactors for ultrasensitive detection of exosomes. Under the optimal conditions, it exhibited a good linearly of exosomes ranging from 10 to 104 exosomes particle μL-1 with limit of detection down to 5.01 exosome particle μL-1. Furthermore, the high ratio of ECL signal and minor change of ECL intensity indicated the good specificity, stability, and repeatability of this ECL biosensor. Given the good performance for exosome analysis, this ultrasensitive ECL biosensor has a promising application in the clinical diagnosis of early cancers.

A Calibration Strategy for Silicon Nanowire Field-Effect Transistor Biosensors and Its Application in Ultra-Sensitive, Label-Free Biosensing.

The silicon nanowire field-effect transistor (SiNW FET) has been developed for over two decades as an ultrasensitive, label-free biosensor for biodetection. However, inconsistencies in manufacturing and surface functionalization at the nanoscale have led to poor sensor-to-sensor consistency in performance. Despite extensive efforts to address this issue through process improvements and calibration methods, the outcomes have not been satisfactory. Herein, based on the strong correlation between the saturation response of SiNW FET biosensors and both their feature size and surface functionalization, we propose a calibration strategy that combines the sensing principles of SiNW FET with the Langmuir-Freundlich model. By normalizing the response of the SiNW FET biosensors (ΔI/I0) with their saturation response (ΔI/I0)max, this strategy fundamentally overcomes the issues mentioned above. It has enabled label-free detection of nucleic acids, proteins, and exosomes within 5 min, achieving detection limits as low as attomoles and demonstrating a significant reduction in the coefficient of variation. Notably, the nucleic acid test results exhibit a strong correlation with the ultraviolet-visible (UV-vis) spectrophotometer measurements, with a correlation coefficient reaching 0.933. The proposed saturation response calibration strategy exhibits good universality and practicability in biological detection applications, providing theoretical and experimental support for the transition of mass-manufactured nanosensors from theoretical research to practical application.

Application of EXODUS system combined with allosteric DNA nanoswitches in the detection of miR-107 among plasma exosomes of Parkinson's disease patients

This study aimed to achieve rapid detection of Parkinson's disease (PD) plasma exosome miR-107. A case-control design was used to collect ten Parkinson's disease and ten healthy control plasma samples from the Department of Laboratory Medicine, Tongji Hospital, Tongji Medical College of Huazhong University of Science and Technology from December 2023 to January 2024. Exosome detection via the ultrafast-isolation system (EXODUS) was used to isolate plasma exosomes. The nanoparticle tracking analysis technology and electron microscopy were used to identify exosome particle size and morphology. The Qiagen miRNeasy Micro Kit was used to extract RNA. The microRNA-activated conditional looping of engineered switches (miRacles) was used to detect miR-107, and the relative expression was analyzed by agarose gel electrophoresis. Thermo Fisher RevertAid RT Reverse Transcription Kit was used to perform reverse transcription of RNA, and real-time PCR was used to detect miR-107. The independent samples t-test was used for comparison between groups. EXODUS system completed the isolation of exosomes from 500 μl plasma within 1.5 hours. The exosome concentration (mean±SD) was (4.82±2.02)×1010 particles/ml in the control group and (5.08±2.34)×1010 particles/ml in the PD group. There was no significant difference in exosome concentration between PD patients and healthy controls (t=-0.168, P=0.872). The morphology of exosomes was confirmed by electron microscopy. The miRacles nanoswitch could detect fM-level miR-107 and also effectively distinguish miR-107 from its family members, including miR-15a and miR-16. Agarose gel electrophoresis showed that the mean±SD of relative grey value content was 1.00±0.26 in the control group and 1.86±0.21 in the PD group. The miR-107 in the PD group was significantly higher than that in the control (t=-8.143, P<0.001), which was consistent with the result of real-time PCR. EXODUS combined with miRacles could achieve rapid, non-enzymatic and cheap detection of plasma exosomal miR-107 in PD patients.

Advancements in Nano-Mediated Biosensors: Targeting Cancer Exosome Detection

Advancements in Nano-Mediated Biosensors: Targeting Cancer Exosome Detection

Exosomal membrane proteins analysis using a silicon nanowire field effect transistor biosensor

Exosomes are of great significance in clinical diagnosis, due to their high homology with parental generation, which can reflect the pathophysiological status. However, the quantitative and classification detection of exosomes is still faced with the challenges of low sensitivity and complex operation. In this study, we develop an electrical and label-free method to directly detect exosomes with high sensitivity based on a Silicon nanowire field effect transistor biosensor (Si-NW Bio-FET). First, the impact of Debye length on Si-NW Bio-FET detection was investigated through simulation. The simulation results demonstrated that as the Debye length increased, the electrical response to Si-NW produced by charged particle at a certain distance from the surface of Si-NW was greater. A Si-NW Bio-FET modified with specific antibody CD81 on the nanowire was fabricated then used for detection of cell line-derived exosomes, which achieved a low limit of detection (LOD) of 1078 particles/mL in 0.01 × PBS. Furthermore, the Si-NW Bio-FETs modified with specific antibody CD9, CD81 and CD63 respectively, were employed to distinguish exosomes derived from human promyelocytic leukemia (HL-60) cell line in three different states (control group, lipopolysaccharide (LPS) inflammation group, and LPS + Romidepsin (FK228) drug treatment group), which was consistent with nano-flow cytometry. This study provides a highly sensitive method of directly quantifying exosomes without labeling, indicating its potential as a tool for disease surveillance and medication instruction.

Multiplexed detection platform implemented with magnetic encoding and deep learning-based decoding for quantitative analysis of exosomes from cancers

Although a number of biosensing technologies have been reported for the detection of cancer-derived exosomes used as early diagnosis markers for cancers, it is necessary to identify them with various biomarkers to distinguish the stages and types of cancers owing to the extreme heterogeneity of cancer. Here, we developed a new multiplexed assay platform for the detection of exosomes using magnetic encoded microparticles (MEMPs), which can recognize multiple proteins expressed on exosomes, and a deep learning-based decoding algorithm. This platform, in which the accuracy of the decoding algorithm was evaluated to be 93 %, was applied to detect exosomes from four types of cancer cell lines and plasma from patients with cancer using three cancer biomarkers: PD-L1 (Programmed Death-Ligand 1), EpCAM (Epithelial cell adhesion molecule), and EGFR (Epidermal growth factor receptor). The limit of detections (LODs) of this platform when applied to the detection of exosomes from MDA-MB-231 cell line were calculated as 4.03 × 106 mL−1 for PD-L1, 1.00 ×107 mL−1 for EpCAM, and 7.17×106 mL−1 for EGFR, respectively. In a clinical study, four types of samples from patients with cancer (n = 92) showed higher signals than those of healthy controls (n = 18). Based on these results, we confirmed that this platform can distinguish patients with cancer from healthy individuals.

The clinical value of rapidly detecting urinary exosomal lncRNA RMRP in bladder cancer with an RT-RAA-CRISPR/Cas12a method

Background and aimsBladder cancer (BCa) is a highly aggressive malignancy of the urinary system. Timely detection is imperative for enhancing BCa patient prognosis. Materials and methodsThis study introduces a novel approach for detecting long non-coding RNA (lncRNA) Mitochondrial RNA Processing Endoribonuclease (RMRP) in urine exosomes from BCa patients using the reverse transcription recombinase-aided amplification (RT-RAA) and clustered regularly interspaced short palindromic repeats and associated Cas12a proteins (CRISPR/Cas12a) technique. Various statistical methods were used to evaluate its diagnostic value for BCa. ResultsThe specificity of urine exosomal RMRP detection for BCa diagnosis was enhanced by using RT-RAA combined with CRISPR/Cas12a. The testing process duration was reduced to 30 min, which supports rapid detection. Moreover, this approach allows the identification of target signals in real-time using blue light, facilitating immediate detection. In clinical sample analysis, this methodology exhibited a high level of diagnostic efficacy. This was evidenced by larger area under the curve values with receiver operating characteristic curve analysis compared with using traditional RT-qPCR methods, indicating superior diagnostic accuracy and sensitivity. Furthermore, the combined analysis of RMRP expression in urine exosomes detected by RT-RAA-CRISPR/Cas12a and NMP-22 expression may further enhance diagnostic accuracy. ConclusionsThe RT-RAA-CRISPR/Cas12a technology is a swift, sensitive, and uncomplicated method for nucleic acid detection. Because of its convenient and non-invasive sampling approach, user-friendly operation, and reproducibility, this technology is very promising for automated detection and holds favorable application possibilities within clinical environments.

Aptamer-Functionalized Magnetic Ti3C2 Based Nanoplatform for Simultaneous Enrichment and Detection of Exosomes.

Exosomes are nanovesicles secreted by cells, which play a crucial role in various pathological processes. Exosomes have shown great promise as tumor biomarkers because of the abundant secretion during tumor formation. The development of a convenient, efficient, and cost-effective method for simultaneously enriching and detecting exosomes is of utmost importance for both basic research and clinical applications. In this study, an aptamer-functionalized magnetic Ti3C2 composite material (Fe3O4@Ti3C2@PEI@DSP@aptamer@FAM-ssDNA) is prepared for the simultaneous enrichment and detection of exosomes. CD63 aptamers are utilized to recognize and capture the exosomes, followed by magnetic separation. The exosomes are then released by cleaving the disulfide bonds of DSP. Compared to traditional methods, Fe3O4@Ti3C2@PEI@DSP@aptamer@FAM-ssDNA exhibited superior efficiency in enriching exosomes while preserving their structural and functional integrity. Detection of exosome concentration is achieved through the fluorescence quenching of Ti3C2 and the competitive binding between the exosomes and a fluorescently labeled probe. This method exhibited a low detection limit of 4.21 × 104 particlesmL-1, a number that is comparable to the state-of-the-art method in the detection of exosomes. The present study demonstrates a method of simultaneous enrichment and detection of exosomes with a high sensitivity, accuracy, specificity, and cost-effectiveness providing significant potential for clinical research and diagnosis.

Empowering Exosomes with Aptamers for Precision Theranostics.

As information messengers for cell-to-cell communication, exosomes, typically small membrane vesicles (30-150nm), play an imperative role in the physiological and pathological processes of living systems. Accumulating studies have demonstrated that exosomes are potential biological candidates for theranostics, including liquid biopsy-based diagnosis and drug delivery. However, their clinical applications are hindered by several issues, especially their unspecific detection and insufficient targeting ability. How to upgrade the accuracy of exosome-based theranostics is being widely explored. Aptamers, benefitting from their admirable characteristics, are used as excellent molecular recognition elements to empower exosomes for precision theranostics. With high affinity against targets and easy site-specific modification, aptamers can be incorporated with platforms for the specific detection of exosomes, thus providing opportunities for advancing disease diagnostics. Furthermore, aptamers can be tailored and functionalized on exosomes to enable targeted therapeutics. Herein, this review emphasizes the empowering of exosomes by aptamers for precision theranostics. A brief introduction of exosomes and aptamers is provided, followed by a discussion of recent progress in aptamer-based exosome detection for disease diagnosis, and the emerging applications of aptamer-functionalized exosomes for targeted therapeutics. Finally, current challenges and opportunities in this research field are presented.

High-Q WGM microcavity-based optofluidic sensor technologies for biological analysis.

High-quality-factor (Q) optical microcavities have attracted extensive interest due to their unique ability to confine light for resonant circulation at the micrometer scale. Particular attention has been paid to optical whispering-gallery mode (WGM) microcavities to harness their strong light-matter interactions for biological applications. Remarkably, the combination of high-Q optical WGM microcavities with microfluidic technologies can achieve a synergistic effect in the development of high-sensitivity optofluidic sensors for many emerging biological analysis applications, such as the detection of proteins, nucleic acids, viruses, and exosomes. They can also be utilized to investigate the behavior of living cells in human organisms, which may provide new technical solutions for studies in cell biology and biophysics. In this paper, we briefly review recent progress in high-Q microcavity-based optofluidic sensor technologies and their applications in biological analysis.

Automatic Electrochemiluminescence Method for the Detection of Cancerous Exosomes Incorporating Specific Aptamer-Magnetic Beads and Signal Nanoprobes.

Exosomes, as an emerging biomarker, have exhibited remarkable promise in early cancer diagnosis. Here, a highly sensitive, selective, and automatic electrochemiluminescence (ECL) method for the detection of cancerous exosomes was developed. Specific aptamer-(EK)4 peptide-tagged magnetic beads (MBs-(EK)4-aptamer) were designed as a magnetic capture probe in which the (EK)4 peptide was used to reduce the steric binding hindrance of cancerous exosomes with a specific aptamer. One new universal ECL signal nanoprobe (CD9 Ab-PEG@SiO2ϵRu(bpy)32+) was designed and synthesized by using microporous SiO2 nanoparticles as the carrier for loading ECL reagent Ru(bpy)32+, polyethylene glycol (PEG) layer, and anticluster of differentiation 9 antibody (CD9 Ab). A "sandwich" biocomplex was formed on the surface of the magnetic capture probe after mixing the capture probe, target exosomes, and ECL signal nanoprobe, and then it was introduced into an automated ECL analyzer for rapid and automatic ECL measurement. It was found that the designed signal nanoprobe shows a 270-fold improvement in the signal-to-noise ratio than that of the ruthenium complex-labeled CD9 antibody signal probe. The relative ECL intensity was proportional to MCF-7 exosomes as a model in the range of 102 to 104 particle/μL, with a detection limit of 11 particle/μL. Furthermore, the ECL method was employed to discriminate cancerous exosomes based on fingerprint responses using the designed multiple magnetic capture probes and the universal ECL signal nanoprobe. This work demonstrates that the utilization of a designed automated ECL tactic using the MBs-(EK)4-aptamer capture probe and the CD9 Ab-PEG@SiO2ϵRu(bpy)32+ signal nanoprobe will provide a unique and robust method for the detection and discrimination of cancerous exosomes.