Detection Of Exosomal miRNAs Research Articles

Recent Advances in Exosomal miRNA Biosensing for Liquid Biopsy.

As a noninvasive detection technique, liquid biopsy plays a valuable role in cancer diagnosis, disease monitoring, and prognostic assessment. In liquid biopsies, exosomes are considered among the potential biomarkers because they are important bioinformation carriers for intercellular communication. Exosomes transport miRNAs and, thus, play an important role in the regulation of cell growth and function; therefore, detection of cancer cell-derived exosomal miRNAs (exo-miRNAs) gives effective information in liquid biopsy. The development of sensitive, convenient, and reliable exo-miRNA assays will provide new perspectives for medical diagnosis. This review presents different designs and detection strategies of recent exo-miRNA assays in terms of signal transduction and amplification, as well as signal detection. In addition, this review outlines the current attempts at bioassay methods in liquid biopsies. Lastly, the challenges and prospects of exosome bioassays are also considered.

DNA Zipper Mediated Membrane Fusion for Rapid Exosomal MiRNA Detection.

Accurate and reliable detection of exosomal miRNA can serve as a promising method for early diagnosis of disease and evaluation of therapeutic effects. However, current exosomal miRNA detection methods commonly involve exosome enrichment, containing RNA extraction, and qRT-PCR based quantification, which are expensive and time-consuming. Herein, we develop a DNA zipper-mediated membrane fusion approach for rapid exosomal miRNA detection and cancer diagnosis. First, a lipid vesicle probe containing miR21-targeting molecular beacons (MBs) is constructed and further loaded with zipper DNA constructs (ZDCs) on its surface. Meanwhile, complementary zipper DNA constructs (cZDCs) are introduced on the exosome of interest. Upon mixing them together, zipping between ZDC and cZDC induces the membrane fusion of exosomes and vesicle probes, triggering the recognition of exosomal miR21 by contained MBs and fluorescence emission that can be conveniently detected within 30 min. Importantly, with the assistance of flow cytometry, miR21-overexpressed tumor exosomes derived from either cell culture medium or clinical patient serums can be distinguished from exosomes secreted from normal cells. This approach provides a convenient way to accurately detect the exosomal miRNA, which may hold great potential in liquid biopsy for early cancer diagnosis and monitoring the therapeutic effects during the treatments.

Ratiometric Fluorescent Biosensor Based on Self-Assembled Fluorescent Gold Nanoparticles and Duplex-Specific Nuclease-Assisted Signal Amplification for Sensitive Detection of Exosomal miRNA.

The sensitive detection of cancer-associated exosomal microRNAs shows enormous potential in cancer diagnosis. Herein, a ratiometric fluorescent biosensor based on self-assembled fluorescent gold nanoparticles (Au NPs) and duplex-specific nuclease (DSN)-assisted signal amplification was fabricated for sensitive detection of colorectal cancer (CRC)-associated exosomal miR-92a-3p. In this biosensing system, the hairpin DNA modified with sulfhydryl and fluorescent dye Atto-425 at both ends is conjugated to fluorescent Au NPs through Au-S bonds, resulting in the quenching of Atto-425. The miR-92a-3p can open the hairpin of DNA and forms an miR-92a-3p/DNA heteroduplex, triggering the specific cleavage of DSN for the DNA in the heteroduplex. As a result, Atto-425 leaves the fluorescent Au NPs and recovers the fluorescence emission. The released miR-92a-3p can hybridize with another hairpin DNA and lead to a stronger fluorescence recovery of Atto-425 to form a signal amplification cycle. The stable fluorescence of Au NPs and the changing fluorescence of Atto-425 constitute a ratiometric fluorescent system reflecting the concentration of miR-92a-3p. This biosensor exhibits excellent specificity and can distinguish CRC patients from healthy individuals by detecting miR-92a-3p extracted from clinical exosome samples, showing the potential in CRC diagnosis.

Dancing in local space: rolling hoop orbital amplification combined with local cascade nanozyme catalytic system to achieve ultra-sensitive detection of exosomal miRNA

The exosomal miRNA (exo-miRNA) derived from tumor cells contains rich biological information that can effectively aid in the early diagnosis of disease. However, the extremely low abundance imposes stringent requirements for accurate detection techniques. In this study, a novel, protease-free DNA amplification strategy, known as “Rolling Hoop Orbital Amplification” (RHOA), was initially developed based on the design concept of local reaction and inspired by the childhood game of rolling iron ring. Benefiting from the local space constructed by the DNA orbital, the circular DNA enzyme rolls directionally and interacts efficiently with the amplification element, making it nearly 3-fold more productive than conventional free-diffusion amplification. Similarly, the localized cascade nanozyme catalytic system formed by bridging DNA probes also exhibits outperformed than free ones. Therefore, a localized energized high-performance electrochemiluminescence (ECL) biosensor was constructed by bridging cascading nanozymes on the electrode surface through DNA probes generated by RHOA, with an impressive limit of detection (LOD) of 1.5 aM for the detection of exosomal miRNA15a-5p and a stable linearity over a wide concentration range from 10− 2 to 108 fM. Thus, this work is a focused attempt at the localized reaction, which is expected to provide a reliable method for accurately detecting of exo-miRNAs.Graphical

A ratiometric fluorescent biosensor based on self-fluorescent MOF and target-triggered rolling circle amplification for sensitive detection of exosome-derived miRNA

MicroRNAs (miRNAs) are considered as biomarkers and display great potential in the diagnosis of diseases. As a colorectal cancer (CRC)-associated miRNA biomarker, miR-92a-3p possesses higher concentration in exosomes than in serum, causing it more feasible to diagnose CRC by measuring the concentration of miR-92a-3p inside exosomes. Herein, a rationally-engineered ratiometric fluorescent biosensor is proposed to detect the concentration of exosomal miR-92a-3p. The metal-organic framework (MOF-525) with self-fluorescence serves as both a fluorescent reference and a quencher of the fluorescence of single-stranded reporter by adsorption. The presence of miR-92a-3p triggers the looping of 5P-template strand and the further periodic rolling circle amplification. The periodic long strands and the reporters can form double strands to prevent the reporters from being adsorbed by MOF-525. The concentration of miR-92a-3p is positively correlated with the Δreporter/MOF-525 fluorescence intensity ratio. The biosensor exhibits a detection range of 0.1–10 pM and can differentiate miR-92a-3p from mismatched RNA sequences. The accuracy and practicality of the proposed biosensor for exosomal miRNA detection were evaluated by comparing with the conventional RT-qPCR. The as-obtained results are close to those of the RT-qPCR. This ratiometric fluorescent biosensor holds the potential for the sensitive detection of exosomal miRNA.

Construction of simple and sensitive pancreatitis related microRNA detection strategy via self-priming triggered cascade signal amplification.

Pancreatic diseases, such as pancreatitis and pancreatic cancer, remain the most threatening gastrointestinal diseases with a high mortality due to atypical symptoms. MicroRNA plays crucial roles in regulating metastasis and cell proliferation of pancreatic cancer, constituting important biomarkers for the early diagnosis of pancreatic cancers. Herein, we develop a sensitive and simple exosomal miRNA detection method with only a dual-hairpin-probe. In detail, the dual-hairpin-probe is constructed through combination of two functional sections for both target miRNA identification and signal amplification. With only one probe, the method possesses the capability to avoid interferences from concentration changes of other probes, and exhibits a higher stability which is demonstrated through the obtained low coefficients of variation (CV) of 6.73%. With let-7a as detection target, the LOD of the established method is determined to be 243 aM, while maintaining a high discriminating capability towards let-7a homogenous miRNAs.

Ultrasensitive detection of exosomal miRNA with PMO-graphene quantum dots-functionalized field-effect transistor biosensor

SummaryCompared with the conventional DNA probe immobilization on the planar surface, nanoparticles-based DNA probes enable more RNA molecules to be anchored to the sensor surface, thereby improving the detection sensitivity. In this work, we report phosphorodiamidate morpholino oligomers (PMO)-graphene quantum dots (GQDs)-functionalized reduced graphene oxide (RGO) field effect transistor (FET) biosensors for ultrasensitive detection of exosomal microRNAs. After the RGO FET sensor was fabricated, polylysine (PLL) film was deposited onto the RGO surface. GQDs-PMO hybrid was prepared and covalently bound to PLL surface, enabling detection of exosomal microRNAs (miRNAs). The method achieved a detection limit as low as 85 aM and high specificity. Furthermore, the FET sensor was able to detect exosomal miRNAs in plasma samples and distinguish breast cancer samples from healthy samples. Compared with other methods, we use GQDs to further improve the sensitivity of FET, making it a potential tool for early diagnosis of breast cancer.

DNA barcode-based detection of exosomal microRNAs using nucleic acid lateral flow assays for the diagnosis of colorectal cancer.

Colorectal cancer (CRC) is the third most common cancer and the second leading cause of cancer-related deaths worldwide. The standard methods for diagnosing CRC, endoscopy and tissue biopsy, are invasive and time-consuming. Herein, we propose a novel method for the accurate and non-invasive diagnosis of CRC based on the analysis of exosomes that are circulating in biological fluids using a DNA barcode-based nucleic acid lateral flow assay (NALFA). Our technology combines reverse transcription using a stem-loop primer with DNA barcode-based NALFA. A colorimetric signal is generated only in the presence of the target exosomal miRNA, which can be determined even with the naked eye. The proposed system successfully detected miR-92a and miR-141, which are overexpressed in CRC exosomes. Moreover, when applied to plasma samples from CRC patients, our system simultaneously detected multiple markers in one strip. By combining these markers, we achieved high analytical performance with a sensitivity and a specificity of 95.24% and 100.0%, respectively, demonstrating that the proposed assay can be a simple diagnostic platform for the detection of exosomal miRNA.

Three-dimensional hierarchical plasmonic nano-architecture based label-free surface-enhanced Raman spectroscopy detection of urinary exosomal miRNA for clinical diagnosis of prostate cancer

The urinary exosomal miRNAs are recently emerging prostate cancer (PC)-associated biomarkers for the early-stage diagnosis and prognosis due to their non-invasiveness, inherent stability and the representation of the status of the originated cells. However, developing a urinary exosomal miRNA detection method with high accuracy is challenging because of the low abundance and high sequence homology of miRNAs. Herein, we present a quantitative and label-free miRNA sensing platform using surface-enhanced Raman scattering (SERS) based on three-dimensional (3D) hierarchical plasmonic nano-architecture to detect urinary exosomal miRNAs. This hierarchical nanostructure is constructed by self-assembly between target-complementary DNA probes-conjugated gold nanoparticles and head-flocked gold nanopillars in the presence of the target miRNAs, creating numerous 3D plasmonic hot-spots inducing exceedingly high amplification of SERS signals. This 3D SERS biosensor achieved ∼10 aM detection limits for the target miRNAs (miR-10a and miR-21), which is over 1000-fold more sensitive than previously reported miRNA sensors without the requirement of any labelling or pre-treatment steps. Finally, the clinical validation using urinary samples revealed that our 3D SERS sensor discriminates PC patients from healthy control with high diagnostic accuracy (0.93) based on the differential expression level of urinary exosomal miRNAs. These outputs demonstrate that our SERS sensor based on 3D hierarchical nano-architecture can offer facile, accurate and rapid methods to measure miRNA expression and is helpful for the diagnosis of various diseases.

Cubic DNA nanocage-based three-dimensional molecular beacon for accurate detection of exosomal miRNAs in confined spaces

In situ nondestructive bioanalysis of targets in nanoscale confined space, e.g. exosomes, poses a high challenge to analytical technologies, especially to molecular fluorescent probes, because it is required to enter the confined space to recognize the target, and maintain independent and stable signal output. The unexpected fluorescence quenching and fluorescence resonance energy transfer (FRET) caused by high-frequency Brownian motion and collision in confined space are the main limiting factors. Herein, we constructed a well-defined and programmable cubic DNA nanocage-based three-dimensional molecular beacon (ncMB), which successfully broke through the above dilemma, and realized the detection of miRNA in exosomes. Specifically, steric hindrance and electrostatic repulsion derived from the unique three-dimensional structure of ncMB result in a barrier between fluorescent probes, thus eliminating unexpected fluorescence quenching during single exosomal miRNA detection and unexpected FRET during dual exosomal miRNA detection. Benefiting from the excellent anti-fluorescence and anti-FRET performance of ncMB, compared with traditional molecular beacons (MB), the detected fluorescence signal in exosomes can be improved by an order of magnitude. Moreover, ncMB is proven to have powerful programmability and anti-interference capability. Overall, it is believed that the ncMB can eliminate the signal distortion that was usually associated with commonly used MB, especially in the confined space. The ncMB is considered as a powerful and versatile tool for accurate in situ signal output in exosomes and maybe other confined spaces.

Photoelectrochemical Detection of Exosomal miRNAs by Combining Target-Programmed Controllable Signal Quenching Engineering.

MicroRNAs extracted from exosomes (exosomal miRNAs) have recently emerged as promising biomarkers for early prognosis and diagnosis. Thus, the development of an effective approach for exosomal miRNA monitoring has triggered extensive attention. Herein, a sensitive photoelectrochemical (PEC) biosensing platform is demonstrated for exosomal miRNA assay via the target miRNA-powered λ-exonuclease for the amplification strategy. The metal-organic framework (MOF)-decorated WO3 nanoflakes heterostructure is constructed and implemented as the photoelectrode. Also, a target exosomal miRNA-activatable programmed release nanocarrier was fabricated, which is responsible for signal control. Hemin that acted as the electron acceptor was prior entrapped into the programmed control release nanocarriers. Once the target exosomal miRNAs-21 was introduced, the as-prepared programmed release nanocarriers were initiated to trigger the release of hemin, which enabled the quenching of the photocurrent. Under the optimized conditions, the level of exosomal miRNAs-21 could be accurately tracked ranging from 1 fM to 0.1 μM with a low detection limit of 0.5 fM. The discoveries illustrate the possibility for the rapid and efficient diagnosis and prognosis prediction of diseases based on the detection of exosomal miRNAs-21 and would provide feasible approaches for the fabrication of an efficient platform for clinical applications.

Spatial Confinement-Derived Double-Accelerated DNA Cascade Reaction for Ultrafast and Highly Sensitive In Situ Monitoring of Exosomal miRNA and Exosome Tracing.

Exosomal microRNAs (miRNAs) are reliable biomarkers of disease progression, allowing for non-invasive detection. However, detection of exosomal miRNAs in situ remains a challenge due to low abundance, poor permeability of the lipid bilayers, and slow kinetics of previous methods. Herein, an accelerated DNA nanoprobe was implemented for fast, in situ monitoring of miRNA in exosomes by employing a spatial confinement strategy. This nanoprobe not only detects miRNA in exosomes but also distinguishes tumor exosomes from those derived from normal cells with high accuracy, paving the way toward exosomal miRNA bioimaging and disease diagnosis. Furthermore, the fast response allows for this nanoprobe to be successfully utilized to monitor the process of exosomes endocytosis, making it also a tool to explore exosome biological functions.

Detection of multiplex exosomal miRNAs for clinically accurate diagnosis of Alzheimer’s disease using label-free plasmonic biosensor based on DNA-Assembled advanced plasmonic architecture

Alzheimer’s disease (AD), the most common neurologic disorder, is characterized by progressive cognitive impairment. However, the low clinical significance of the currently used core AD biomarkers amyloid-beta and tau proteins remains a challenge. Recently, exosomes, found in human biological fluids, are gaining increasing attention because of their clinical significance in diagnosing of various diseases. In particular, blood-derived exosomal miRNAs are not only stable but also provide information regarding the different characteristics according to AD progression. However, quantitative and qualitative detection is difficult due to their characteristics, such as small size, low abundance, and high homology. Here, we present a DNA-assembled advanced plasmonic architecture (DAPA)-based plasmonic biosensor to accurately detect exosomal miRNAs in human serum. The designed nanoarchitecture possesses two narrow nanogaps that induce plasmon coupling; this significantly enhances its optical energy density, resulting in a 1.66-fold higher refractive-index (RI) sensitivity than nanorods at localized surface plasmon resonance (LSPR). Thus, the proposed biosensor is ultrasensitive and capable of selective single-nucleotide detection of exosomal miRNAs at the attomolar level. Furthermore, it identified AD patients from healthy controls by measuring the levels of exosomal miRNA-125b, miRNA-15a, and miRNA-361 in clinical serum samples. In particular, the combination of exosomal miRNA-125b and miRNA-361 showed the best diagnostic performance with a sensitivity of 91.67%, selectivity of 95.00%, and accuracy of 99.52%. These results demonstrate that our sensor can be clinically applied for AD diagnosis and has great potential to revolutionize the field of dementia research and treatment in the future.

Simultaneous and multiplex detection of exosomal microRNAs based on the asymmetric Au@Au@Ag probes with enhanced Raman signal

Simultaneous and quantitative detection of multiple exosomal microRNAs (miRNAs) was successfully performed by a surface-enhanced Raman scattering (SERS) assay consisting of Raman probes and capture probes. In this design, the asymmetric core-shell structured Au@Au@Ag nanoparticles were first synthesized by layer-by-layer self-assembly method and modified with different Raman molecules and recognition sequences (polyA-DNA) to prepare the surface-enhanced Raman probes. Then, the streptavidin-modified magnetic beads were used to immobilize the biotinylated DNA capture sequences (biotin-DNA) to obtain capture probes. In the presence of target exosomal miRNAs, the Raman probes and capture probes could bind to the target exosomal miRNAs in the partial hybridization manner. Thus, the developed SERS sensor could indicate the target miRNAs levels in the buffer solution. Using breast cancer-related miRNAs as model targets, the limits of detection of this sensor were determined to be 1.076 fmol/L for synthetic miR-21, 0.068 fmol/L for synthetic miR-126, and 4.57 fmol/L for synthetic miR-1246, respectively. Such SERS sensors were further employed to detect the miR-21 in 20% human serum and the extraction solution of exosomes, respectively. Therefore, simultaneous and multiplex detection of cancer-related exosomal miRNAs by this assay could provide new opportunities for further biomedical applications.

Carbon Nanotube Field-Effect Transistor Biosensor for Ultrasensitive and Label-Free Detection of Breast Cancer Exosomal miRNA21.

Tumor-derived exosomal miRNAs may have important functions in the onset and progression of cancers and are potential biomarkers for early diagnosis and prognosis monitoring. Yet, simple, sensitive, and label-free detection of exosomal miRNAs remains challenging. Herein, an ultrasensitive, label-free, and stable field-effect transistor (FET) biosensor based on a polymer-sorted high-purity semiconducting carbon nanotube (CNT) film is reported to detect exosomal miRNA. Different from conventional CNT FETs, the CNT FET biosensors employed a floating gate structure using an ultrathin Y2O3 as an insulating layer, and assembled Au nanoparticles (AuNPs) on Y2O3 as linkers to anchor probe molecules. A thiolated oligonucleotide probe was immobilized on the AuNP surface of the sensing area, after which miRNA21 was detectable by monitoring the current change before and after hybridization between the immobilized DNA probe and target miRNA. This method achieved both high sensitivity (LOD: 0.87 aM) and high specificity. Furthermore, the FET biosensor was employed to test clinical plasma samples, showing significant differences between healthy people and breast cancer patients. The CNT FET biosensor shows the potential applications in the clinical diagnosis of breast cancer.

A portable system for isothermal amplification and detection of exosomal microRNAs

Exosomal microRNAs (miRNAs) play a key role in cell-cell communication to regulate gene expression in target cells and have great potential as biomarkers for disease diagnosis. This paper reports an on-chip exosomal miRNA amplification and detection system for rapid analysis of exosomal miRNAs. The compact system consists of two connected flow cells for processing exosomes and detecting miRNAs, respectively. The miRNAs extracted from exosomes were quantitatively measured using the on-chip exponential amplification reaction (EXPAR) assay. The sensor chip was designed to store multiple oligonucleotide templates for the EXPAR, mix sample and reagent, and simultaneously analyze multiple exosomal miRNAs of interest. To facilitate the miRNA analysis, a portable detection instrument was built on an IoT platform using a low-cost microcontroller to execute the EXPAR assay, collect fluorescent images, and analyze amplification curves. Here, we studied the miRNA profiles carried by exosomes derived from three different phenotypes of tissue macrophages. The affordable instrument, rapid assay, multiplexed analysis, as well as disposable sensor chip, would boost the development of point-of-care liquid biopsy tests using exosomal miRNAs.

A microfluidic surface-enhanced Raman scattering (SERS) sensor for microRNA in extracellular vesicles with nucleic acid-tyramine cascade amplification

Exosomal microRNA (miRNA) is an ideal candidate of noninvasive biomarker for the early diagnosis of cancer. Sensitive and accurate sensing of abnormal exosomal miRNA plays essential role for clinical promotion due to its close correlation with tumor proliferation and progression. Herein, a microfluidic surface-enhanced Raman scattering (SERS) sensor was proposed for an on-line detection of exosomal miRNA based on rolling circle amplification (RCA) and tyramine signal amplification (TSA) strategy. The microfluidic chip consists of a magnetic enrichment chamber, a serpentine fluidic mixer and a plasmonic SERS substrate functionalized with capture probes. The released miRNA activates the capture probe, triggers RCA reaction, and generates a large number of single-stranded DNA products to drive the catalysis of nanotags deposition via TSA, producing numerous “hot spots” to enhance the SERS signals. In merit of the microfluidics chip and nucleic acid-tyramine cascade amplification, the developed SERS sensor significantly improves the sensitivity for the exosomal miRNA assay, resulting in a limit of detection (LOD) as low as 1 pmol/L and can be successfully applied in the analysis of exosomes secreted from breast tumor cells, which demonstrates the potential utility in practical applications.

Peptide Nucleic Acid-Functionalized Nanochannel Biosensor for the Highly Sensitive Detection of Tumor Exosomal MicroRNA.

Compared with free miRNAs in blood, miRNAs in exosomes have higher abundance and stability. Therefore, miRNAs in exosomes can be regarded as an ideal tumor marker for early cancer diagnosis. Here, a peptide nucleic acid (PNA)-functionalized nanochannel biosensor for the ultrasensitive and specific detection of tumor exosomal miRNAs is proposed. After PNA was covalently bound to the inner surface of the nanochannels, the detection of tumor exosomal miRNAs was achieved by the charge changes on the surface of nanochannels before and after hybridization (PNA-miRNA). Due to the neutral characteristics of PNA, the efficiency of PNA-miRNA hybridization was improved by significantly reducing the background signal. This biosensor could not only specifically distinguish target miRNA-10b from single-base mismatched miRNA but also achieve a detection limit as low as 75 aM. Moreover, the biosensor was further used to detect exosomal miRNA-10b derived from pancreatic cancer cells and normal pancreatic cells. The results indicate that this biosensor could effectively distinguish pancreatic cancer tumor-derived exosomes from the normal control group, and the detection results show good consistency with those of the quantitative reverse-transcription polymerase chain reaction method. In addition, the biosensor was used to detect exosomal miRNA-10b in clinical plasma samples, and it was found that the content of exosomal miRNA-10b in cancer patients was generally higher than that of healthy individuals, proving that the method is expected to be applied for the early diagnosis of cancer.

Hydrogel-based hybridization chain reaction (HCR) for detection of urinary exosomal miRNAs as a diagnostic tool of prostate cancer

Although urinary exosomal microRNAs (miRNAs) have recently emerged as potential biomarkers, clinical applications are still limited due to their low concentration in small volumes of clinical samples. Therefore, the development of a non-invasive, specific diagnostic tool, along with profiling exosomal miRNA markers from urine, remains a significant challenge. Here, we present hydrogel-based hybridization chain reaction (HCR) for multiplex signal amplification to detect urinary exosomal miRNAs from human clinical samples. We succeeded in identifying small amounts (~amol) of exosomal miRNAs from 600 μL of urine with up to ~35-fold amplification and enhanced detection limits by over an order of magnitude for two miRNA biomarker candidates, hsa-miR-6090 and hsa-miR-3665. Furthermore, we proposed ratiometric analysis without requiring normalization to a reference miRNA and validated the clinical diagnostic potential toward differentiating prostate cancer patients from healthy controls. Our hydrogel-based HCR could serve as a new diagnostic platform for a non-invasive liquid biopsy before burdensome tissue biopsy of various diseases, including prostate cancer screening, complementing the PSA test.

An electrochemical biosensor based on DNA “nano-bridge” for amplified detection of exosomal microRNAs

Exosomal miRNAs, as potential biomarkers in liquid biopsy for cancer early diagnosis, have aroused widespread concern. Herein, an electrochemical biosensor based on DNA “nano-bridge” was designed and applied to detect exosomal microRNA-21 (miR-21) derived from breast cancer cells. In brief, the target miR-21 can specifically open the hairpin probe 1(HP1) labeled on the gold electrode (GE) surface through strand displacement reaction. Thus the exposed loop region of HP1 can act as an initiator sequence to activate the hybridization chain reaction (HCR) between two kinetically trapped hairpin probes: HP2 immobilized on the GE surface and biotin labeled HP3 in solution. Cascade HCR leads to the formation of DNA “nano-bridge” tethered to the GE surface with a great deal of “piers”. Upon addition of avidin-modified horseradish peroxidase (HRP), numerous HRP were bound to the formed “nano-bridge” through biotin-avidin interaction to arouse tremendous current signal. In theory, only a single miR-21 is able to trigger the continuous HCR between HP2 and HP3 until all of the HP2 are exhausted. Therefore the proposed biosensor achieved ultrahigh sensitivity toward miR-21 with the detection limit down to 168 amol/L, as well as little cross-hybridization even at the single-base-mismatched level. Successful attempts were also made in the detection of exosomal miR-21 obtained from the MCF-7 of breast cancer cell line. To our knowledge, this is the first attempt to built horizontal DNA nano-structure on the electrode surface for exosomal miRNAs detection. In a word, the high sensitivity, selectivity, low cost make the proposed method hold great potential application for early point-of-care (POC) diagnostics of cancer.