Multiplexed Detection Of miRNAs Research Articles

An enzyme-free and label-free multiplex detection of miRNAs by entropy-driven circuit coupled with capillary electrophoresis

MicroRNAs (miRNAs) are currently recognized as important biomarkers for the early diagnosis and prognostic treatment of cancer. Herein, we developed a simple and label-free method for the multiplex detection of miRNAs, based on entropy-driven circuit (EDC) amplification and non-gel sieving capillary electrophoresis-LED induced fluorescence detection (NGCE-LEDIF) platform. In this system, three different lengths of fuel chains were designed to catalyze three EDC, targeting miRNA-21, miRNA-155, and miRNA-10b, respectively. In the presence of target miRNA, the EDC cycle amplification reaction was triggered, generating numerous stable double-strands products (F-DNA/L-DNA). Since the three miRNAs correspond to three different lengths of F-DNA/L-DNA, they can be easily isolated and detected by NGCE. This strategy has good sensitivity, with detection limits of 68 amol, 292.2 amol, and 394 amol for miRNA-21, miRNA-155, and miRNA-10b, respectively. Additionally, this method has good specificity and can effectively distinguish single-base mismatches of miRNA. The recoveries of the three miRNAs in deproteinized healthy human serum ranged from 91.28 % to 108.4 %, with a relative standard deviation (RSD) of less than 7.9 %. This method was further applied to detect cellular miRNAs in human breast cancer (MCF-7) cell extracts, revealing an up-regulation of miRNA-21, miRNA-155, and miRNA-10b in MCF-7 cells. The successful spiked recovery in human serum and RNA extraction from MCF-7 cells underscores the practicality of this method. Therefore, this strategy has broad application prospects in biomedical research.

Amplification-Free Analysis of Bladder Cancer MicroRNAs on Wrinkled Silica Nanoparticles with DNA-Functionalized Quantum Dots.

Bladder cancer (BC) occurrence and progression are accompanied by alterations in microRNAs (miRNAs) expression levels. Simultaneous detection of multiple miRNAs contributes to the accuracy and reliability of the BC diagnosis. In this work, wrinkled silica nanoparticles (WSNs) were applied as the microreactor for multiplex miRNAs analysis without enzymes or nucleic acid amplification. Conjugated on the surface of WSNs, the S9.6 antibody was adopted as the universal module for binding DNA/miRNA duplexes, regardless of their sequence. Furthermore, single-stranded DNA (ssDNA) was labeled with quantum dots (QDs) for identifying a given miRNA to form QDs-ssDNA/miRNA, which enabled the specific capture of the corresponding QDs on the wrinkled surface of WSNs. Based on the detection of fluorescence signals that were ultimately focused on WSNs, target miRNAs could be sensitively identified to a femtomolar level (5 fM) with a wide dynamic range of up to 6 orders of magnitude. The proposed strategy achieved high specificity to obviously distinguish single-base mutation sequences and possessed multiplex assay capability. Moreover, the assay exhibited excellent practicability in the multiplex detection of miRNAs in clinical serum specimens.

Flexible-Arranged Biomimetic Array Integrated with Parallel Entropy-Driven Circuits for Ultrasensitive, Multiple, and Reliable Detection of Cancer-Related MicroRNAs.

With the emergence of microRNA (miRNA) as a promising biomarker in cancer diagnosis, it is significant to develop multiple analyses of miRNAs. However, it still faces difficulties in ensuring the sensitivity and accuracy during multiplex detection owing to the low abundance and experimental deviation of miRNAs. In this work, a flexible-arranged biomimetic array integrated with parallel entropy-driven circuits (EDCs) was developed for ultrasensitive, multiplex, reliable, and high-throughput detection of miRNAs. The biomimetic array was fabricated by arrangement of various photonic crystals (PCs) for adjustable photonic band gaps (PBGs) and specific fluorescence enhancement. Meanwhile, two cancer-related miRNAs and one reference miRNA were introduced as multiple analytes as a proof-of-concept. The parallel EDCs with negligible crosstalk were designed based on the modular property. Because of the one-to-one match between the emitted fluorescence of parallel EDCs and the PBGs of the flexible-arranged biomimetic array, the generated fluorescence signal triggered by target miRNAs can be enhanced on the corresponding domain of the array. Furthermore, the amplified signal of the array was detected with high-throughput scanning, which could reveal specific information on cancer-related miRNAs as well as reference miRNA, enhancing the abundance and reliability of the analysis. The proposed array has the merits of a modular design, flexible deployment, simple operation (nonenzymatic and isothermal), improved accuracy, high sensitivity, and multiplex analysis, showing potential in disease diagnosis.

Programmable DNA barcode-encoded exponential amplification reaction for the multiplex detection of miRNAs.

Multiple analysis of miRNAs is essential for the early diagnosis and monitoring of diseases. Here, a programmable, multiplex, and sensitive approach was developed for one-pot detection of miRNAs by melting temperature encoded sequences and exponential isothermal amplification (E-EXPAR). In the presence of target miRNAs, the corresponding templates initiate the cycles of nicking and polymerization/displacement, generating numerous barcode strands with unique encoding sequences. Subsequently, generated barcode strands hybridize with fluorescent probes and quench the fluorophore by a triplet of G base through a photo-induced electron transfer mechanism. Finally, a melting curve analysis is performed to quantify miRNAs by calculating the rate of fluorescence change at the corresponding melting temperature. Based on this, miRNA-21, miRNA-9, and miRNA-122 were detected with the detection limits of 3.3 fM, 2.9 fM, and 1.7 fM, respectively. This E-EXPAR was also employed to simultaneously detect three miRNAs in biological samples, showing consistent results with RT-qPCR. Overall, this study provides a programmable and universal platform for multiplex analysis of miRNAs, and holds great promise as an alternative to the multiplex analysis in clinical diagnostics and prognostics for nucleic acid detection.

An ultrasensitive and multiplexed miRNA one-step real time RT-qPCR detection system and its application in esophageal cancer serum

MicroRNAs (miRNAs) are increasingly recognized as promising biomarkers for early disease diagnosis and prognosis. Therefore, the need for rapid, robust methods for multiplex miRNA detection in biological research and clinical diagnosis is crucial. This study introduces a novel multiplex miRNA detection method, SMOS-qPCR (Sensitive and Multiplexed One-Step RT-qPCR). The method integrates multiplexed reverse transcription and TaqMan-based qPCR into a single tube, employing a one-step operation on a real-time PCR system. We investigated the effect of 3′ end phosphorylation of the Linker, Linker concentration and probe concentration on the SMOS-qPCR, resulted in a wide linear range from 1 fM to 0.1 zM (R2 ≥ 0.99 for each miRNA), surpassing the capabilities of stem-loop RT-qPCR and SYBR Green One-step RT-qPCR. The method showed excellent performance in distinguishing mature miRNA from miRNA precursor, and successfully detected four miRNAs in a single tube without cross-interference. Its high specificity enables precise differentiation of less than 1% nonspecific signal. Finally, we demonstrated the effectiveness of the SMOS-qPCR system in detecting circulating miRNAs in serum samples, distinguishing between esophageal cancers and health individuals with high AUC values (>0.940). In conclusion, the proposed SMOS-qPCR system offers a straightforward and promising approach for miRNA profiling in future clinical applications.

Stem-loop RT-qPCR system for multiplex miRNA profiling and its application in wound healing-specific biomarker identification

MiRNAs are biomarkers widely used in research but their clinical application is still challenging due to their low expression levels. Current methods for miRNA detection involve separate transcription and quantification for each target, which is costly and unsuitable for large sample sizes. This study provides a strategy for designing and screening miRNA-specific stem-loop reverse transcription (RT) primers, which enable the simultaneous transcription of three miRNAs and U6, and the concurrent detection of miRNA and U6 in the same transcript using TaqMan probes labeled with different dyes. The strategy was successfully employed to establish multiplex RT-PCR and dual-quantitative PCR (qPCR) quantification systems for 21 differentially expressed miRNAs during wound healing. The corresponding system can accurately quantify the cell culture samples containing miR-7a-5p mimic, miR-7a-5p inhibitor, or negative control. In summary, our results demonstrate that this strategy could efficiently accomplish the design, screening, and analysis of stem-loop RT primers for multiplex miRNA detection. Compared with the commercially customized miRNA assay kits, our system showed a higher degree of automation, more accurate qPCR assay capabilities, and lower assay costs, which could provide practical value for clinical diagnosis.

Multiplex metal enhanced fluorescence (MEF) effect on porous Au nanorod for highly sensitive multi-microRNA (miRNA) detection

MicroRNA (miRNA) is an important biomarker for early diagnosis or progression of a disease because the type and amount of its release can vary depending on the type or condition of the cell. However, since microRNAs exist at low concentrations in the body with several microRNAs released, it is necessary to develop a biosensor that can sensitively measure multiple miRNAs. In this study, a metal-enhanced fluorescence (MEF)-based highly sensitive nanobiosensor for measuring multiple miRNAs was developed. To extend the plasmonic-based MEF phenomenon to fluorescent materials with various emission wavelengths, porous gold (Au) nanorods with a wide absorption wavelength were synthesized. Using this, two different miRNAs were simultaneously measured sensitively and selectively. The proposed sensing method is a highly sensitive, rapid, simple, and one-step way for multiplex detection of miRNAs. It can be used for early diagnosis of cancer and other nucleic acid-related diseases.

Multiplexed and accurate quantification strategy for miRNA based on specific terminal-mediated PCR with equivalent amplification.

MicroRNAs (miRNAs) are recognized as potential biomarkers for the early diagnosis and prognosis of different diseases. Multiplexed and accurate miRNA quantification methods with equivalent detection efficiency are particularly crucial due to their complex biological functions and lack of a unified internal reference gene. Here, a unique multiplexed miRNA detection method, named Specific Terminal-Mediated miRNA PCR (STEM-Mi-PCR), was developed. It mainly includes a linear reverse transcription step using tailored-designed target specific capture primers, followed by an exponential amplification process using two universal primers to execute the multiplex assay. For proof of concept, four miRNAs were used as models to develop a multiplexed detection assay within one tube simultaneously and then evaluate the performance of the established STEM-Mi-PCR. The sensitivity of the 4-plexed assay was approximately 100 aM with an equivalent amplification efficiency (95.67±8.58%), and had no cross-reactivity each other with high specificity. Quantification of different miRNAs in twenty patients' tissues shown variation from approximately pM to fM concentration level, demonstrating the possibility of practical application of the established method. Moreover, this method was extraordinarily capable of single nucleotide mutation discrimination in different let-7 family members with no more than 0.7% nonspecific detection signal. Hence, the STEM-Mi-PCR we proposed here paves an easy and promising way for miRNA profiling in future clinical applications.

Encoded Fusion-Mediated MicroRNA Signature Profiling of Tumor-Derived Extracellular Vesicles for Pancreatic Cancer Diagnosis.

MicroRNAs (miRNAs) in tumor-derived extracellular vesicles (tEVs) are important cancer biomarkers for cancer screening and early diagnosis. Multiplex detection of miRNAs in tEVs facilitates accurate diagnosis but remains a challenge. Herein, we propose an encoded fusion strategy to profile the miRNA signature in tEVs for pancreatic cancer diagnosis. A panel of encoded-targeted-fusion beads was fabricated for the selective recognition and fusion of tEVs, with the turn-on fluorescence signals of molecule beacons for miRNA quantification and barcode signals for miRNA identification using readily accessible flow cytometers. Using this strategy, six types of pancreatic-cancer-associated miRNAs can be profiled in tEVs from 2 μL plasma samples (n = 36) in an isolation-free and lysis-free manner with only 2 h of processing, offering a high accuracy (98%) to discriminate pancreatic cancer, pancreatitis, and healthy donors. This encoded fusion strategy exhibits great potential for multiplex profiling of miRNA in tEVs, offering new avenues for cancer diagnosis and screening.

Signal differentiation models for multiple microRNA detection: a critical review.

MicroRNAs (miRNAs) are a class of small, single-stranded non-coding RNAs which have critical functions in various biological processes. Increasing evidence suggested that abnormal miRNA expression was closely related to many human diseases, and they are projected to be very promising biomarkers for non-invasive diagnosis. Multiplex detection of aberrant miRNAs has great advantages including improved detection efficiency and enhanced diagnostic precision. Traditional miRNA detection methods do not meet the requirements of high sensitivity or multiplexing. Some new techniques have opened novel paths to solve analytical challenges of multiple miRNA detection. Herein, we give a critical overview of the current multiplex strategies for the simultaneous detection of miRNAs from the perspective of two different signal differentiation models, including label differentiation and space differentiation. Meanwhile, recent advances of signal amplification strategies integrated into multiplex miRNA methods are also discussed. We hope this review provides the reader with future perspectives on multiplex miRNA strategies in biochemical research and clinical diagnostics.

Self-Generation of Distinguishable Fluorescent Probes via a One-Pot Process for Multiple MicroRNA Detection by Liquid Chromatography.

To address the challenge of signal production and separation for multiple microRNA (miRNA) detection, in this work, a "one-pot" process to self-generate distinguishable fluorescent probes was developed. Based on a long and short probe amplification strategy, the generated G-quadruplex fluorescent dye-free probes can be separated and detected by a high-performance liquid chromatography-fluorescence platform. The free hairpin probes enriched in guanine with different lengths and base sequences were designed and could be opened by the target miRNAs (miRNA-10b, miRNA-21, and miRNA-210). Cleaved G-quadruplex probes with fluorescent signal could be generated in a one-pot process after a duplex-specific nuclease-based cleavage, and the detection of multiple miRNAs could be realized in one run. No solid nanomaterials were applied in the assay, which avoided the blocking of the column. Moreover, without modification of expensive fluorescein, the experimental cost was greatly reduced. The one-pot reaction process also eliminated tedious preparation steps and suggested feasibility of automation. The limits of detection of miRNA-10b, miRNA-21, and miRNA-210 were 2.19, 2.20, and 2.75 fM, respectively. Notably, this method was successfully applied to multiplex detection of miRNAs in serum samples from breast cancer patients within 30 min.

Miniature Hierarchical DNA Hybridization Circuit for Amplified Multiplexed MicroRNA Imaging.

Accurate diagnosis requires the development of multiple-guaranteed DNA circuits. Nevertheless, for reliable multiplexed molecular imaging, existing DNA circuits are limited by poor cell-delivering homogeneity due to their cumbersome and dispersive reactants. Herein, we developed a compact-yet-efficient hierarchical DNA hybridization (HDH) circuit for in situ simultaneous analysis of multiple miRNAs, which could be further exploited for specifically discriminating cancer cells from normal ones. By integrating the traditional hybridization chain reaction and catalytic hairpin assembly reactants into two highly organized hairpins, the HDH circuit is fitted with condensed components and multiple response domains, thus permitting the programmable multiple microRNA-guaranteed sequential activations and the localized cascaded signal amplification. The synergistic multi-recognition and amplification features of the HDH circuit facilitate the magnified detection of multiplex endogenous miRNAs in living cells. The in vitro and cellular imaging experimental results revealed that the HDH circuit displayed a reliable sensing performance with high selective cell-identification capacity. We anticipate that this compact design can provide a powerful toolkit for accurate diagnostics and pathological evolution.

Detection of MicroRNA Expression Dynamics Using LNA/DNA Nanobiosensor.

The investigation of complex biological processes requires effective tools for probing the spatiotemporal dynamics of individual cells. Single-cell gene expression analysis, such as RNA in situ hybridization and single-cell PCR, has been demonstrated in various biological applications (Tautz and Pfeifle, Chromosoma 98(2):81-5, 1989; Stahlberg and Bengtsson, Methods 50(4):282-288, 2010; Sanchez-Freire et al., Nat Protoc 7(5):829-838, 2012). However, existing techniques require cell lysis or fixation. The dynamic information and spatiotemporal regulation of the biological process cannot be obtained with these methods. Real-time gene expression analysis in living cells remains an outstanding challenge in the field. Here, we described a single-cell gene expression analysis method in living mammalian cells using a locked nucleic acid/DNA (LNA/DNA) nanobiosensor. This LNA/DNA nanobiosensor consists of a fluorophore-labeled detecting strand and a quenching strand. The fluorophore-labeled LNA probe is designed to hybridize with the target microRNA (miRNA) specifically and displace from the quenching strand, allowing the fluorophore to fluorescence. Large-scale single-cell dynamic gene expression monitoring can be performed using time-lapse microscopy to study spatiotemporal distribution and heterogeneity in gene expression. Multiplex detection of miRNAs can be achieved using different fluorophore-labeled LNA/DNA nanobiosensors. This LNA/DNA protocol is fast, generally applicable, and easily accessible.

Ultrasensitive and multiplexed miRNA detection system with DNA-PAINT

MiRNAs hold great potential as biomarkers for the early detection and monitoring of diseases based on their differential expression profiles. Therefore, the sensitive, specific and accurate detection of miRNAs represents an emerging new tool to improve diagnosis and treatment of several diseases, cancer in particular. DNA origami-based miRNA detection is particularly advantageous as it allows to incorporate multiple attachment sites to capture different target miRNAs at the nanoscale. In this work, we present a DNA origami nanoarray system providing distance-dependent recognition of miRNAs by applying super-resolution microscopy technique; DNA-PAINT (point accumulation for imaging in nanoscale topography). The sensor can detect up to 4 miRNAs either separately or in combination based on the relative distance to the boundary markers on the structure using a single imager strand. The detection is highly sensitive, with a limit of detection down to the low femtomolar range (11 fM - 388 fM) and has a large dynamic range up to 10 nM without need for amplification. Moreover, our detection system can discriminate single base mismatches with low false positive rates. Using our strategy, we demonstrate the detection of endogenous miRNAs from cell extracts of cancer cell lines and plasma from breast cancer patients. Overall, we developed an ultrasensitive and amplification-free, DNA-PAINT imaging-based miRNA detection method using DNA origami nanoarray system for the detection of breast-cancer associated miRNAs which potentially provides a sensitive and accurate alternative to the current multiplexed diagnostic technologies.

Polychromatic Quantum Dot Array to Compose a Community Signal Ensemble for Multiplexed miRNA Detection.

We herein describe a polychromatic quantum dot array (PQDA) to compose a community signal ensemble enabling accurate and precise quantification of miRNAs in a multiplexed manner. Advanced multicomponent ultrahigh-resolution patterning technique achieved by capsulation-assisted transfer printing following self-assembly-based poly(methyl methacrylate) (PMMA) patterning is utilized to manufacture the PQDA, which is designed to discharge a target miRNAs-specific set of fluorescent quantum dots (QDs) through the activity of duplex-specific nuclease (DSN). On the basis of the community signal ensemble produced by the discharged QD profiles, target miRNAs are very specifically identified down to a femtomolar level (1.27 fM) in a multiplexed manner over a wide dynamic range of up to 6 orders of magnitude. The practical diagnostic capability of this strategy is also demonstrated by reliably identifying breast cancer-specific miRNAs from heterogeneous cancer cell lysates.

Shape-coded hydrogel microparticles integrated with hybridization chain reaction and a microfluidic chip for sensitive detection of multi-target miRNAs

The abnormal expressed miRNAs have been suggested as important biomarkers for disease diagnosis and prognosis. It is imperative to develop rapid, cost-effective, sensitive, and multiplexed detection of miRNAs. Taking advantages of hydrogel microparticles and the microfluidic techniques, a simple enzyme-free method based on shape-coded hydrogel microparticles combined with hybridization chain reaction (HCR) and a microfluidic chip was developed for multiplexed miRNA detection. Cascades of hybridization between carboxyfluorescein (FAM)-labeled hairpin probes were triggered to form long dsDNA products in hydrogel microparticles, which led to deposition of FAM in them. The proposed method exhibited good sensitivity with the limits of detection (LODs) of 27.8 pM and 24.7 pM for miRNA 21 and miRNA let-7a, respectively. Furthermore, one-base-mismatched sequences can be discriminated, which indicated the high specificity of the approach. Moveover, the proposed method were applied to parallel detection of multi-target miRNAs in real biological samples with favorable results. Finally, by integration of the proposed method with a microfluidic chip, the detection time was significantly shortened, the detection throughput was further enlarged, and the whole operations were greatly simplified.

Multiplexed miRNA detection based on target-triggered transcription of multicolor fluorogenic RNA aptamers

We herein describe a new multicolor fluorogenic RNA aptasensor to accomplish multiplexed detection of miRNAs. The stem-loop primer (SL primer) entailing a fluorogenic RNA aptamer (FRA) antisense sequence is designed to anneal to target miRNA at its 3′ overhang, which would be reverse transcribed by reverse transcriptase (RT) to produce the cDNA sequence followed by the degradation of target miRNA. The T7 promoter-containing primer (T7 primer) is then annealed to the 3’ end of the extended cDNA sequence and the following RT-promoted extension in both directions produces the T7 promoter-containing double-stranded DNA (T7 dsDNA). T7 RNA polymerase finally transcribes the T7 dsDNA to produce a large number of RNA transcripts containing FRA sequence, which would produce intense fluorescence signals by forming fluorescent complexes with cognate fluorogens, reflecting the amount of target miRNAs. Based on this unique design principle employing the SL primers to encode several different FRAs with distinct fluorescence profiles, target miRNAs were very specifically determined in a multiplexed manner down to a subpicomolar level. The practical applicability of this technique was also verified by reliably quantifying target miRNAs in serum and human cancer cell lysates.

Metal sulfide nanoparticle-based dual barcode-triggered DNAzyme cascade for multiplex miRNA detection in a single assay.

Single miRNAs are not specific and accurate enough to meet the strict diagnosis requirements in practice. Therefore, simultaneous monitoring of multiplexed miRNA in biological samples can not only improve the accuracy and specificity of bioassays but also avoid the squandering of valuable biological specimens. Herein, we designed a metal sulfide nanoparticle-based dual barcode-triggered DNAzyme cascade strategy for the sensitive and simultaneous multiplex miRNA detection in a single assay. Firstly, the capture probes (H1, H2) specifically recognize targets (miRNA-21, miRNA-141), exposing the stem of H1 and H2. Then, with the introduction of a detection probe (CuS-H3, ZnS-H4), the exposed H1 and H2 catalyze the hairpin assembly (CHA) reaction, realizing target miRNA recycling, and forming H1/H3-CuS and H2/H4-ZnS complexes. Subsequently, the formed H1/H3-CuS and H2/H4-ZnS complexes are encoded on magnetic beads through the biotin/streptavidin interaction. The CuS and ZnS nanoparticles captured by magnetic beads release thousands of Cu2+ and Zn2+via the cation exchange reaction. Finally, the released Cu2+ and Zn2+ specially activate the DNAzyme of the catalytic and molecular beacon (CAMB) system. The CAMB system affords an amplified fluorescence signal output by cycling and regenerating the metal ion-dependent DNAzyme to realize multiple enzymatic turnovers. Benefiting from target recycling, nanoparticle amplification, and catalytic and molecular beacon amplification, there is substantial amplification and the target miRNAs can be detected at 0.06 fM (miRNA-21) and 0.048 fM (miRNA-141) in a single assay. Furthermore, the high selectivity and accuracy of the assay were proved by practical analysis of different cancer cells, which exhibited good practicability in multiplex miRNA detection in clinical sera. The results indicate that the proposed strategy holds great potential for the sensitive detection of multiplex cancer biomarkers and offers the opportunity for future applications in clinical diagnosis.

Ultrasensitive multiplexed miRNA detection based on a self-priming hairpin-triggered isothermal cascade reaction.

We herein describe an ultrasensitive isothermal strategy to detect miRNAs in a multiplexed manner by utilizing a self-priming hairpin-triggered cascade reaction and the adsorption properties of graphene oxide (GO). In principle, a self-priming hairpin probe (SHP) was designed to be opened through binding to the target miRNA and rearranged to serve as a primer. The following extension displaced the target miRNA to be recycled for opening another SHP and produced a double-stranded (ds) SHP with a longer stem region. The nicking enzyme recognition site within the ds SHP was then subjected to continuous repeated nicking and extension reactions, consequently producing a large amount of the trigger sequence. In the second reaction phase, the trigger also transformed another single-stranded (ss) target template probe (TTP) into ds TTP and simultaneously produced numerous target mimic strands (Target') in the same manner, which could activate the first reaction phase, mimicking the target miRNA. Since the ss portions of the two probes were all transformed to the ds forms (ds SHP and ds TTP), they are resistant to the adsorption by graphene oxide (GO) and then emitted intense fluorescence after the application of GO while the ss forms of the two probes produced a negligible fluorescence signal without the target miRNAs. Based on this unique design principle, we were able to simultaneously identify multiple target miRNAs very sensitively down to attomolar levels (42.63 aM for miRNA let-7a, 13.08 aM for miRNA-141, and 10.14 aM for miRNA-98) within 30 min.

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.