Detection Of Multiple miRNAs Research Articles

Autonomously driving multiplexed hierarchical hybridization chain reaction of a DNA cobweb sensor for monitoring intracellular microRNA

Here, we present an autonomously driving multiplexed hierarchical hybridization chain reaction (MH-HCR) of a DNA cobweb sensor for monitoring intracellular microRNAs. The amplifier of the sensor consists of an enzyme-free amplification, two-step and multiple round hybridization chain reaction (HCR) circuit, where the target miRNA-triggered output of the first HCR stage (HCR1) serves as input to generate the following HCR stage (HCR2) and then produces n cycles (HCR1-HCR2) amplifying processes to result in a DNA cobweb sensor. By introducing a pair of fluorescence resonance energy transfer (FRET) probes, this MH-HCR-based DNA cobweb sensor enables selective and sensitive detection of miRNAs down to 1 fM. In situ imaging of miRNAs in several living cells was successfully achieved. Compared with other nonlinear HCRs, the initiation sequence is enclosed in the stable multiple capture probe, avoiding interference reaction of the capture probe with a hairpin containing DNA fluorescent agent to generate false signals. This MH-HCR amplifier provides a general and robust method for the highly sensitive and selective detection of multiple miRNAs and has the potential for early cancer diagnosis.

High-Throughput Detection of Multiple miRNAs and Methylated DNA by Droplet Digital PCR.

Altered miRNA expression and DNA methylation have highly active and diverse roles in carcinogenesis. Simultaneous detection of the molecular aberrations may have a synergistic effect on the diagnosis of malignancies. Herein, we develop a high-throughput assay for detecting multiple miRNAs and DNA methylation using droplet digital PCR (ddPCR) coupled with a 96-microwell plate. The microplate-based ddPCR could absolutely and reproducibly quantify 15 miRNAs and 14 DNA methylation sites with a high sensitivity (one copy/µL and 0.1%, respectively). Analyzing sputum and plasma of 40 lung cancer patients and 36 cancer-free smokers by this approach identified an integrated biomarker panel consisting of two sputum miRNAs (miRs-31-5p and 210-3p), one sputum DNA methylation (RASSF1A), and two plasma miRNAs (miR-21-5p and 126) for the diagnosis of lung cancer with higher sensitivity and specificity compared with a single type of biomarker. The diagnostic value of the integrated biomarker panel for the early detection of lung cancer was confirmed in a different cohort of 36 lung cancer patients and 39 cancer-free smokers. The high-throughput assay for quantification of multiple molecular aberrations across sputum and plasma could improve the early detection of lung cancer.

FeII 4L4 Tetrahedron-Assisted Three-Way Junction Probe for Multiple miRNA Detection.

MicroRNAs (miRNAs) modulate a variety of cellular signaling pathways and play a vital role in cell-to-cell communication. The overlapped expression of a certain miRNA is commonly reported to be related to cancers. Therefore, combined detection of multiple miRNAs is of great significance for cancer diagnosis. Herein, we developed a FeII4L4 tetrahedron-assisted three-way junction (3WJ) probe, which exhibited a higher stability than the normal 3WJ probe, for multiple miRNA detection. In this method, the simultaneous existence of miRNA-21 and miRNA-144 triggers the release of the Y3 sequence in the FeII4L4 tetrahedron-assisted 3WJ probe, which in turn triggers subsequent CRISPR-Cas12a-assisted rolling circle amplification. Based on this, simultaneous detection of miRNA-21 and miRNA-144 was achieved. Furthermore, we also applied this method to the detection of miRNAs in clinical samples and achieved good agreement with quantitative real-time polymerase chain reaction (qRT-PCR), indicating its significant potentials in early diagnosis and treatment of cancer.

NEase-based amplification for detection of miRNA, multiple miRNAs and circRNA

MiRNAs are particularly desirable biomarkers for the diagnosis of many cancers, but the methods for simultaneous detection of multiple miRNAs are rarely published. In addition, circRNAs are a novel class of non-coding RNAs and it has been accepted that circRNAs participate in regulating various biological processes. Herein, we design a method using NEase which has highly selectivity and activity to amplify fluorescence signal, so single miRNA, circRNA and multiple miRNAs could be simply quantified through our proposed assay. Furthermore, this method also can detect miRNA level in different cell lines.

A pico-HPLC-LIF system for the amplification-free determination of multiple miRNAs in cells

MicroRNAs are a class of important biomarkers, and the simultaneous detection of multiple miRNAs can provide valuable information about many diseases and biological processes. Amplification-free determination has been developed for the analysis of multiple miRNAs because of its characteristic low cost and high fidelity. Herein, a method for the amplification-free analysis and simultaneous detection of multiple miRNAs based on a so-called pico-HPLC-LIF system is described. In this process, a bare open capillary with an inner diameter of 680nm is used as a separation column for a sample volume of several hundreds of femtoliters (300fL), followed by separation and detection. The technique has a zeptomolar limit of detection. The method was applied to detect cellular miRNA from adenocarcinomic human alveolar basal epithelial (A549) cell extracts, and the simultaneous detection of the mir-182, miR-155, and let-7a was achieved. The results showed that the expression of mir-182 and miR-155 was up-regulated and that of let-7a was down-regulated in A549 cells. This method for multiple miRNAs detection is expected to have broad applications in miRNA-based disease diagnosis, prognosis, treatment, and monitoring.

Target-triggered entropy-driven amplification system-templated silver nanoclusters for multiplexed microRNA analysis

MicroRNAs (miRNAs) are important biomarkers for the diagnosis, prognosis, and treatment of human diseases. Sensitive and selective detection of multiple miRNAs simultaneously will greatly facilitate the early and accurate diagnosis of cancers. Herein, a novel entropy-driven amplification system-templated silver nanoclusters sensing platform was developed for the multiplexed analysis of tumor-associated miRNAs. The sensing platform was constructed by coupling target-triggered entropy-driven catalysis with luminescence adjustable DNA-templated silver nanoclusters (Ag NCs). In the presence of target miRNA, the sensing platform initiates the branch migration and strand displacement of the complex, which has a six-base cytosine loop for stabilizing the luminous Ag NCs. The target is cyclically generated for new catalysis while turning off the fluorescence of Ag NCs; this is accompanied by a significantly amplified optical readout. In this study, two different complex-stabilized Ag NCs systems were proposed, the yellow-emitting Ag NCs and red-emitting Ag NCs biosensors enabled the analysis of miRNA-141 and miRNA-155 with detection limits of 6.1 pM and 8.7 pM, respectively. Impressively, owing to the excellent selectivity, flexibility, and narrow-band excitation of the platform, the multiplexed synchronous detection of miRNA-141 and miRNA-155 were achieved in buffer, biological cell lysates and human serum samples with satisfactory results. The simple, flexible, and convenient strategy provides a powerful tool for multiple biomarkers analysis and related clinical applications.

Zeptomolar-level one-pot simultaneous detection of multiple colorectal cancer microRNAs by cascade isothermal amplification

Multi-microRNA (miRNA) detection would greatly facilitate early diagnosis of colorectal cancer (CRC). Here a convenient cascade isothermal amplification approach incorporating a G-quadruplex molecular beacon (G4MB) was established for achieving one-pot detection of multiple CRC miRNAs (miRNA-21, miRNA-92a, miRNA-31); this strategy incorporated a Bsu DNA polymerase (Bsu pol)-induced strand-displacement reaction and a Lambda exonuclease (λexo)-aided recycling reaction. In the presence of target miRNA, the G-rich stem structure was opened and became available for hybridization with the primer to initiate synthesis of Bsu pol-catalyzed double-stranded DNA (dsDNA) that displaced the miRNA target and released it, allowing it to participate in subsequent amplification cycles. Meanwhile, the dsDNA was gradually digested into fragments by λexo from the 5′ phosphorylated end, releasing the newly synthesized DNA strand for participation in subsequent cycles that led to amplification of the fluorescent signal. This approach provided a low limit of detection (LOD) of zeptomolar-level, 85.8 zM, 77.6 zM, 78.9 zM for miRNA-21, miRNA-92a, miRNA-31, respectively. It could distinguish the mismatched targets and achieved three miRNA targets detection run in parallel in one-pot within 2 h. Thus, this fast, simple, and convenient strategy holds great promise as a clinical application for the detection of multiple miRNAs in clinical CRC samples.

Express and sensitive detection of multiple miRNAs via DNA cascade reactors functionalized photonic crystal array

Array based detection techniques with fluorescence signal reading is a powerful tool for multiple targets analysis. However, when applied fluorescence array for microRNA detection, time-consuming multi-steps surface signal amplification is usually required due to the low abundance of microRNA in total RNA expressions, which impairs detection efficiency and limits its application in point of care test (POCT) manner. Herein, DNA cascade reactors (DCRs) functionalized photonic crystal (PC) array was fabricated for express and sensitive detections of miRNA-21 and miRNA-155. DCRs were assembled by interval conjugation of self-quenched hairpin DNA probes to single strand DNA nanowire synthesized by rolling circle amplification, which generated cascade DNA hybridization reactions in response to target miRNA with instant fluorescence recovery signal. PC array patterns with multi-structure colors further amplified fluorescence with their respective photonic bandgaps (PBGs) matching with the emission peaks of fluorescence molecules labelled on DCRs. The as-prepared DCRs functionalized PC array demonstrated express and sensitive simultaneous detections of miRNA-21 and miRNA-155 with hundreds fM detection limits only in 15 min, and was successfully applied in fast quantifications of low abundance miRNAs from cell lysates and spiked miRNAs from human serum, which would hold great potential for disease diagnosis and therapeutic effect monitoring with a POCT manner.

Simultaneous and ultrasensitive detection of multiple microRNAs by single-molecule fluorescence imaging.

Cell status changes are typically accompanied by the simultaneous changes of multiple microRNA (miRNA) levels. Thus, simultaneous and ultrasensitive detection of multiple miRNA biomarkers shows great promise in early cancer diagnosis. Herein, a facile single-molecule fluorescence imaging assay was proposed for the simultaneous and ultrasensitive detection of multiple miRNAs using only one capture anti-DNA/RNA antibody (S9.6 antibody). Two complementary DNAs (cDNAs) designed to hybridize with miRNA-21 and miRNA-122 were labelled with Cy3 (cDNA1) and Cy5 (cDNA2) dyes at their 5′-ends, respectively. After hybridization, both miRNA-21/cDNA1 and miRNA-122/cDNA2 complexes were captured by S9.6 antibodies pre-modified on a coverslip surface. Subsequently, the Cy3 and Cy5 dyes on the coverslip surface were imaged by the single-molecule fluorescence setup. The amount of miRNA-21 and miRNA-122 was quantified by counting the image spots from the Cy3 and Cy5 dye molecules in the green and red channels, respectively. The proposed assay displayed high specificity and sensitivity for singlet miRNA detection both with a detection limit of 5 fM and for multiple miRNA detection both with a detection limit of 20 fM. Moreover, it was also demonstrated that the assay could be used to detect multiple miRNAs simultaneously in human hepatocellular cancer cells (HepG2 cells). The proposed assay provides a novel biosensing platform for the ultrasensitive and simple detection of multiple miRNA expressions and shows great prospects for early cancer diagnosis.

In situ multiplex detection of serum exosomal microRNAs using an all-in-one biosensor for breast cancer diagnosis.

Herein, a simple all-in-one biosensor based on a DNA three-way junction has been constructed for in situ simultaneous detection of multiple miRNAs by competitive strand displacement. In our design, three oligonucleotides (Y1, Y2 and Y3) of a Y-type scaffold were extended at their 5' ends by introducing three single-stranded recognition sequences with quenchers (BHQ1, BHQ2 and BHQ2), respectively. Subsequently, three reporter sequences labeled with different fluorophores (FAM, Cy3 and Cy5) were bound to the corresponding recognition sequences to form a multicolour DNA biosensor that gives self-quenched fluorescence. The biosensor can effectively enter into exosomes and then hybridize to the complementary miRNA targets to form longer duplexes and release the reporter sequences, thus activating the readable fluorescence signals for the simultaneous detection of multiple miRNAs in exosomes. As a proof of principle, miR-21, miR-27a and miR-375 were chosen as model targets because of their high expressions in breast cancer cells (MCF-7). Fluorescence signals of MCF-7 exosomes after being treated with the biosensor exhibited positive correlations to their concentrations and the limits of detection were determined to be 0.116 μg mL-1, 0.125 μg mL-1 and 0.287 μg mL-1 for exosomes by detecting three exosomal miRNAs (miR-21, miR-27a and miR-375), respectively. In contrast, there were no obvious correlations between fluorescence intensities and control MCF-10A exosome concentrations. Importantly, by testing multiple exosomal miRNAs using the biosensor in clinical serum samples, breast cancer patients can be effectively differentiated from healthy donors. Consequently, the developed biosensor demonstrates high potential as a routine bioassay for the multiplex quantification of exosomal miRNAs in clinical diagnosis.

Logic-signal-based multiplex detection of MiRNAs with high tension hybridization and multiple signal amplification.

MicroRNAs (miRNAs) are crucial regulators of gene expression. The abnormal expression of miRNA is often closely related to many diseases. However, the accurate clinical profiling of miRNA expression remains a great challenge due to the high similarity and wide variety of miRNA sequence structures. Here, we report a highly specific and sensitive multiplex miRNA detection scheme with high tension hybridization and dual signal amplification based on the recyclable autocatalytic DNAzyme and a light harvesting conjugated polymer. Multiple signals can be read out simultaneously by single excitation through the efficient multiple fluorescence resonance energy transfer (FRET) between the conjugated polymer and different small molecule dyes. In addition, different types of logic gates can also be operated by observing the emission intensities of the labeling dyes with miRNAs as inputs, thus giving rise to a new way for the specific detection of certain miRNAs according to the logic signals. Furthermore, we successfully applied the strategy for multiple miRNA detection in cell lysates and the results agree well with those of qRT-PCR. Thus, we believe that this platform holds great potential for miRNA detection in biological samples.

Triple-Input Molecular AND Logic Gates for Sensitive Detection of Multiple miRNAs.

Abnormal miRNA expressions are closely related to the occurrence and development of cancers. It is of great significance to monitor miRNA expression levels for early diagnosis and therapy of the diseases. This study presents two independent colorimetric strategies for simultaneously monitoring multiple miRNAs based on cross-linking or non-cross-linking aggregations of gold nanoparticles (AuNPs). By introducing a Y shaped DNA structure and two types of DNA modified AuNPs, a triple-input DNA AND logic gate is facilely developed with the cross-linking aggregation of AuNPs as the signal output. To improve the sensitivity and shorten reaction time, the logic gate is modified by further employing a three DNA strands formed duplex and hybridization chain reaction. Non-cross-linking aggregation of AuNPs is used to evaluate the concentration of initial miRNA inputs. This strategy does not require DNA modification of AuNPs and ultrahigh sensitivity is achieved with the amplification of hybridization chain reaction. The present work may provide powerful tools for multiple miRNAs diagnostics and inspire further development of DNA based logic gates.

Intelligent Detection Platform for Simultaneous Detection of Multiple MiRNAs Based on Smartphone.

As a marker of malignant tumors, miRNA is closely related to the occurrence and metastasis of tumors. How to achieve rapid and sensitive real-time detection is important for clinical prevention and treatment of cancer. In this study, an intelligent detection platform based on smartphone image processing technology made point-of-care testing a reality. This new smart approach could detect multiple targets simultaneously and sensitively. Hydrogel microparticles of different coding modes (shapes, numbers) were prepared by flow lithography to detect different miRNAs. After sandwich immunoassays, different shapes of hydrogels showed different fluorescence intensities depending on their targets. Images were captured by a smartphone and then analyzed by image recognition processing software installed on the smartphone. The concentration of miRNA was obtained within 10 s. The entire reaction process did not exceed 2 h. This intelligent and portable detection platform for miRNAs was reliable and the limit of detection reached the femtomole level. This work provided a demonstration of intelligent, portable, real-time detection of tumor markers.

Strand displacement dual amplification miRNAs strategy with FRET between NaYF4:Yb,Tm/Er upconversion nanoparticles and Ti3C2 nanosheets

Due to the low abundance of miRNAs and the coordinated regulation of multiple miRNAs toward some biological processes, a method that can sensitively and simultaneously detect multiple miRNAs is needed. Here, a strand displacement dual amplification (SDDA) strategy was developed for multiple miRNAs analyses using a unique single strand-double strand-single strand DNA (sdsDNA), which generated by the target recognition probe hybridizing with the site region probe. During the process of SDDA, miRNA hybridized to the target recognition domain of sdsDNA, thereby releasing the site, which triggered the first strand displacement reaction. This strand displacement event resulted in the release of the other site, triggering the second strand displacement reaction. Both two strand displacement reactions in each assembly circle were integrated for amplified signals, which were tested by a fluorescence resonance energy transfer (FRET) assay. NaYF4:Yb,Tm/Er UCNPs and Ti3C2 nanosheets were used as the energy donor-acceptor pair. By employing 1:2 ratio of donor-acceptor, the energy transfer efficiency between NaYF4:Yb,Tm/Er UCNPs and Ti3C2 nanosheets could reach 90%, which enhanced signal-to-background ratio. Combining the benefits of dual amplification and efficient energy transfer on miRNAs detection, this assay exhibited a linear range from 5 fM to 100 pM for miRNA-21 and miRNA-10b. Moreover, this assay was applied to estimate the expression of miRNA-21 and miRNA-10b in human hepatoma and hepatocyte cell lines. Therefore, this FRET-based SDDA strategy has been used for highly sensitive detection of multiple miRNAs in cell lysate and facilitates the development of efficient biosensor platform.

Simply Constructed and Highly Efficient Classified Cargo-Discharge DNA Robot: A DNA Walking Nanomachine Platform for Ultrasensitive Multiplexed Sensing.

In this work, a classified cargo-discharge DNA robot with only two DNA strands was designed and driven by an analogous proximity ligation assay (aPLA)-based enzyme cleaving for fast walk to construct a novel electrochemical biosensor for simultaneously ultrasensitive detection of microRNA-155 (miRNA-155) and miRNA-21. Compared with traditional DNA nanomachines, the multifunctional DNA robot possessed simple structure, high self-assembling efficiency and walking efficiency. Once it interacted with target miRNAs, this DNA robot could walk fast on the electrode surface and realize the classified cargoes discharging including beacons methylene blue (MB) and ferrocene (Fc), respectively labeled in the double-stranded DNA (A1-A2) for ultrasensitive detection of multiple miRNAs simultaneously. As a result, the wide linearity ranging from 100 aM to 100 pM and low detection limits of 42.7 and 51.1 aM were obtained for miRNA-155 and miRNA-21 detection, respectively. As a proof of concept, the present strategy initiates a novel and highly efficient walking platform to realize the ultrasensitive detection of biomarkers and possesses potential applications in the clinical diagnosis of disease.

Rare-earth-doped upconversion nanocrystals embedded mesoporous silica nanoparticles for multiple microRNA detection

Simultaneous detection of multiple types of microRNA (miRNA) is of great significance in early cancer diagnosis. In this study, rare-earth-doped CaF2 upconversion nanocrystals (CaF2: RE3+, namely Ho3+ or Tm3+ and Yb3+) are formed within the mesopores of silica nanoparticles (CaF2: RE3+@MSN), in an in-situ manner, by capillary effect and thermo-decomposition method. Comparing to current upconversion nanoparticles, the amorphous and mesoporous nature of CaF2: RE3+@MSN provides abundant active groups and high specific surface area for feasibly immobilizing oligonucleotide probe. The simultaneous and selective detection of multiple miRNAs was achieved based on the photoluminescence spectra of different CaF2: RE3+@MSN conjugated with DNA probes in the same solution. The results indicated that the proposed approach holds significant potential for multiple miRNA detection and offers an opportunity for cancer early diagnosis.

A novel photoelectrochemical biosensor for the sensitive detection of dual microRNAs using molybdenum carbide nanotubes as nanocarriers and energy transfer between CQDs and AuNPs

Herein, a novel photoelectrochemical (PEC) biosensor was developed for the ultrasensitive detection of dual microRNAs (miRNAs), with the detection being based on energy transfer (ET) between carbon quantum dots (CQDs) and gold nanoparticles (AuNPs). The PEC platform consisted of a CQDs@Mo2C nanotube modified ITO electrode. Two hairpin probes (H1 and H2) carrying the Au NPs were used “switch off” and “switch on” the PEC signal of the CQDs, with a close approach of the tagged AuNPs to the CQDs quenching the PEC signal. The introduction of different miRNAs (miRNA-159b and miRNA-166a) altered the interparticle distance between the AuNPs and CQDs, thereby affecting the intensity of the PEC response. This approach allowed the highly sensitive detection of both miRNA-159b and miRNA-166a. The linear range of the biosensor for miRNA-159b and miRNA-166a detection were 0.5–5000 fM, with low detection limits of 0.15 fM and 0.21 fM, respectively. To our knowledge, this is the first reported CQDs-based ET biosensor for the PEC detection of dual miRNAs. Results suggest that this approach offers a promising platform for the ultrasensitive detection of multiple miRNAs.

Resettable and enzyme-free molecular logic devices for the intelligent amplification detection of multiple miRNAs via catalyzed hairpin assembly.

The integration of multi-level DNA logic gates for biological diagnosis is far from being fully realized. In particular, the simplification of logical analysis to implement advanced logic diagnoses is still a critical challenge for DNA computing and bioelectronics. Here, we developed a magnetic bead/DNA system to construct a library of logic gates, enabling the sensing of multiplex target miRNAs. In this assay, the miRNA-catalyzed hairpin assembly (CHA) was successfully applied to construct two/three-input concatenated logic circuits with excellent specificity extended to design a highly sensitive multiplex detection system. Significantly, the CHA-based multiplex detection system can distinguish individual target miRNAs (such as miR-21, miR-155, and miR let-7a) under a logic function control, which presents great applications in the development of rapid and intelligent detection. Another novel feature is that the multiplex detection system can be reset by heating the output system and the magnetic separation of the computing modules. Overall, the proposed logic diagnostics with high amplification efficiency is simple, fast, low-cost, and resettable, and holds great promise in the development of biocomputing, multiparameter sensing, and intelligent disease diagnostics.

Condensing-enriched magnetic photonic barcodes on superhydrophobic surface for ultrasensitive multiple detection.

The ultrasensitive detection of multiple nucleic acids has great significance in a broad range of medical fields since nucleic acid samples are usually of small volumes and highly diluted. Herein, we present a novel magnetic photonic crystal (PhC) barcode-integrated condensing-enriched superhydrophobic platform for the ultrasensitive multiple miRNA detection. Droplets containing targets could suck the hydrogel PhC barcodes without losing their targets due to the difference in wettability between the hydrophilic PhC barcodes and hydrophobic substrate. During the evaporation of water from the droplet microreactors, these targets could be effectively enriched in the barcodes for achieving higher sensitivities. As the encoding signals of the PhC barcodes are the characteristic reflection peaks generated by their ordered physical nanostructures, the barcodes are stable and free from any fluorescent background and photobleaching; thus, they are accurate for distinguishing different targets. In addition, with the integration of magnetic nanoparticles into the hydrogel PhC barcodes, they could be imparted with the features of immobilization during change of medium and flexible movement for controlling the reaction, both of which could facilitate the detection processes. Based on this platform, we have demonstrated that the detection limit of multiple miRNAs is improved by about three orders. Thus, our platform is an ideal alternative for the ultrasensitive simultaneous multiplex analysis in medical fields.

Label-Free and Multiplexed Quantification of microRNAs by Mass Spectrometry Based on Duplex-Specific-Nuclease-Assisted Recycling Amplification.

MicroRNAs (miRNAs) are important biomarker candidates for cancer screening and early detection research. Generally, miRNAs undergo synergistic adjustments in tumor cells. Herein, a mass-spectrometric method based on a duplex-specific-nuclease (DSN)-enzyme-assisted signal-amplification technique was proposed for label-free and multiplexed detection of multiple miRNAs, and applied to the quantification of three miRNAs (i.e., miRNA-141, miRNA-21, and let-7a) in samples of HeLa and MDA-MB231 cell extracts. Experimental results showed that the digestion modes of DSN against three different DNAs complementary to miRNA-141, miRNA-21, and let-7a in their DNA-miRNA heteroduplexes were quite different, verifying the multiplexed-detection capability of the proposed method. Moreover, an advanced calibration model was derived for the quantitative analysis of the complex mass-spectral data measured during the label-free and multiplexed detection of miRNA-141, miRNA-21, and let-7a by the proposed mass-spectrometric method. With the aid of the advanced calibration model, the proposed mass-spectrometric method achieved quite reliable quantitative results for miRNA-141, miRNA-21, and let-7a in samples of HeLa and MDA-MB231 cell extracts, with recovery rates within the range of 89.2 to 111.6%. The limits of detection (LODs) of the proposed mass-spectrometric method for miRNA-141, miRNA-21, and let-7a in standard samples were estimated to be 42, 41, and 95 pM, respectively. Therefore, it is reasonable to expect that the proposed mass-spectrometric method can be a competitive alternative for the label-free and multiplexed detection of multiple miRNAs in clinical diagnosis.