Simultaneous Detection Of Multiple microRNAs Research Articles

Triple ligation-based formation of a G-quadruplex for simultaneous detection of multiple miRNAs.

The simultaneous detection of multiple microRNAs (miRNA) is of great necessity but has not been extensively studied. This prompted our study, which involved the development of a triple ligation-based system for detecting three miRNAs at the same time. We designed a multi-ligation-padlock (MLP) probe that consists of three parts, each of which is complementary to two different miRNAs at the same time. In the presence of all three miRNAs, the probe becomes circularized, but in the absence of even one target, the probe remains linear. The first part of the MLP probe (MLP1) contains a T7 promoter part that can initiate RNA synthesis for any given target condition. However, it also includes a G-quadruplex complementary segment, which can only form a parallel RNA G-quadruplex through rolling circle transcription by the circularized template in the presence of all three targets. In this case, the application of our parallel G-quadruplex sensing fluorescent probe lutidine DESA (LutD) produces a strong signal. However, in the absence of any one of the targets, the RNA G-quadruplex cannot be formed and ultimately the LutD probe does not generate any signal. This difference in the signal intensity represents the presence or absence of all the target miRNAs. With our system, we were able to detect miRNA 21 at levels as low as 1.13 fM, miRNA 146a as low as 1.37 fM, and miRNA 25b as low as 1.51 fM within 45 minutes, confirming that our novel system can selectively and sensitively diagnose triple miRNAs.

Cotton thread-based multi-channel photothermal biosensor for simultaneous detection of multiple microRNAs

The abnormal expression of microRNAs (miRNAs) is associated with various diseases. Developing simple and portable methods for sensitive, rapid and simultaneous detection of multiple miRNAs is critical to achieve accurate and timely diagnosis. Herein, a cotton thread-based multi-channel photothermal biosensor was proposed for simultaneous detection of three breast cancer-related miRNAs including miRNA-10b, miRNA-27a and miRNA-let-7a. Three cotton thread-based channels with one input were designed and the capture probes for detecting different miRNAs were immobilized on the test zones of the corresponding channels. Cu2-xS nanostrings prepared on the basis of hybridization chain reaction (HCR) were taken as the photothermal agents for signal transduction and amplification. The formation of a sandwich structure among the capture probe, target miRNA, and Cu2-xS nanostrings led to the accumulation of the Cu2-xS nanostrings on the test zones and transformed the concentration of miRNA into temperature signal under 808 nm laser irradiation. The temperature changes were quantified by a portable thermal camera and directly reflected the concentration of miRNAs. Under the optimal conditions, the developed multi-channel photothermal biosensor showed excellent specificity and sensitivity with the detection limits of 37 pM, 38 pM and 38 pM for miRNA-10b, miRNA-27a and miRNA-let-7a, respectively. Furthermore, a simultaneous detection of the three miRNAs in cell lysates were achieved and the results were in accordance with that obtained by the quantitative reverse transcription polymerase chain reaction (qRT-PCR), indicating its excellent capacity for practical applications. The developed biosensor provided an important tool for analysis of multiple targets and showed great potential in clinical diagnosis.

DNA nanolantern-mediated catalytic hairpin assembly nanoamplifiers for simultaneous detection of multiple microRNAs

Simultaneous detection of multiple microRNAs (miRNAs) with high sensitivity can give accurate and reliable information for clinical applications. By uniformly anchoring hairpin probes on the surface of DNA nanolantern, a three-dimensional DNA nanostructure contains abundant and adjustable modification sites, highly integrated DNA nanoprobes were designed and developed as catalytic hairpin assembly (CHA)-based signal amplifiers for enzyme-free signal amplification detection of target miRNAs. The nanolantern-based CHA (NLC) amplifiers, which were facilely prepared via a simple “one-pot” annealing method, showed enhanced biostability, improved cell internalization efficiency, accelerated CHA reaction kinetics, and increased signal amplification capability compared to the single-stranded DNA hairpin probes used in traditional CHA reaction. By co-assembling multiple hairpin probes on a DNA nanolantern surface, as-prepared NLC amplifiers were demonstrated to work well for highly sensitive and specific imaging, expression level fluctuation analysis of two miRNAs in living cells, and miRNAs-guided tumor imaging in living mice. The proposed DNA nanolantern-based nanoamplifier strategy might provide a feasible way to promote the cellular and in vivo applications of nucleic acid probes.

DNA functionalized double quantum dots-based fluorescence biosensor for one-step simultaneous detection of multiple microRNAs

The disease diagnosis by detecting single microRNAs (miRNAs) can produce high false positive rate. Herein, a novel fluorescence biosensor method for one-step simultaneous detection of multiple miRNAs was proposed by using single-stranded DNA (ssDNA) functionalized double quantum dots (QDs) and black hole quencher (BHQ)-decorated magnetic nanobeads (MNs). MNs were linked with two black hole quenchers (BHQ1 and BHQ3) via a complementary DNA (cDNA). The ssDNA/cDNA hybridization contributed to the fluorescence quenching of double QDs due to the fluorescence resonance energy transfer (FRET) between double QDs and BHQ. In the presence of target miRNA-33 (miR-33) and miRNA-125b (miR-125b), the ssDNA1 and ssDNA2 were respectively hybridized with miR-33 and miR-125b to form more stable duplexes. Thus, the double QDs were released into supernatant after the magnetic separation, leading to the fluorescence signals recovery at 537 nm and 647 nm. A wide linear range (0.5 nM–320 nM for miR-33 and 0.1 nM–250 nM for miR-125b) and low limits of detection (0.09 nM for miR-33 and 0.02 nM for miR-125b) were achieved. Moreover, our approach has been demonstrated to simultaneously detect miR-33 and miR-125b in cell extracts. With advantages of high sensitivity, strong specificity, low background and low cost, the strategies show great potentials for the detection of various targets in bioanalysis and disease diagnosis.

Multicolor fluorescence encoding of different microRNAs in lung cancer tissues at the single-molecule level.

The simultaneous detection of multiple microRNAs (miRNAs) will facilitate early clinical diagnosis. Herein, we demonstrate the integration of multicolor fluorophore-encoded cascade signal amplification with single-molecule detection for simultaneous measurement of different miRNAs in lung cancer tissues. This assay involves two linear templates and two circular templates without the requirement of any fluorescent-labeled probes. The binding of target miRNAs to their corresponding linear templates initiates the cyclic strand displacement amplification, generating many triggers which can specifically hybridize with the corresponding biotin-labeled AP probes to initiate the apurinic/apyrimidic endonuclease 1-assisted cyclic cleavage reaction for the production of more biotin-labeled primers for each miRNA. The resultant two primers can react with their corresponding circular templates to initiate rolling circle amplification which enables the incorporation of Cy5-dCTP/Cy3-dGTP nucleotides, resulting in the simultaneous production of abundant biotin-/multiple Cy5/Cy3-labeled DNA products. After magnetic separation and exonuclease cleavage, the amplified products release abundant Cy5 and Cy3 fluorescent molecules which can be simply monitored by single-molecule detection, with Cy3 indicating miR-21 and Cy5 indicating miR-155. This assay involves three consecutive amplification reactions, enabling the conversion of extremely low abundant target miRNAs into large numbers of Cy5/Cy3 fluorophore-encoded DNA products which can release abundant fluorescent molecules for the generation of amplified signals. This assay exhibits high sensitivity, good selectivity, and the capability of multiplexed assay. This method can simultaneously quantify miR-155 and miR-21 in living cells and in lung cancer tissues, and it can distinguish the expression of miRNAs between non-small cell lung cancer patients and healthy persons. The accuracy and reliability of the proposed method are further validated by quantitative reverse transcription polymerase chain reaction.

Paper-Based Bipolar Electrode Electrochemiluminescence Platform for Detection of Multiple miRNAs.

This paper introduces a novel potential-resolved paper-based biosensor for simultaneous detection of multiple microRNAs (miRNAs) (taking miRNA-155 and miRNA-126 as examples) based on the bipolar electrode (BPE) electrochemiluminescence (ECL) strategy. The proposed multiple-channel paper-based sensing microfluidic platform was prepared by wax-printing technology, screen-printing method, and in situ Au nanoparticles (AuNPs) growth to form hydrophilic areas, hydrophobic boundaries, waterproof electronic bridge, driving electrode regions, and parallel bipolar electrode regions. CdTe quantum dots (QDs)-H2 and Au@g-C3N4 nanosheets (NSs)-DNA1 were used as dual electrochemiluminescence signal probes, and carboxylated Fe3O4 magnetic nanoparticles existed as carriers. CdTe QDs-H2/S2O82- and Au@g-C3N4 NSs-DNA1/S2O82- could exhibit two strong and stable ECL emissions at a drive voltage of 9 and 12 V, respectively, which can be used as effective potential-resolved signal tags. In addition, the proposed three-dimensional (3D) DNA nanomachine model and the target miRNA cycle strategy were used to achieve double amplification of electrochemiluminescence intensity. More importantly, the combination of the bipolar electrode system and the potential-resolved multitarget electrochemiluminescence method can greatly reduce the spatial interference between substances. The prepared ECL biosensor showed a favorable linear response for the detection of miRNA-155 and miRNA-126 with relatively low detection limits of 5.7 and 4.2 fM, respectively. With excellent sensitivity, the strategy may provide an efficient method for clinical application, especially in detection of trace multiple targets.

MNAzyme-Catalyzed Amplification Assay with Lanthanide Tags for the Simultaneous Detection of Multiple microRNAs by Inductively Coupled Plasma-Mass Spectrometry.

Quantification of multiple disease-related microRNAs (miRNAs) is of great significance for clinical diagnosis. Based on the simultaneous multiple element detection ability of inductively coupled plasma-mass spectrometry (ICP-MS) and good specificity of multicomponent nucleic acid enzymes (MNAzymes), a novel and simple method based on the MNAzyme amplification strategy and lanthanide labeling coupled with ICP-MS detection was proposed for the sensitive and simultaneous detection of three miRNAs (miRNA-21, miRNA-155, and miRNA-10b). Specifically, a probe consisting of streptavidin-modified magnetic beads (SA-MBs) and three DNA substrates labeled with lanthanide tags (159Tb/165Ho/175Lu) was constructed. In the presence of target miRNAs, three pairs of MNAzymes were assembled where each pair was hybridized with the corresponding miRNA, and then the substrates on the SA-MBs were cleaved by the activated MNAzymes, continuously releasing the fragment with lanthanide tags. The released lanthanide tags in the supernatant were collected after magnetic separation and analyzed by ICP-MS, realizing the simultaneous quantification of multiple miRNAs. The correlation of the lanthanide tag signal with the miRNA concentration fitted well in a linear model in the range of 50-1000 pmol L-1 (miRNA-21) and 50-2000 pmol L-1 (miRNA-155 and miRNA-10b). The limits of detection for three miRNAs were 11-20 pmol L-1, with the relative standard deviations of 2.2-2.7%. The recoveries of target miRNAs in the human serum and HepG-2 cells were in the range of 87.2-111% and 93.3-111%, respectively. Overall, the method is ideal for the simultaneous quantification of multiple miRNAs with advantages of low spectral interference, high sensitivity, good selectivity, and strong resistance to the complex matrix.

DNA polymerase/NEase-assisted signal amplification coupled with silver nanoclusters for simultaneous detection of multiple microRNAs and molecular logic operations

Herein, using a novel bifunctional DNA-hybridization-DNA (DHD) probe, a symmetric DNA polymerase/NEase-assisted target signal amplification reaction into a single probe coupled with dual “light-up” colored silver nanoclusters (AgNCs) was introduced for label-free simultaneous detection of multiple microRNAs (miRNAs) for the first time. The DHD probe contained two single-stranded loops complementary to two different miRNA targets. Upon the recognition of the specific target miRNAs (miR-21 or miR-141), a symmetric polymerase/NEase-assisted signal amplification reaction was initiated, which generated massive triggers (trigger 1 and trigger 2). Subsequently, the released triggers hybridized with two different dark AgNCs and formed a partial double-stranded structure; thus, “light-up” red or yellow fluorescence readout was observed. The established biosensing approach was capable of simultaneously detecting multiple miRNAs with a limit of detection (LOD) down to 9.8/6.1 pM for miR-21/miR-141, respectively. This innovative approach displayed acceptable reproducibility and high specificity. Furthermore, the “AND” logic gate was activated by combining the dual-functional DHD probe with the dual “light-up” colored AgNCs, with/without miRNAs as inputs.

SiRNA-directed self-assembled quantum dot biosensor for simultaneous detection of multiple microRNAs at the single-particle level

MicroRNAs are essential post-transcriptional regulators and may act as the noninvasive biomarkers for disease diagnosis. Sensitive and multiplexed detection of microRNAs may facilitate the accurate and early clinical diagnosis, but the available methods are usually compromised by using organic dyes as the signal probes, laborious chemical and enzymatic manipulations, and the complicated reaction schemes. Here we reported a siRNA-directed self-assembled quantum dot (QD) biosensor for facile and simultaneous detection of multiple microRNAs. In this biosensor, the binding of microRNA targets with corresponding QD nanoprobes leads to the formation of siRNA duplexes, which not only induces the spectrally resolved coding of microRNAs, but also facilitates the assembly of QDs:magnetic nanoparticle bioconjugates for the isolation and enrichment of target microRNAs. The disassembled QDs can be sensitively detected by single-molecule detection, enabling quantitatively sensing of microRNAs at the single-particle level. This biosensor employs only QDs as the signal reporters, which can simultaneously detect multiple microRNAs from the same sample and achieves femtomolar sensitivity and single-base mismatch selectivity without the involvement of any target labeling and amplification steps. Moreover, it can be successfully applied for simultaneous detection of circulating microRNAs in clinical serum samples, holding great potential in non-invasive early diagnosis and biomedical researches.

Peptide nucleic acid-based electrochemical biosensor for simultaneous detection of multiple microRNAs from cancer cells with catalytic hairpin assembly amplification

Herein we demonstrated a facile electrochemical method for simultaneous detection of miRNA21 and miRNA155 using a peptide nucleic acids (PNAs)-modified gold electrode coupled with the target-catalyzed hairpin assembly (CHA) strategy. In the presence of the target miRNA, the CHA was triggered selectively between two hairpins with one ferrocene (Fc) or methylene blue (MB) labelled. The resulting redox-active group modified CHA products (Fc-CHA21 or MB-CHA155) were then specifically captured by the PNA probes (PNA21 or PNA155) attached on the surface of a gold electrode, which bring the Fc and MB labels into close proximity to generate apparently enhanced electrochemical signals for sensitive and simultaneous detecting of low amount miRNA21 and miRNA155 in cancer cells. This assay was highly selective for discriminating miRNAs with similar sequences and has detection limits of 2.49 fM and 11.63 fM for miRNA21 and miRNA155, respectively. The feasibility of the method for sensitive determination of miRNA21 and miRNA155 from human cancer cells was also demonstrated. This method thus has great potential to be applied for simultaneous detecting of a variety of miRNA biomarkers for clinic applications due to its simple, sensitive and accurate features.

A simple and universal enzyme-free approach for the detection of multiple microRNAs using a single nanostructured enhancer of surface plasmon resonance imaging.

Here we describe a simple approach for the simultaneous detection of multiple microRNAs (miRNAs) using a single nanostructured reagent as surface plasmon resonance imaging (SPRi) enhancer and without using enzymatic reactions, sequence specific enhancers or multiple enhancing steps as normally reported in similar studies. The strategy involves the preparation and optimisation of neutravidin-coated gold nanospheres (nGNSs) functionalised with a previously biotinylated antibody (Ab) against DNA/RNA hybrids. The Ab guarantees the recognition of any miRNA sequence adsorbed on a surface properly functionalised with different DNA probes; at the same time, gold nanoparticles permit to detect this interaction, thus producing enough SPRi signal even at a low ligand concentration. After a careful optimisation of the nanoenhancer and after its characterisation, the final assay allowed the simultaneous detection of four miRNAs with a limit of detection (LOD) of up to 0.5pM (equal to 275attomoles in 500μL) by performing a single enhancing injection. The proposed strategy shows good signal specificity and permits to discriminate wild-type, single- and triple-mutated sequences much better than non-enhanced SPRi. Finally, the method works properlyin complex samples (total RNA extracted from blood) as demonstrated by the detection of four miRNAs potentially related to multiple sclerosis used as case study. This proof-of-concept study confirms that the approach provides the possibility to detect a theoretically unlimited number of miRNAs using a simple protocol and an easily prepared enhancing reagent, and may further facilitate the development of affordable multiplexing miRNA screening for clinical purposes.

Hybridization-initiated exonuclease resistance strategy for simultaneous detection of multiple microRNAs

Multiple miRNA detection is often limited by sample, time-consuming, and complicated procedures. To address such challenges, we present a relatively simple and amplification-free fluorescent strategy based on hybridization-initiated exonuclease resistance for simultaneous detection of multiple miRNAs in a single tube. Single-stranded linear DNA probes were designed with dual roles of capture and reporter DNA, i.e. each DNA probe was labeled with biotin at the 3′-end for signal readout and amino at the 5′-end for probe immobilization. After target miRNAs specifically hybridized with the corresponding probes, the formation of double-stranded probe-target duplex protected the biotin on the probe from Exonuclease I digestion, thus resulting in high fluorescent intensities through the reaction between biotin and streptavidin-phycoerythrin. Coupling with the 3-plex fluorescent microsphere-based assay system, herein we successfully demonstrated the simultaneous and quantitative measurement of three sequence-specific miRNAs at concentration range of 2.5 pM to 1.25 nM and detection limits of 2 pM. To meet high throughput and rapid turnaround time requirements, we have also exemplified that, even shortening the total reaction time within 1 h, wider linear response range and lower detection limit were guaranteed. We further applied this assay to detect endogenous target miRNA levels from five kinds of cancer cell line and one normal cell line HEK 293T. This simple and rapid strategy will hold great potential for monitoring of multiple miRNAs biomarkers in biomedical research and early clinical diagnosis.

Integration of isothermal amplification with quantum dot-based fluorescence resonance energy transfer for simultaneous detection of multiple microRNAs.

MicroRNAs (miRNAs) are small non-coding RNAs that regulate important physiological processes, and their dysregulation is associated with various human diseases. Simultaneous sensitive detection of multiple miRNAs may facilitate early clinical diagnosis. In this research, we demonstrate for the first time the integration of hyperbranched rolling circle amplification (HRCA) with quantum dot (QD)-based fluorescence resonance energy transfer (FRET) for the simultaneous detection of multiple microRNAs with a single-color QD as the donor and two fluorescent dyes as the acceptors. We used miR-21 and miR-221 as target miRNAs. We designed two circular templates which may specifically hybridize with miR-21 and miR-221, respectively, for the initiation of the HRCA reaction. The products of the HRCA reaction may hybridize with both capture probes and reporter probes to form the biotinylated acceptor-labeled sandwich hybrids. The resultant sandwich hybrids can assemble on the surface of the QD, enabling efficient FRET between the QD and the acceptors, with the Cy3 signal indicating the presence of miR-21 and the Texas Red signal indicating the presence of miR-221. This assay has significant advantages of simplicity and low cost. The HRCA reaction can be performed under isothermal conditions with the same reverse primer for different target miRNAs, and the products of the HRCA reaction for both miR-21 and miR-221 can specifically hybridize with the same capture probes. This assay exhibits excellent specificity and high sensitivity with a detection limit of 7.2 × 10-16 M for miR-21 and 1.6 × 10-17 M for miR-221, and it can be used for simultaneous detection of multiple miRNAs in human cancer cells, holding great potential in biomedical research and clinical diagnosis.

A versatile size-coded flow cytometric bead assay for simultaneous detection of multiple microRNAs coupled with a two-step cascading signal amplification.

A versatile flow cytometric bead assay is developed for the sensitive and simultaneous detection of multiple microRNAs that combines an elegant enzyme-mediated cascading signal amplification on magnetic beads with a novel bead size-encoding mechanism.

Simultaneous detection of multiple microRNAs for expression profiles of microRNAs in lung cancer cell lines by capillary electrophoresis with dual laser-induced fluorescence

MicroRNAs (miRNAs) are small, endogenous, single-stranded, noncoding RNAs. Circulating miRNAs are being considered as promising disease biomarkers. Indeed, single miRNAs have been associated with a wide variety of disease conditions and can target multiple mRNAs; therefore, several miRNAs may be simultaneously involved in disease progression and development. In this study, we developed a capillary electrophoresis with dual laser-induced fluorescence (CE with dual LIF) method using two color laser excitations for simultaneous determination of multiple miRNAs. Target miRNAs were hybridized with 6-FAM- or Cy5-labeled DNA probes for simultaneous determination of multiple miRNAs at excitation wavelengths of 488 and 635nm. The hybridized miRNAs were separated using CE with dual LIF and detected within 13min at excitation wavelengths of 488 and 635nm without any interference or crosstalk. Additionally, the proposed approach was used successfully to detect and evaluate levels of several endogenous miRNAs from lung cancer cell lines. These results showed the potential of CE with dual LIF for fast, specific, simultaneous analysis of multiple miRNAs in cell extracts, biofluids, and tissues.