Selective Detection Of miRNA Research Articles

Label-Free Fluorescent DNA Dendrimers for microRNA Detection Based On Nonlinear Hybridization Chain Reaction-Mediated Multiple G-Quadruplex with Low Background Signal.

Various fluorescent sensing systems for miRNA detection have been developed, but they mostly contain enzymatic amplification reactions and label procedures. The strict reaction conditions of tool enzymes and the high cost of labeling limit their potential applications, especially in complex biological matrices. Here, we have addressed the difficult problems and report a strategy for label-free fluorescent DNA dendrimers based on enzyme-free nonlinear hybridization chain reaction (HCR)-mediated multiple G-quadruplex for simple, sensitive, and selective detection of miRNAs with low-background signal. In the strategy, a split G-quadruplex (3:1) sequence is ingeniously designed at both ends of two double-stranded DNAs, which is exploited as building blocks for nonlinear HCR assembly, thereby acquiring a low background signal. A hairpin switch probe (HSP) was employed as recognition and transduction element. Upon sensing the target miRNA, the nonlinear HCR assembly of two blocks (blocks-A and blocks-B) was initiated with the help of two single-stranded DNA assistants, resulting in chain-branching growth of DNA dendrimers with multiple G-quadruplex incorporation. With the zinc(II)-protoporphyrin IX (ZnPPIX) selectively intercalated into the multiple G-quadruplexes, fluorescent DNA dendrimers were obtained, leading to an exponential fluorescence intensity increase. Benefiting from excellent performances of nonlinear HCR and low background signal, this strategy possesses the characteristics of a simplified reaction operation process, as well as high sensitivity. Moreover, the proposed fluorescent sensing strategy also shows preferable selectivity, and can be implemented without modified DNA blocks. Importantly, the strategy has also been tested for miRNA quantification with high confidence in breast cancer cells. Thus, this proposed strategy for label-free fluorescent DNA dendrimers based on a nonlinear HCR-mediated multiple G-quadruplex will be turned into an alternative approach for simple, sensitive, and selective miRNA quantification.

MoS2 Nanoprobe for MicroRNA Quantification Based on Duplex-Specific Nuclease Signal Amplification.

MicroRNAs (miRNAs) play significant regulatory roles in physiologic and pathologic processes and are considered as important biomarkers for disease diagnostics and therapeutics. Simple, fast, sensitive, and selective detection of miRNAs, however, is challenged by their short length, low abundance, susceptibility to degradation, and homogenous sequence. Here, we report a novel design of nanoprobes for highly sensitive and selective detection of miRNAs based on MoS2-loaded molecular beacons (MBs) and duplex-specific nuclease (DSN)-mediated signal amplification (DSNMSA). We show that MoS2 nanosheets not only exhibit high affinity toward MBs but also act as an efficient quencher for absorbed MBs. The strong fluorescence-quenching ability of MoS2 in combination with cyclic DSNMSA contributes to the superior sensitivity of our method, with a limit of detection 4 orders of magnitude lower than that of traditional hybridization methods. Moreover, the nanoprobes also show high selectivity for discriminating homogenous miRNA sequences with one-base differences because of the discrimination ability of MBs and DSN. Furthermore, we demonstrate that the MoS2-loaded MB nanoprobes can be utilized for multiplexed detection of miRNAs. Given its high sensitivity and specificity, as well as the multiplexed function; this novel method as an effective tool shows a great promise for simultaneous quantitative analysis of multiple miRNAs in biomedical research and clinical diagnosis.

A new fluorescence turn-on nanobiosensor for the detection of micro-RNA-21 based on a DNA–gold nanocluster

In this study, DNA/gold nanoclusters (AuNCs) were used to develop an AuNC-based turn-on fluorescence probe for the analysis of mi-RNA-21, which is a potential screening biomarker for cancer detection. AuNCs on a DNA scaffold were prepared through a one-pot wet-chemical route and evaluated by transmission electron microscopy and dynamic light scattering. Experiments revealed that the fluorescence intensity of the DNA–AuNCs showed a gradual increase with the addition of the target species in a concentration range from 1pM to 10 nM. The method had a detection limit of 0.7 pM and was able to discriminate the target species from mismatched mi-RNAs very efficiently. The method was used for the determination of mi-RNA spiked human plasma samples, and was evaluated as a promising nanobiosensor for application in the selective detection of mi-RNA in various biomedical and clinical tests.

MicroRNA detection by an amplification-free biosensor based on controllable solid-state electrochemiluminescence quenched by charge transfer

A simple and amplification-free solid-state electrochemiluminescence (ECL) biosensor was developed for the sensitive and selective detection of miRNA.

An enzyme free electrochemical biosensor for sensitive detection of miRNA with a high discrimination factor by coupling the strand displacement reaction and catalytic hairpin assembly recycling.

An isothermal, enzyme free, ultra-specific and ultra-sensitive protocol for electrochemical detection of miRNAs is proposed based on the toehold-mediated strand displacement reaction (SDR) and non-enzymatic catalytic hairpin reaction (CHA) recycling. The SDR was first triggered only in the presence of target miRNA and this process also affects other miRNA interferences having similar target sequences, thus guaranteeing a high discrimination factor and could be used in rare content miRNA detection with various amounts of interferences having similar target sequences. The output protector strand then triggered enzyme free CHA amplification and generates plenty of hairpin self-assembly products. This process in turn influences SDR equilibrium to move to the right and generates large amounts of protector output to ensure analysis sensitivity. Compared with traditional CHA, our proposed method greatly improved the signal to noise ratio and shows excellent performance in rare miRNA detection with miRNA analogue interference. Under the optimal experimental conditions and using square wave voltammetry, the established biosensor could detect target miRNA-21 down to 30 fM (S/N = 3) with a dynamic range from 100 fM to 2 nM, and discriminate rare target miRNA-21 from mismatched miRNA with high selectivity. This method holds great promise in miRNA detection from human cancer cell lines and would be a versatile and powerful tool for clinical molecular diagnostics.

Polycationic Probe-Guided Nanopore Single-Molecule Counter for Selective miRNA Detection.

MicroRNAs (miRNAs) are a class of noncoding RNAs that are being explored as a new type of disease biomarkers. The nanopore single-molecule sensor offers a potential noninvasive tool to detect miRNAs for diagnostics and prognosis applications. However, one of the challenges that limits its clinical applications is the presence of a large variety of nontarget nucleic acids in the biofluid extracts. Upon interacting with the nanopore, nontarget nucleic acids produce "contaminative" nanopore signals that interfere with target miRNA discrimination, thus severely lowering the accuracy in target miRNA detection. We have reported a novel method that utilizes a designed polycationic peptide-PNA probe to specifically guide the target miRNA migration toward the nanopore, whereas any nontarget nucleic acids without the probe bound is rejected by the nanopore. Consequently, nontarget species are driven away from the nanopore and only the target miRNA can be detected at low concentration. This method is also able to discriminate miRNAs with single-nucleotide difference by using PNA to capture miRNA. Considering the significance and impact of this substantial advance for the future miRNA detection in biofluid samples, we prepared this detailed protocol, by which the readers can view the experimental procedure, data analysis, and resulting explanation.

A three-line lateral flow biosensor for logic detection of microRNA based on Y-shaped junction DNA and target recycling amplification

A rapid, sensitive, and accurate detection strategy for microRNA 16 (miR-16) was developed, which combined the convenience of lateral flow biosensors (LFBs), the design flexibility of Y-shaped junction DNA probe, and the enhancement ability of endonuclease-assisted target recycling amplification. The systemis composed of a molecular beacon (MB) probe, an assistant probe, and endonuclease Nt.BbvCI, which plays the role of signal translation and amplification. In the presence of the target microRNAs (miRNAs), three chains of nucleic acid could hybridize with each other to form a Y-shaped junction structure, which could be recognized by the endonuclease Nt.BbvCI. The MB probe was efficiently cleaved by endonuclease and produced two new DNA fragments, while the regenerated assistant probe and target were hybridized to another MB probe and entered into the next cycle of the amplification. In this way, the detection of the readily biodegradable miRNA was turned into the detection of two DNA fragments in the LFB. Meanwhile, the detection of two different DNAs would improve the accuracy and effectively avoid false results. The amplified products containing DNA fragments were then applied to the lateral flow nucleic acid biosensor (LFNAB) with two test zones, on which specific DNA probes were designed. The formed DNA-DNA/gold nanoparticle (GNP) conjugates were captured and accumulated to produce two red bands in two test zones. The logic judgment of the two test zones provided more accurate and convincing results. Under optimal conditions, the visual detection limit of miR-16 in aqueous solutions was 0.1pM, which is 100-1000 times lower than that of visual or colorimetric methods in the literature. It could be used for on-field and point-of-care testing and meet the urgent demand of sensitive and selective miRNA detection in remote rural areas without costly equipment. The system displayed good universality, compatibility, high specificity, and stability of miRNA detection, which revealed significant potentiality in biomedical diagnosis. Graphical abstract A simple and rapid approach for microRNA-16 (miR-16) detection was developed based on Y-shaped junction DNA probe and the endonuclease-assisted target recycling amplification using lateral flow biosensors.

Multiplexed miRNA northern blots via hybridization chain reaction.

Northern blots enable detection of a target RNA of interest in a biological sample using standard benchtop equipment. miRNAs are the most challenging targets as they must be detected with a single short nucleic acid probe. With existing approaches, it is cumbersome to perform multiplexed blots in which several RNAs are detected simultaneously, impeding the study of interacting regulatory elements. Here, we address this shortcoming by demonstrating multiplexed northern blotting based on the mechanism of hybridization chain reaction (HCR). With this approach, nucleic acid probes complementary to RNA targets trigger chain reactions in which fluorophore-labeled DNA hairpins self-assemble into tethered fluorescent amplification polymers. The programmability of HCR allows multiple amplifiers to operate simultaneously and independently within a blot, enabling straightforward multiplexing. We demonstrate simultaneous detection of three endogenous miRNAs in total RNA extracted from 293T and HeLa cells. For a given target, HCR signal scales linearly with target abundance, enabling relative and absolute quantitation. Using non-radioactive HCR, sensitive and selective miRNA detection is achieved using 2′OMe-RNA probes. The HCR northern blot protocol takes ∼1.5 days independent of the number of target RNAs.

Ultrasensitive detection of microRNAs using catalytic hairpin assembly coupled with enzymatic repairing amplification.

A novel isothermal nucleic acid amplification technology is developed by coupling catalytic hairpin assembly with enzymatic repairing amplification as a highly sensitive and selective platform for miRNA detection.

Highly sensitive and selective miRNA detection based on a closed ring probe and multiple signal amplification.

A highly sensitive and selective miRNA detection approach based on a small circular probe and dual signal amplification has been reported. We successfully applied the assay for quantitative let-7a detection in cell lysates and the results agreed well with those of qRT-PCR.

Label-Free and Enzyme-Free Homogeneous Electrochemical Biosensing Strategy Based on Hybridization Chain Reaction: A Facile, Sensitive, and Highly Specific MicroRNA Assay.

Homogenous electrochemical biosensing strategies have attracted substantial attention, because of their advantages of being immobilization-free and having rapid response and improved recognition efficiency, compared to heterogeneous biosensors; however, the high cost of labeling and the strict reaction conditions of tool enzymes associated with current homogeneous electrochemical methods limit their potential applications. To address these issues, herein we reported, for the first time, a simple label-free and enzyme-free homogeneous electrochemical strategy based on hybridization chain reaction (HCR) for sensitive and highly specific detection of microRNA (miRNA). The target miRNA triggers the HCR of two species of metastable DNA hairpin probes, resulting in the formation of multiple G-quadruplex-incorporated long duplex DNA chains. Thus, with the electrochemical indicator Methylene Blue (MB) selectively intercalated into the duplex DNA chain and the multiple G-quadruplexes, a significant electrochemical signal drop is observed, which is dependent on the concentration of the target miRNA. Thus, using this "signal-off" mode, a simple, label-free and enzyme-free homogeneous electrochemical strategy for sensitive miRNA assay is readily realized. This strategy also exhibits excellent selectivity to distinguish even single-base mismatched miRNA. Furthermore, this method also exhibits additional advantages of simplicity and low cost, since both expensive labeling and sophisticated probe immobilization processes are avoided. Therefore, the as-proposed label-free and enzyme-free homogeneous electrochemical strategy may become an alternative method for simple, sensitive, and selective miRNA detection, and it has great potential to be applied in miRNA-related clinical diagnostics and biochemical research.

Nanomaterials-Based Fluorimetric Methods for MicroRNAs Detection

MicroRNAs (miRNAs) are small endogenous non-coding RNAs of ~22 nucleotides that play important functions in the regulation of many biological processes, including cell proliferation, differentiation, and death. Since their expression has been in close association with the development of many diseases, recently, miRNAs have been regarded as clinically important biomarkers and drug discovery targets. However, because of the short length, high sequence similarity and low abundance of miRNAs in vivo, it is difficult to realize the sensitive and selective detection of miRNAs with conventional methods. In line with the rapid development of nanotechnology, nanomaterials have attracted great attention and have been intensively studied in biological analysis due to their unique chemical, physical and size properties. In particular, fluorimetric methodologies in combination with nanotechnology are especially rapid, sensitive and efficient. The aim of this review is to provide insight into nanomaterials-based fluorimetric methods for the detection of miRNAs, including metal nanomaterials, quantum dots (QDs), graphene oxide (GO) and silicon nanoparticles.

Highly sensitive and selective detection of miRNA: DNase I-assisted target recycling using DNA probes protected by polydopamine nanospheres.

Based on the protective properties of polydopamine nanospheres for DNA probes against nuclease digestion, we have developed a DNase I-assisted target recycling signal amplification method for highly sensitive and selective detection of miRNA.

Target-fueled DNA walker for highly selective miRNA detection.

Artificial DNA motifs as architectural scaffolds have been widely used to assemble a variety of nanoscale devices. Synthetic DNA nanostructures have accomplished mechanical switching in response to external stimuli, suggesting the promise of constructing a walking device that is being used in the field of biosensors. Here, we design a novel miRNA-responsive DNA walker biosensor based on strand displacement cascades and an enzymatic recycling cleavage strategy. By using miRNA as a driving force, the DNA walkers can be activated to move along the track and generate specific signals for let-7a with a high signal-to-noise ratio. This biosensor exhibits excellent analytical performance toward the sensing of let-7a with great specificity for resolving one nucleotide variation and a detection limit of 58 fM. Such an ultraselective sensor shows that DNA nanostructures have great potential in providing platforms for applications in the fields of biosensing, clinical diagnostics and environmental sample analysis.

A G-quadruplex DNA-based, label-free and ultrasensitive strategy for microRNA detection.

MicroRNAs (miRNAs) have been considered to be potent biomarkers for early disease diagnosis and for cancer therapy. The rapid and selective detection of miRNAs without reverse transcription and labelling is highly desired. Herein, we report a simple and label-free miRNA detection method that is based on the Duplex-Specific Nuclease (DSN)-Assisted simple target miRNA recycling procedure. The interaction of the G-quadruplex DNA structure with N-methyl mesoporphyrin IX (NMM) led to a label-free signal output. Under the optimised conditions, this method allowed for simple, rapid, and sequence-specific detection of miR-141 over a dynamic range from 1 fM to 100 nM with a linear range from 1 pM to 100 nM. Moreover, our method offered an excellent capacity to discriminate between miRNA family members with just one mismatched nucleotide. This simple and label-free strategy holds great potential in applications in biomedical research and in early clinical diagnostics.

Ultrasensitive detection of microRNA with isothermal amplification and a time-resolved fluorescence sensor

MicroRNAs (miRNAs) play important roles in a plethora of biological and cellular processes. The levels of miRNAs can be useful biomarkers for cellular events or disease diagnosis, thus the method for sensitive and selective detection of miRNAs is imperative to miRNA discovery, study, and clinical diagnosis. Here we develop a novel method to quantify miRNA expression levels as low as attomolar sensitivity by two-stage exponential amplification reaction (EXPAR) and a time-resolved fluorescence sensor in real samples. The method reveals superior sensitivity with a detection limit of miRNA of 0.1aM under pure conditions. The method also shows the high selectivity for discriminating differences between miRNA family members, thus providing a promising alternative to standard approaches for quantitative detection of miRNA.

Highly Sensitive and Selective Strategy for MicroRNA Detection Based on WS2 Nanosheet Mediated Fluorescence Quenching and Duplex-Specific Nuclease Signal Amplification

MicroRNAs (miRNAs) play vital roles in physiologic and pathologic processes and are significant biomarkers for disease diagnostics and therapeutics. However, rapid, low-cost, sensitive, and selective detection of miRNAs remains a challenge because of their short length, sequence homology, and low abundance. Herein, we report for the first time that WS2 nanosheet can exhibit differential affinity toward short oligonucleotide fragment versus ssDNA probe and act as an efficient quencher for adsorbed fluorescent probes. This finding is utilized to develop a new strategy for simple, sensitive, and selective detection of miRNA by combining WS2 nanosheet based fluorescence quenching with duplex-specific nuclease signal amplification (DSNSA). This assay exhibits highly sensitive and selective with a detection limit of 300 fM and even discriminate single-base difference between the miRNA family members. The result indicates that this simple and cost-effective strategy holds great potential application in biomedical research and clinical diagnostics.

Designing a Polycationic Probe for Simultaneous Enrichment and Detection of MicroRNAs in a Nanopore

The nanopore sensor can detect cancer-derived nucleic acid biomarkers such as microRNAs (miRNAs), providing a noninvasive tool potentially useful in medical diagnostics. However, the nanopore-based detection of these biomarkers remains confounded by the presence of numerous other nucleic acid species found in biofluid extracts. Their nonspecific interactions with the nanopore inevitably contaminate the target signals, reducing the detection accuracy. Here we report a novel method that utilizes a polycationic peptide-PNA probe as the carrier for selective miRNA detection in the nucleic acid mixture. The cationic probe hybridized with microRNA forms a dipole complex, which can be captured by the pore using a voltage polarity that is opposite the polarity used to capture negatively charged nucleic acids. As a result, nontarget species are driven away from the pore opening, and the target miRNA can be detected accurately without interference. In addition, we demonstrate that the PNA probe enables accurate discrimination of miRNAs with single-nucleotide difference. This highly sensitive and selective nanodielectrophoresis approach can be applied to the detection of clinically relevant nucleic acid fragments in complex samples.

A novel fluorescence probe of dsDNA-templated copper nanoclusters for quantitative detection of microRNAs

We have developed a facile and label-free method for sequence-specific microRNA detection using fluorescent dsDNA-templated copper nanoclusters as signal indicators. It is designed such that, the miRNA targets are transferred to the oligonucleotide reporters and act as the scaffold for the synthesis of fluorescent copper nanoclusters via an isothermal exponential amplification reaction, in which the unimolecular DNA designed for miRNA targets is employed as the amplification template with polymerases and nicking enzymes as mechanical activators. Under the selected conditions, the proposed assay allowed sensitive and selective detection of miRNAs with a linear range from 1 pM to 10 nM.

Naked Eye Detection of Lung Cancer Associated miRNA by Paper Based Biosensing Platform

This letter demonstrates a biosensing platform for naked eye detection of miRNA, fabricated using a poly(vinylidene fluoride) thin paper impregnated with positively charged poly(3-alkoxy-4-methylthiophene) as luminescent reporters. The miRNA assay is based on the formation of a duplex and a triplex species between the "reporter and miRNA" and "reporter and miRNA-peptide nucleic acid (PNA) hybrid", which yields two significantly different optical signals, thereby facilitating naked eye detection. This letter illustrates the successful validation of the proposed methodology via a mir21 assay (miRNA sequence associated with lung cancer). Furthermore, this facile platform enables rapid, sensitive, and selective detection of miRNA, at clinically relevant concentration levels as well as single base pair mismatch, without requiring complex and expensive instrumentation.