Specific Detection Of microRNAs Research Articles

AND Logic-Gate-Based Dual-Locking Probe System for the Sensitive Detection of microRNA and APE1.

MicroRNA (miRNA) and apurinic/apyrimidinic endonuclease 1 (APE1) have been reported to be closely associated with cancers, making them potential crucial biomarkers and therapeutic targets. However, focusing on the detection of a single target is not conducive to the diagnosis and prognosis assessment of diseases. In this study, an AND logic-gate-based dual-locking hairpin-mediated catalytic hairpin assembly (DL-CHA) was developed for sensitive and specific detection of microRNA and APE1. By addition of a lock to each of the hairpins, with APE1 and microRNA serving as keys, fluorescence signals could only be detected in the presence of simultaneous stimulation by APE1 and miRNA-224. This indicated that the biosensor could operate as an AND logic gate. DL-CHA exhibited advantages such as a low background, rapid response, and high logic capability. Therefore, the biosensor serves as a novel approach to cancer diagnosis with significant potential applications.

Thermus thermophilus Argonaute-based signal amplifier for highly sensitive and specific microRNA detection.

The prokaryote-derived gene defense system as a new generation of nucleic acid detection tool exhibits impressive performance in the field of molecular diagnosis. Prokaryotic Argonaute (Ago) is a CRISPR-associated protein that is guided by a short DNA (gDNA) and then efficiently cleaves gDNA-complementary nucleic acids and presents unique characteristics that are different from the CRISPR/Cas system. However, the application of Ago in biosensing is still relatively scarce, and many properties of Ago need to be further clarified. In this study, we aim to systematically explore the properties of Thermus thermophilus Argonaute (TtAgo), including the dependence of TtAgo activity on guide DNA (gDNA) length, substrates' length, and the position of gDNA complementary region on the substrate. Based on these properties, we constructed an exonuclease III-assisted target-recycled amplification system (exoAgo) for sensitive miRNA detection. The result showed that exoAgo can be used for miRNA profiling with a detection limit of 12.2pM and single-base-resolution and keep good performance for the detection of complex samples, which indicates that Ago has great application potential in the detection of nucleic acids. In conclusion, this study will provide guidance for further development and utilization of Ago in the field of biosensing.

Primer exchange reaction-amplified protein-nucleic acid interactions for ultrasensitive and specific microRNA detection

Protein-nucleic acid interactions are not only fundamental to genetic regulation and cellular metabolism, but molecular basis to artificial biosensors. However, such interactions are generally weak and dynamic, making their specific and sensitive quantitative detection challenging. By using primer exchange reaction (PER)-amplified protein-nucleic acid interactions, we here design a universal and ultrasensitive electrochemical sensor to quantify microRNAs (miRNAs) in blood. This PER-miR sensor leverages specific recognition between S9.6 antibodies and miRNA/DNA hybrids to couple with PER-derived multi-enzyme catalysis for ultrasensitive miRNA detection. Surface binding kinetic analysis shows a rational Kd (8.9 nM) between the miRNA/DNA heteroduplex and electrode-attached S9.6 antibody. Based on such a favorable affinity, the programmable PER amplification enables the sensor to detect target miRNAs with sensitivity up to 90.5 aM, three orders of magnitude higher than that without PER in routine design, and with specificity of single-base resolution. Furthermore, the PER-miR sensor allows detecting multiple miRNAs in parallel, measuring target miRNA in lysates across four types of cell lines, and differentiating tumor patients from healthy individuals by directly analyzing the human blood samples (n = 40). These advantages make the sensor a promising tool to enable quantitative sensing of biomolecular interactions and precision diagnostics.

A hairpin probe-mediated exponential amplification reaction for highly sensitive and specific detection of microRNAs.

In this work, we propose a hairpin probe-mediated exponential amplification reaction (HEAR) strategy that combines DNA strand displacement with a "who triggers, who gets generated" mode, providing excellent single-base discrimination and a reduced background signal. The detection limit is 19 aM, which is reduced by 3 orders of magnitude compared to traditional exponential amplification approaches. This one-pot strategy also exhibits a wide dynamic range, high specificity and short detection time. It is expected to become a powerful tool for clinical diagnosis.

Interfacial DNA Framework-Enhanced Background-to-Signal Transition for Ultrasensitive and Specific Micro-RNA Detection.

Interfacial DNA self-assembly is fundamental to solid nucleic acid biosensors, whereas how to improve the signal-to-noise ratio has always been a challenge, especially in the charge-based electrochemical DNA sensors because of the large noise from the negatively charged DNA capture probes. Here, we report a DNA framework-reversed signal-gain strategy through background-to-signal transition for ultrasensitive and highly specific electrical detection of microRNAs (miRNAs) in blood. By using a model of enzyme-catalyzed deposition of conductive molecules (polyaniline) targeting to DNA, we observed the highest signal contribution per unit area by the highly charged three-dimensional (3D) tetrahedral DNA framework probe, relative to the modest of two-dimensional (2D) polyA probe and the lowest of one-dimensional (1D) single-stranded (ss)DNA probe, suggesting the positive correlation of background DNA charge with signal enhancement. Using such an effective signal-transition design, the DNA framework-based electrochemical sensor achieves ultrasensitive miRNAs detection with sensitivity up to 0.29 fM (at least 10-fold higher than that with 1D ssDNA or 2D polyA probes) and high specificity with single-base resolution. More importantly, this high-performance sensor allows for a generalized sandwich detection of tumor-associated miRNAs in the complex matrices (multiple cell lysates and blood serum) and further distinguishes the tumor patients (e.g., breast, lung, and liver cancer) from the normal individuals. These advantages signify the promise of this miRNA sensor as a versatile tool in precision diagnosis.

One-step isothermal amplification strategy for microRNA specific and ultrasensitive detection based on nicking-assisted entropy-driven DNA circuit triggered exponential amplification reaction

Sensitive and specific detection of microRNAs (miRNAs) is of critical significance for early diagnosis of cancers such as pancreatic cancer with atypical initial symptoms and high mortality. Despite exponential amplification reaction (EXPAR) is an attractive isothermal amplification method for detecting miRNAs, it faces the problems of the dependence difference and low specificity. To address such challenges, herein, a nicking-assisted entropy-driven DNA circuit triggered exponential amplification reaction (NAED-EXPAR) was firstly employed for ultrasensitive and specific detection of miRNA in “one-pot” manner at constant temperature. Nicking-assisted entropy-driven DNA circuit can specifically recognize the target miRNA, leading to continuous disassembly of DNA substrates via intramolecular toehold-mediated branch migration. During the reaction, the catalytic circuit can consume excess fuel DNA strands to produce a large number of primers. Then the newly formed primers can trigger EXPAR for highly efficient signal amplification. Mechanism analysis shows that the amplification efficiency of NAED-EXPAR is superior than that of single EXPAR. For miR-21, the detection limit of NAED-EXPAR can reach 100 aM, which is at least five orders of magnitude higher than the standard EXPAR that directly uses the target as primer. NAED-EXPAR shows improved specificity for identifying single nucleotide variations and enables sensitive and accurate analysis of miR-21 in human cancer cell lines. This method is expected to offer a new approach for the reliable quantification of miRNAs in complex biological matrices and provide valuable information for early cancer diagnosis.

CRISPR/Cas13-Based Approaches for Ultrasensitive and Specific Detection of microRNAs

MicroRNAs (miRNAs) have a prominent role in virtually every aspect of cell biology. Due to the small size of mature miRNAs, the high degree of similarity between miRNA family members, and the low abundance of miRNAs in body fluids, miRNA expression profiling is technically challenging. Biosensors based on electrochemical detection for nucleic acids are a novel category of inexpensive and very sensitive diagnostic tools. On the other hand, after recognizing the target sequence, specific CRISPR-associated proteins, including orthologues of Cas12, Cas13, and Cas14, exhibit collateral nonspecific catalytic activities that can be employed for specific and ultrasensitive nucleic acid detection from clinically relevant samples. Recently, several platforms have been developed, connecting the benefits of enzyme-assisted signal amplification and enzyme-free amplification biosensing technologies with CRISPR-based approaches for miRNA detection. Together, they provide high sensitivity, precision, and fewer limitations in diagnosis through efficient sensors at a low cost and a simple miniaturized readout. This review provides an overview of several CRISPR-based biosensing platforms that have been developed and successfully applied for ultrasensitive and specific miRNA detection.

Universal and Programmable Rolling Circle Amplification-CRISPR/Cas12a-Mediated Immobilization-Free Electrochemical Biosensor.

The development of a sensing platform with high sensitivity and specificity, especially programmability and universal applicability, for the detection of clinically relevant molecules is highly valuable for disease monitoring and confirmation but remains a challenge. Here, for the first time, we introduce the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system into an immobilization-free electrochemical biosensing platform for sensitively and specifically detecting the disease-related nucleic acids and small molecules. In this strategy, a modular rolling circle amplification (RCA) is designed to transform and amplify the target recognition event into the universal trigger DNA strand that is used as the trigger to activate the deoxyribonuclease activity of CRISPR/Cas12a for further signal amplification. The cleavage of the target-activated blocker probe allows the methylene blue-labeled reporter probes to be captured by the reduced graphene oxide-modified electrode, leading to an obviously increased electrochemical signal. We only need to simply tune the sequence for target recognition in RCA components, and this strategy can be flexibly applied to the highly sensitive and specific detection of microRNAs, Parvovirus B19 DNA, and adenosine-5'-triphosphate and the calculated limit of detection is 0.83 aM, 0.52 aM, and 0.46 pM, respectively. In addition, we construct DNA logic circuits (YES, NOT, OR, AND) of DNA inputs to experimentally demonstrate the modularity and programmability of the stimuli-responsive RCA-CRISPR/Cas12a system. This work broadens the application of the CRISPR/Cas12a system to the immobilization-free electrochemical biosensing platform and provides a new thinking for developing a robust tool for clinical diagnosis.

A self-quenching fluorescence probe-mediated exponential isothermal amplification system for highly sensitive and specific detection of microRNAs.

We designed an efficient self-quenching fluorescence probe and constructed this probe-mediated exponential isothermal amplification system for miRNA detection. Owing to the significant improvement in the detective signal-to-background ratio, a wide dynamic range of 9 orders of magnitude and a limit of detection as low as 0.08 aM can be easily achieved in a single step. Furthermore, benefiting from the additional advantages of high specificity and biocompatibility, the proposed method has been demonstrated to be capable of accurately quantifying miRNA biomarkers in serum, which will provide promising perspectives for clinical diagnosis.

Ultrasensitive and specific microRNA detection via dynamic light scattering of DNA network based on rolling circle amplification

In this paper, rolling circle amplification (RCA) -based self-assembly DNA network was developed for ultrasensitive and specific detection of microRNA (miRNA) with dynamic light scattering (DLS) technology. A dumbbell-shaped padlock probe was designed, which could be connected with itself as a template under the action of T4 DNA ligase to form a dumbbell-shaped sealed probe. In the presence of the target miRNA, the miRNA bound to the toehold domain of the sealed probe. The sealed probe was activated into a circular probe to start RCA reaction by a spontaneous chain displacement reaction. The DNA functionalized AuNPs probe was hybridized with the RCA products to form RCA-DNA-AuNPs polymer with network, whose diameter was measured by DLS. Not only did this method greatly improve the sensitivity of detection, but the specificity of detection increased by using sealed probe as well. The limit of detection (LOD) was 0.11 fM. Furthermore, this strategy has been successfully used to analyze target miRNA in cancer cell samples, which confirmed that RCA-DLS analysis has broad application prospects in early clinical diagnosis.

A PNA-DNA2 Triple-Helix Molecular Switch-Based Colorimetric Sensor for Sensitive and Specific Detection of microRNAs from Cancer Cells.

Peptide nucleic acids (PNAs), the synthetic DNA mimics that can bind to oligonucleotides to form duplexes, triplexes, and quadruplexes, could be advantageous as probes for nucleic acid sequences owing to their unique physicochemical and biochemical properties. We have found that a homopurine PNA strand could bind to two homopyrimidine DNA strands to form a PNA-DNA2 triplex. Moreover, the cyanine dye DiSC2 (5) could bind with high affinity to this triplex and cause a noticeable color change. On the basis of this phenomenon, we have designed a label-free colorimetric sensing platform for miRNAs from cancer cells by using a PNA-DNA2 triple-helix molecular switch (THMS) and DiSC2 (5). This sensing platform can detect miRNA-21 specifically with a detection limit of 0.18 nM, which is comparable to that of the THMS-mediated fluorescence sensing platform. Moreover, this colorimetric platform does not involve any chemical modification or enzymatic signal amplification, which boosts its applicability and availability at the point of care in resource-limited settings. The universality of this approach can be simply achieved by altering the sequences of the probe DNA for specific targets.

DSN/TdT recycling digestion based cyclic amplification strategy for microRNA assay

Sensitive and specific detection of microRNAs (miRNAs) is of great significance for early cancer diagnosis. Here we report a simple and sensitive fluorescence signal amplification strategy that based on DSN/TdT recycling digestion for miRNA detection. DSN initiates DNA digestion on 3′-phosphate-primer/miRNA heteroduplex which causes miRNA recycle. The digested DNA strands with 3′-OH ends enable TdT to synthesize a polydeoxyguanylic tails on the 3′-end. The DNAs with polydeoxyguanylic tails are converted to double-stranded-DNA prior to initiation of DSN/TdT recycling digestion. With the cooperation of TdT and DSN, a new round of digestion and extension is triggered, leading to massive fluorophores separating and signal amplification. The amplification strategy produces large amounts of 3′-OH probes that can be used directly for dsDNA enrichment and DSN digestion. Moreover, both DSN digestion and TdT extension are sequence-independent reaction without the need of complex sequences design. In addition, this strategy is utilized to analyze miRNA samples from MCF-7 cell lysates and Cu (II) ion samples, indicating its potential application in actual sample analysis. The method shows a promising analytical platform for DNA nicking-related studies and tumor biomarkers measuring in clinical diagnostics.

Sensitive and specific microRNA detection by RNA dependent DNA ligation and rolling circle optical signal amplification

MicroRNAs (miRNAs) have been regarded as potential biomarkers in early diagnosis of cancer. Since the high sequence similarity among miRNA family members, biosensing miRNAs with single-base resolution is still a challenge, particularly when the different base is located at the terminal of miRNA. Herein, we developed two real-time fluorescence monitoring methods for miRNA detection utilizing efficient PBCV-1 DNA ligase mediated target miRNA dependent DNA ligation, followed by rolling circle signal amplification. Compared to duplex-specific nuclease (DSN) enhanced rolling circle transcription (RCT) system, nicking endonuclease (NEase) assisted rolling circle amplification (PRCA-NESA) can provide higher amplification efficiency, and achieve a limit-of-detection of 0.5 amol for miR-17 in 10 μL sample. More importantly, benefiting from the unique characteristics of PBCV-1 DNA ligase, we designed an asymmetric PRCA-NESA method, which can greatly discriminate the single-base difference at either 5′- or 3′-terminals of miRNAs. MiR-17 from various tumor cells also can be reliably detected. In conclusion, our strategy exploited the application potential of PBCV-1 DNA ligase in biosensing, and provided a new idea to highly specific miRNA detection, thereby would possess a promising potential for further application in biomedical research and early cancer diagnosis.

Multiplexed fluorometric determination for three microRNAs in acute myocardial infarction by using duplex-specific nuclease and MoS2 nanosheets.

Circulating microRNAs are of diagnostic value for acute myocardial infarction (AMI). This study describes a fluorometric assay for multiplexed detection of the AMI biomarkers microRNA-499, microRNA-133a and microRNA-1. The assay involves the following two steps: (a) duplex-specific nuclease (DSN)-mediated signal amplification using aptamer-based sensor; (b) MoS2 nanosheets-based multiplexed fluorometric signal detection, and fluorometric signals have excitation/emission maxima at 492/518nm for microRNA-499, 565/580nm for microRNA-133a and 649/663nm for microRNA-1. The assay has detection limits of around 100 fM for all three microRNAs. The assay is highly specific and rapid. It demonstrates that the expressions of microRNA-499, microRNA-133a and microRNA-1 are significantly higher in AMI patients. The ROC curves allow a clear distinction to be made between AMI group and non-AMI group. Graphical abstractSchematic representation of a multiplexed fluorometric method based on duplex-specific nuclease (DSN)-mediated signal amplification and MoS2 nanosheets-based fluorometric signal detection (DSN-MoS2) for rapid, sensitive and specific detection of microRNAs in AMI.

Electrochemical Biosensor for MicroRNA Detection Based on Cascade Hybridization Chain Reaction

AbstractThe development of effective strategies for sensitive and specific detection of microRNAs will facilitate clinical diagnostics. Here, we utilize cascade hybridization chain reaction as an enzyme‐free isothermal amplified strategy, and construct label‐free electrochemical biosensors for microRNAassay. MicroRNA initiated autonomous assembly of DNA nanostructures through the toehold‐mediated strand displacement reaction. The assembly behavior was examined by gel electrophoresis and morphology of branched DNA nanostructure was verified by atomic force microscope. This cascade hybridization chain reaction leads to extended growth of DNA chains and higher amplification efficiency compared with the regular hybridization chain reaction. The performance of the sensors was characterized by electrochemical impedance spectroscopy, differential pulse voltammetry, and chronocoulometric analysis. The prepared biosensor exhibits high sensitivity and a lower detection limit of 11 pM for the determination of microRNA‐21. This electrochemical assay based on cascade hybridization chain reaction is specific and can distinguish between highly homologous targets such as fully complementary target microRNA‐21, single, or two‐base mismatched sequences and other family members. These findings open the possibility of using cascade hybridization chain reaction as possible amplified electrochemical sensing platforms.

Signal-on electrochemiluminescence biosensor for microRNA-319a detection based on two-stage isothermal strand-displacement polymerase reaction

MicroRNAs play crucial role in regulating gene expression in organism, thus it is very necessary to exploit an efficient method for the sensitive and specific detection of microRNA. Herein, a signal-on electrochemiluminescence biosensor was fabricated for microRNA-319a detection based on two-stage isothermal strand-displacement polymerase reaction (ISDPR). In the presence of target microRNA, amounts of trigger DNA could be generated by the first ISDPR. Then, the trigger DNA and the primer hybridized simultaneously with the hairpin probe to open the stem of the probe, and then the ECL signal will be emitted. In the presence of phi29 DNA polymerase and dNTPs, the trigger DNA could be displaced to initiate a new cycle which was the second ISDPR. Due to the two-stage amplification, this method presented excellent detection sensitivity with a low detection limit of 0.14 fM. Moreover, the applicability of the developed method was demonstrated by detecting the change of microRNA-319a content in the leaves of rice seedlings after the rice seeds were incubated with chemical mutagen of ethyl methanesulfonate.

Electrochemical strategy for ultrasensitive detection of microRNA based on MNAzyme-mediated rolling circle amplification on a gold electrode

The authors describe an electrochemical strategy for ultrasensitive and specific detection of microRNA (miRNA). It is based on both multicomponent nucleic acid enzyme (MNAzyme) amplification and rolling circle amplification (RCA). In the presence of target miRNAs, partial enzyme A (partzyme A) and partial enzyme B (partzyme B) are assembled to form active MNAzymes. Once formed, the MNAzymes catalyze the cleavage of the hairpin substrates to liberate biotinylated fragments which hybridized with the capture probes immobilized on a gold electrode. The RCA is then initiated to form a product that binds many detection probes. Finally, the amperometric signal (best acquired at a working voltage of 0.22 V vs. Ag/AgCl) is obtained by employing the streptavidinylated alkaline phosphatase as the enzyme. This biosensor has a 1.66 fM detection limit, and a dynamic range that extends from 10 fM to 1 nM. It displays specificity down to single mismatch discrimination of target miRNAs and good reproducibility. It was successfully applied to the determination of miRNA in total RNA samples extracted from human breast adenocarcinoma MCF-7 cells.

C222: The detection of specific microRNAs in urine as potential biomarker of bladder cancer

C222: The detection of specific microRNAs in urine as potential biomarker of bladder cancer

Visible-light induced photoelectrochemical biosensor for the detection of microRNA based on Bi2S3 nanorods and streptavidin on an ITO electrode

We demonstrate a photo-electrochemical biosensor for the sensitive and specific detection of microRNA using Bi2S3 nanorods as a photoactive material and streptavidin as the unit that inhibits photocurrent. Bi2S3 nanorods were synthesized hydrothermally in organic phase and displayed excellent light-to-current conversion efficiency. The Bi2S3 was deposited on an indium tin oxide (ITO) slice and then modified with gold nanoparticles onto which biotinylated hairpin probe DNA was deposited as a monolayer. Following hybridization between the biotinylated probe DNA and the target microRNA, the stem-loop structure of the probe DNA was unfolded and the biotin directed outwards into the solution. Streptavidin was then added to bind to biotin via the strong streptavidin-biotin interactions. This causes the photocurrent of the modified ITO to decrease due to steric hindrance that blocks the transfer of electrons from added ascorbic acid to the surface of the electrode. The method has a detection limit as low as 3.5 fM of microRNA and can excellently discriminate even singly mismatched microRNA. The method was successfully applied to investigate the effect of abscisic acid on the expression level of microRNA-159a in seeds of Arabidopsis thaliana. We conclude that the assay presented here has a large potential as a method for quantification of microRNA and for studying the epigenetic regulation of flowering plants.

Target-Responsive, DNA Nanostructure-Based E-DNA Sensor for microRNA Analysis

Because of the short size and low abundance of microRNAs, it is challenging to develop fast, inexpensive, and simple biosensors to detect them. In this work, we have demonstrated a new generation (the third generation) of E-DNA sensor for the sensitive and specific detection of microRNAs. Our third generation of E-DNA sensor can sensitively detect microRNA target (microRNA-141) as low as 1 fM. The excellent specificity has been demonstrated by its differential ability to the highly similar microRNA analogues. In our design, the use of DNA tetrahedron ensures the stem-loop structure in well controlled density with improved reactivity. The regulation of the thermodynamic stability of the stem-loop structure decreases the background signal and increases the specificity as well. The enzymes attached bring the electrocatalytic signal to amplify the detection. The combination of these effects improves the sensitivity of the E-DNA sensor and makes it suitable to the microRNA detection. Finally, our third generation of E-DNA sensor is generalizable to the detection of other micro RNA targets (for example, microRNA-21).