Duplex-specific Nuclease Research Articles

Self-Adaptive Activation of DNAzyme Nanoassembly for Synergistically Combined Gene Therapy.

DNAzymes represents a promising toolbox for gene silencing but face challenges related to poor substrate accessibility in specific cells. Herein, a compact DNA nanoassembly, incorporating multimeric therapeutic DNAzyme, was prepared for selective delivery of gene-silencing DNAzyme with requisite cofactors and auxiliary chemo-drugs. By virtue of the sequence-conservative duplex-specific nuclease, the endogenous miRNA catalyzes the successive and site-specific cleavage of DNA nanoassembly substrate (nominated as the localized RNA walking machine) and thus ensures the liberation/activation of therapeutic agents with high accuracy and efficacy. The miR-10b-stimulated DNAzyme was designed to downregulate the TWIST transcription factor, an upstream promotor of miR-10b, thus acquiring the self-sufficient downregulation of TWIST/miR-10b signaling nodes (self-adaptive negative feedback loop) for abrogating tumor metastasis and chemo-resistance issues.

Silver Nanoclusters-Decorated Porous Microneedles Coupling Duplex-Specific Nuclease-Assisted Signal Amplification for Sampling and Detection of MicroRNA in Interstitial Fluid.

MicroRNAs (miRNAs) in dermal interstitial fluid (ISF) have recently been recognized as clinically promising biomarkers for the diagnosis and prognosis of cancer. However, the detection poses significant challenges, primarily due to the low abundance of miRNAs and the limitations of current sampling techniques. To address this issue, we develop novel porous microneedles (PMNs) array-based sensor composed of poly(vinyl alcohol) porous hydrogel and DNA-templated silver nanoclusters (AgNCs) to facilitate the enrichment and highly sensitive detection of ISF miRNA. Leveraging the capillary action facilitated by its unique porous structure and the swelling properties of the hydrogel, the PMNs array can efficiently extract 2.7 ± 0.3 mg of ISF within 5 min. Additionally, the interconnected pores within the PMNs array contribute to an increased specific surface area, thereby offering a convenient platform for the decoration of DNA-templated AgNCs. The immobilized large amount of AgNCs effectively capture the target miRNA from the extracted ISF, resulting in miRNA-induced fluorescence quenching of AgNCs. Subsequently, the introduction of the duplex-specific nuclease leads to the cleavage of DNA in DNA-RNA heteroduplexes, which release miRNA to interact with other AgNCs. This process of target recycling triggers a further reduction in fluorescence intensity, thereby enabling sensitive detection of the low-abundant miRNA down to 1.6 pM. Both in vitro and in vivo experiments validate the efficacy of the AgNCs immobilized PMNs array for the detection of miRNA biomarkers in ISF within minutes. These results indicate that the proposed PMNs array-based sensor holds great potential for the development of noninvasive personalized diagnostic strategies.

Biosensor development for diabetes diagnosis: Determining relevant miRNA using a newly developed N-annulated perylene fluorescent dye

MicroRNAs (miRNAs) have emerged as essential biomarkers for disease diagnosis, and several techniques are available to determine type 2 diabetes (T2D) relevant miRNAs. However, detecting circulating miRNAs can be challenging due to their small size, low abundance, and high sequence similarity, often requiring sensitive detection approaches combined with additional amplification processes. Laser-induced fluorescence (LIF) is a classic analytical method suitable for sensitively detecting trace amounts of nucleotide acid. Duplex-specific nuclease (DSN)-mediated amplification recently gained attention due to its catalytic activity based on target recycling, demonstrating a promising approach for miRNA amplification. This work developed a novel N-annulated perylene fluorescent dye to create a biosensor to analyze the miRNA (miR-223) relevant to T2D. The amine-reactive fluorescent dye assists the amidation reaction for nucleotide labeling, giving the oligonucleotide probe a high fluorescence quantum yield and sufficient water solubility. By combining the locked nucleic acid (LNA) modified oligonucleotide fluorescent probe to enhance the stability of LNA/RNA hybrids, thereby improving the DSN-mediated target miR-223 recycling for signal amplification, the proposed biosensor can highly selectively determine miR-223 with a limit of detection (LOD, S/N = 3) of 9.5 pM. When applied to real-world samples, the biosensor demonstrated its potential to distinguish between T2D patients and healthy controls.

Duplex-specific-nuclease-assisted graphene field-effect transistor biosensor: A novel platform for preamplification-free detection of cancer related miRNA

The pivotal role of microRNA (miRNA) in cancer diagnosis and monitoring is well recognized, yet current detection methods remain complex and costly, given the rarity of tumor miRNA in the bloodstream. This study proposes a simple and elegant miRNA sensing platform utilizing duplex-specific nuclease (DSN) and ultrasensitive graphene field-effect transistor (GFET) biosensors. The DSN-assisted GFET enables preamplification-free detection of miR-21, a promising cancer biomarker, at dilutions down to 25 aM. Along with the successful validation of its excellent specificity using clinical plasma samples without RNA extraction process, this innovative approach holds great promise for improving cancer diagnostics and monitoring.

Sequence-Unconstrained DNA Computing: DSN cycling and PER circuitry for dynamic miRNAs analysis and multifunctional logic operations

DNA strand displacement and enzyme-driven catalysis are pivotal in regulating molecular interactions and constructing molecular logic circuits. However, their application in intricate hierarchical cascading networks has been hindered by sequence crosstalk restrictions on the multiple inputs and outputs. In this study, we present a sequence-unconstrained strategy to implement versatile molecular logic circuits, relying on duplex-specific nuclease (DSN)-assisted cycling and primer exchange reaction (PER) amplification circuitry (DSN-PER). Employing linear capture probe (CP) conjugated with magnetic beads (MBs) significantly minimizes crosstalk reactions compared to the previous molecular beacon probe (MBP) for DNA barcoding. Importantly, the sequence design of “barcodes” is fully independent of target binding sites on MBs, allowing for the sensitive detection of a broad range of microRNAs (miRNAs) using the DSN-PER method. Specifically, this platform supports the programming of common logic computations (YES, NOT, OR, INHIBIT, AND, and NOR) and hierarchical circuit operation (OR-AND) in response to a specific pattern of varying miRNAs. These versatile logic circuits offer unique features with flexible sequence design, potentially broadening the applications of dynamic nucleic acid nanotechnologies in multiplexed bio-computation, intelligent diagnostics, and sophisticated DNA nanodevices.

Brainstem transcriptomic changes in male Wistar rats after acute stress, comparing the use of duplex specific nuclease (DSN)

In this work, we have analyzed the transcriptomic changes in the brainstem of male Wistar rats 2 h after an acute stress exposure. We performed duplex-specific nuclease normalization of cDNA libraries and compared the results back-to-back for the first time. Based on our RNAseq data, we selected reference genes for RT-qPCR that are best suited for acute stress experiments. Most genes were upregulated. We detected a massive shift in neuropeptide Crh, Trh,Cga, Tshb, Uts2b, Tac4, Lep and neuropeptide receptor Hcrtr1, Sstr5, Bdkrb2, Crhr2 signaling, as well as glutamate Grin3b, Grm2 and GABA Gpr156, acetylcholine Chrm4,Chrne, adrenergic Adra2b receptors expression. A strong increase in the expression of intermediate filaments Krt83/Krt86/Krt80/Krt84/Krt87/Krt4/Krt76 and motor proteins Myo7a, Klc3 was detected. Remarkably, in the absence of astrocyte activation, we also observed signs of microglial activation at this time point. Both expression of anti-inflammatory cytokines Il13, Ccl24 and pro-inflammatory cytokine receptors Il9r, Il12rb1, Tnfrsf14, Tnfrsf13c, Tnfrsf25, Tnfrsf1b were increased. In the Wnt signaling pathway, we observed increased expression of ligands-receptors Wnt1, Wnt11, Ror2 and also negative regulators Notum, Sfrp5, Sost. RNAseq results after DSN treatment correlated at a high level with RNAseq results without DSN, but there was a proportion of genes that shifted their logFC values. They are mostly rare transcripts TPM 1–10 with higher 0.5–0.9 GC content.

Near-infrared light induced cathodic photoelectrochemical biosensor for duplex-specific nuclease mediated detection of miRNA166-a

The photoelectrochemical (PEC) biosensor is a new sensing platform which has the merits of low background signal, low cost, easy miniaturization and so on. However, at present, most PEC biosensors utilize visible light or ultraviolet light as the excitation light source. This type of light has high energy and may cause some light damage to the detected object and some photoelectric materials, thus limiting its application scope. Herein, based on NaYF4:Yb,Tm/BiOBr0.8I0.2 a cathode PEC biosensor that can be excited by near-infrared light was constructed to avoid the occurrence of anode false positives and light damage. Through two cycles of the miRNA cycle assisted by Duplex-Specific Nuclease (DSN) and the dissolved oxygen cycle assisted by gold nanoparticles during the detection process, the detection signal was amplified and miRNA166-a was sensitively detected. By using up-conversion materials, the near-infrared light of 980 nm can be converted into ultraviolet light and visible light, thus exciting the photogenerated carriers of BiOBr0.8I0.2. The photocurrent reaction shows a positive correlation with the quantity of gold nanoparticles present on the electrode surface, which are derived from the reporter molecules produced by the hybridization of miRNA166-a and H1. It has a good linear relationship and selectivity towards miRNA166-a, and the detection limit is 0.32 fM. This sensing method provides a cathode PEC biosensor, which can be excited by near-infrared light. The overall light damage of the system is low, and the possibility of false positive of anode PEC biosensor can be avoided, which has certain significance.

Electrochemical Biosensing Platform Based on Toehold-Mediated Strand Displacement Reaction and DSN Enzyme-Assisted Amplification for Two-Target Detection.

Simultaneous detection of multiple targets is of great significance for accurate disease diagnosis. Herein, based on duplex-specific nuclease (DSN) assisted signal amplification and the toehold-mediated strand displacement reaction (TSDR), we constructed an electrochemical biosensor with high sensitivity and high specificity for dual-target detection. MiRNA-141 and miRNA-133a were used as the targets, and ferrocene (Fc) and methylene blue (MB) with significant peak potential differentiation were used as the electrochemical signal probes. The elaborately designed hairpin probe H1, which was fixed on the electrode surface, could be hybridized with the target miRNA-141 to perform signal amplification by the DSN-assisted enzyme cleavage cycle; thus, miRNA-141 could be detected by Fc signal changes at 0.41 V. The hairpin H1 can also combine with the MB-labeled signal probe (SP) output from miRNA-133a-induced TSDR, and the detection of miRNA-133a can be realized according to the response signal generated by MB at -0.26 V. The two sensing lines are independent of each other, and there is no mutual interference in the detection process. Therefore, two independent detection lines could be connected in series, and the simultaneous detection of two targets can be achieved on a single electrode. This novel detection strategy provides a new way to simultaneously detect different biomarkers.

DSN signal amplification strategy based nanochannels biosensor for the detection of miRNAs

MiRNA-21 is recognized as an important biological marker for the diagnosis, treatment, and prognosis of breast cancer. Here, we have created a nanochannel biosensor utilizing the duplex-specific nuclease (DSN) signal amplification strategy to achieve the detection of miRNAs. In this system, DNA as the capture probe was covalently immobilized on the surface of nanochannels, which hybridized with the target miRNA and forms RNA/DNA duplexes. DSN could cleave the probe DNA in RNA/DNA duplexes, recycling target miRNA, which may again hybridized with other DNA probes. After N cycles, most of the DNA probes had been cleaved, and the content of miRNA could be quantified by detecting changes in surface charge density. This biosensor can distinguish miR-21 from non-complementary miRNAs and one-base mismatched miRNAs, with reliable detection limits as low as 1 fM in PBS. In addition, we had successfully applied this method to analysis of total RNA samples in MCF-7 cells and HeLa cells, and the nanochannels had also shown excellent responsiveness and strong anti-interference ability. This new method is expected to contribute to miRNA detection in clinical diagnostics, providing a unique approach to detecting and distinguishing disease-associated molecules.

Single Particle Analysis-Enhanced DNA Walking Machine for Sensitive miRNA Detection.

DNA walking machines have achieved significant breakthroughs in areas such as biosensing, bioimaging, and early cancer diagnosis, facilitated by the self-assembly of DNA or its combination with other materials, such as magnetic beads and metal nanoparticles. However, current DNA walking machine strategies are constantly challenged by inadequate analytical sensitivity, while sophisticated signal amplification procedures are often indispensable. Single-particle inductively coupled plasma mass spectrometry (SP-ICPMS) provides superior sensitivity and can effectively discriminate between background noise and detected signals due to the large number of metal atoms in a nanoparticle and the concentrating effect of single nanoparticle detection. In this study, we present a novel approach utilizing single nanoparticle counting and duplex-specific nuclease (DSN)-assisted signal amplification to construct a 3D DNA walking machine for detecting the aggressive prostate cancer (PCa) biomarker miRNA-200c. The proposed strategy showed an improvement in sensitivity with a detection limit (LOD) of 0.93 pM (28 amol) and was successfully applied in human serum samples. To the best of our knowledge, this is the first report of the DNA walking machine with single nanoparticle counting study.

Construction of a 3D Quantum Dot Nanoassembly with Two-Step FRET for One-Step Sensing of Human Telomerase RNA in Breast Cancer Cells and Tissues.

Telomerase is an important biomarker for early diagnosis of cancers, but current telomerase assays usually rely on measuring the extension products of telomerase substrates, which increases the assay complexity. More evidence indicates that human telomerase RNA (hTR), as a core component of telomerase, is positively correlated with the telomerase activity. Herein, we demonstrate the development of a duplex-specific nuclease (DSN)-propelled 3D quantum dot (QD) nanoassembly with two-step Föster resonance energy transfer (FRET) for the one-step sensing of hTR in breast cancer cells and tissues. This assay involves only one hairpin probe modified with a Cy5 at the sixth base from the 5'-biotin end and a BHQ2 at the 3'-terminus, which integrates three functions of target recognition, target recycling amplification, and signal readout. The anchoring of the hairpin probe on the 605QD surface results in the formation of a 3D 605QD-Cy5-probe-BHQ2 nanoassembly in which two-step FRET occurs among the 605QD, Cy5, and BHQ2 quencher. Notably, the formation of 605QD-Cy5-probe-BHQ2 nanoassembly facilitates the reduction of background signal and the increase of signal-to-background ratio due to its dense, highly oriented nucleic acid shell-induced steric hindrance effect. This assay can achieve one-step and rapid detection of hTR with a detection limit of 2.10 fM, which is the simplest and most rapid hTR assay reported so far. Moreover, this assay can efficiently distinguish single-base mismatched sequences, and it can discriminate the hTR level between breast cancer patients and healthy donors with a high accuracy of 100%, with great prospects for early diagnosis of cancers.

CRISPR-Cas12a-activated proximity catalytic hairpin assembly strategy for sensitive gene analysis in Streptococcus mutans

The cnm gene in Streptococcus mutans (S. mutans) mediates bacterial binding to extracellular matrix and invasion within host cells, and has been found to be correlated with the occurrence of brain microbleeds. Hence, it is crucial to develop a reliable method to evaluate cnm-positive S. mutans. Herein, we propose a novel diagnostic platform utilizing Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a system for accurate identification of the cnm gene, along with proximity catalytic hairpin assembly (CHA) for enhanced signal amplification, for the detection and analysis of the cnm gene in S. mutans. When the cnm gene is present, it activates trans-cleavage activity of Cas12a to trigger the proximity CHA. In contrast to conventional CHA, the hairpin probes in proximity CHA were linked in a sequential manner using a liner ssDNA sequence. Additionally, the CHA process was facilitated by the Duplex-Specific Nuclease (DSN), which enhanced the efficiency and speed of the reaction. The approach exhibits several notable benefits, including i) the target recognition capability of crRNA in CRISPR-Cas system enhances the detection specificity; ii) Cas12a-crRNA is independent on the PAM during recognizing target dsDNA; iii) the use of the DSN and the proximity design of the H1-H2 complex has significantly enhanced the reactive efficiency of proximity CHA. As a result, the method exhibits a low limit of detection of 47 aM and a high selectivity to cnm gene. Moreover, the approach has been effectively employed in clinical settings to accurately differentiate S. mutans from a combination of microorganisms.

Sensitive and reliable miRNA analysis based on cyclic reverse transcription and CRISPR-Cas12a-assisted signal cycle

MicroRNAs (miRNAs), a category of small molecules that possess significant regulatory capabilities, have been extensively employed as biomarkers in the domain of biosensing to facilitate the early detection of diverse ailments. However, sensitive and accurate miRNA detection remains a huge challenge due to the high similarity between the homologous sequences and low abundance. Therefore, it is essential to develop methods with high sensitivity and specificity for miRNA detection. In this study, we present the development of a signal cycle-based platform that utilizes cyclic reverse transcription (CRT) and CRISPR-Cas12a to enable the precise and sensitive detection of microRNAs. The CRT mechanism facilitates precise target recognition in the presence of target miRNA, thereby converting miRNA signals to DNA signals. The trans-cleavage activity of the Cas12a protein is triggered by the formation of complete hairpin-shaped CRT products; this results in the cleavage of the DNA section contained in the H probe, while the RNA section (“4”@MBs) remains loaded onto the surface of magnetic beads (MB). By binding with the “reporter” sensor, the “4” sequences create an RNA/DNA duplex that the duplex-specific nuclease (DSN) can recognize. The “reporter” probe is thus metabolized, leading to the reappearance of the fluorescence signal. By capitalizing on the exceptional fidelity and selectivity of CRISPR/Cas12a, as well as the substantial impact of triggered enzymatic cycle amplification, this approach demonstrated remarkable sensitivity and specificity in miRNA detection, even in a complex environment containing 10% fetal bovine serum (FBS) and a serum sample. In contrast, a detection limit of 3.2 fM is conceivable. Furthermore, this approach maintained a notable degree of stability, which was anticipated to result in the detection of miRNAs in an effective and sensitive manner.

An integrated liposome-based microfluidic strategy for rapid colorimetric analysis: A case study of microRNA-21 detection

In this study, a novel integrated liposome-based microfluidic platform combined with a smartphone was designed for the rapid colorimetric detection of microRNA-21 (miRNA-21) in real samples. The flowing surface-functionalized liposomes were first captured by nucleic acid-functionalized Au nanoparticles in the microfluidic chip. In the presence of miRNA-21, the DNA strand modified on the surface of Au nanoparticles hybridized with the target to form double-stranded products and was cleaved by duplex-specific nuclease (DSN) enzyme, causing the liposomes to be re-released. Then, as the liposomes in the colorimetric module were lysed and the “cellular” contents were released, a step-by-step “glucose-glucose oxidase-3,3′,5,5′-tetramethylbenzidine (TMB)” colorimetric reaction process catalyzed by the G-quadruplex/hemin was triggered. The grayscale values were recorded and recognized by the smartphone camera for miRNA-21 analysis. The advantages of the present strategy included the portability of smartphone-based colorimetric assay, the encapsulation and transport of reactants by liposomes and the low solvent usage of microfluidic chip. Under optimal conditions, this assay exhibited a wide linear range from 1 pM to 1 nM (r2 = 0.9981), and the limit of detection of miRNA-21 was as low as 0.27 pM. Moreover, the high specificity of this strategy allowed its successful application to the rapid analysis of miRNA-21 in real blood serum samples of people with type 2 diabetes.

Multiplexed discrimination of SARS-CoV-2 variants via duplex-specific nuclease combined MALDI-TOF MS.

The frequent mutations in SARS-CoV-2 significantly increase the virus's pathogenicity and transmissibility while also diminishing the effectiveness of vaccines. Consequently, assays capable of rapidly and simultaneously identifying multiple SARS-CoV-2 variants are essential for large-scale applications that aim to monitor the evolution of the virus. In this work, we propose a method combining duplex-specific nuclease (DSN)-assisted cyclic amplification with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) detection, enabling the simultaneous identification of multiple SARS-CoV-2 variants at high-throughput. Due to the high specificity of DSN, single-base mutations can be resolved by the method. With ultra-sensitive detection by MALDI-TOF MS, a limit of detection of 100 pM viral RNA fragment was demonstrated. The assay was used for simultaneous identification and typing of SARS-CoV-2 Alpha, Beta, and Delta variants. The whole assay can be accomplished within 3 h, and the amplification is performed under constant temperature, making the technique simple in operation and efficient. It is also feasible to extend the technique to the detection of many other variants of the virus. We expect that the method can add value to the rapid screening of viral variants and can play an important role in pandemic control.

CRISPR-Cas12a based target recognition initiated duplex-specific nuclease enhanced fluorescence and colorimetric analysis of cell-free DNA (cfDNA)

The significant role of cell-free DNA (cfDNA) for disease diagnosis, including cancer, has garnered a lot of attention. The challenges of creating target-specific primers and the possibility of false-positive signals make amplification-based detection methods problematic. Fluorescent biosensors based on CRISPR-Cas have been widely established, however they still require an amplification step before they can be used for detection. To detect cfDNA, researchers have created a CRISPR-Cas12a-based nucleic acid amplification-free fluorescent biosensor that uses a combination of fluorescence and colorimetric signaling improved by duplex-specific nuclease (DSN). DSN-assisted signal recycling is initiated in H1@MBs when the target cfDNA activates the CRISPR-Cas12a complex, leading to the degradation of single-strand DNA (ssDNA) sequences. This method has an extremely high detection limit for the BRCA-1 breast cancer gene. In addition to measuring viral DNA in a field-deployable and point-of-care testing (POCT) platform, this fast and highly selective sensor can be used to evaluate additional nucleic acid biomarkers.

A simultaneous strategy with multiple-signal amplification and self-calibration for ultrasensitive assay of miRNA-21 based on 3D MNPs-IL-rGO-AuNPs

MicroRNA-21 (miRNA-21) is a significant biomarker for the development and progression of diverse cancers but is present in relatively low concentrations. Detecting such low-abundance molecules accurately can be challenging, especially in early-stage cancers where the concentration may be even lower. Herein, a self-calibration biosensing platform based on 3D novel MNPs-IL-rGO-AuNPs nanocomposites was successfully established for the ultrasensitive detection of miRNA-21. Duplex-specific nuclease (DSN) was introduced to recognize perfectly matched duplexes and trigger target recycling, enhancing the specificity and sensitivity of the biosensor. DSN-assisted target recycling, in conjunction with magnetic separation enrichment and high-performance MNPs-IL-rGO-AuNPs, collectively formed a multiple-signal amplification strategy. The obtained biosensor could output dual signals in both electrochemical and fluorescent modes, enabling self-correcting detection to enhance the accuracy. The obtained dual-mode biosensor prepared exhibited a wide detection range from 5 fM to 100 nM with a remarkably low LOD of 1.601 fM. It accomplished the sensitive evaluation of miRNA-21 in total RNA extracted from various human cancer cell lines and normal cell lines. Additionally, the greatly satisfactory outcomes in the analysis of human serum samples suggested that the proposed biosensor was a powerful screening candidate in early clinical diagnosis of cancer.

Programmably engineered stochastic RNA nanowalker for ultrasensitive miRNA detection.

A programmably engineered stochastic RNA nanowalker powered by duplex-specific nuclease (DSN) is developed. By utilizing poly-adenine-based spherical nucleic acids (polyA-SNA) to accurately regulate the densities of DNA tracks, the nanowalker showcases its capability to identify miRNA-21, miRNA-486, and miRNA-155 with quick kinetics and attomolar sensitivity, positioning it as a promising option for cancer clinical surveillance.

Twice-walk strategy based on three-dimensional DNA walking machine driven by duplex-specific nuclease.

A twice-walk strategy based on a three-dimensional (3D) cleat-equipped DNA walking machine with a high signal amplification efficiency was investigated for ultrasensitive detection of miRNA. Impressively, addition of duplex-specific nuclease (DSN) just once drove the twice-walk strategy, making the strategy simpler. With the advantages of being simple, rapid and ultrasensitive, the biosensor offers potential for use in early clinical diagnosis.

Simultaneous detection of breast cancer biomarkers HER2 and miRNA-21 based on duplex-specific nuclease signal amplification.

The detection of a single biomarker is prone to false negative or false positive results. Simultaneous analysis of two biomarkers can greatly improve the accuracy of diagnosis. In this work, we designed a new method for coinstantaneous detection of two breast cancer biomarkers miRNA-21 and HER2 using the properties of duplex-specific nuclease (DSN). Cy5-labeled DNA1 and FAM-labeled DNA2 are used as signal probes to distinguish the two signals. When the sample contains the targets HER2 and miRNA-21, HER2 binds to the HER2 aptamer on the double-stranded DNA2, while miRNA-21 binds to the complementary DNA1. Then, DSN enzyme is added to cut the DNA probes adsorbed on the HER2 aptamer and miRNA-21, releasing the fluorescent groups, which can be readsorbed to the empty sites, thus repeating the cutting of the probes and producing an exponential signal amplification with two distinct fluorescent signals. The detection limits of miRNA-21 and HER2 are 1.12 pM and 0.36 ng mL-1, respectively, with linear ranges of 5 pM to 50 pM and 1 ng mL-1 to 15 ng mL-1. The method was validated in real biological samples, providing a new approach for synchronous analysis of important markers in breast cancer.