Hairpin Substrates Research Articles

One-step cascade amplification system based on entropy-driven catalysis and DNAzyme triggered DNA walker for label-free detection of acetamiprid

Herein, an electrochemical aptasensor for highly sensitive detection of acetamiprid (ACE) was constructed based on a one-step cascade amplification strategy. This innovative strategy integrated DNA walker containing DNAzyme sequence into entropy-driven catalysis (EDC) system. The trigger strand was released by aptamer-specific binding to ACE, initiating the EDC amplification circuit and delivering DNA walker strands. The dangling DNA walker continuously bound and cleaved hairpin substrate to form G-quadruplex fragments with the assistance of Mg2+. The G-quadruplex fragments folded and captured hemin to form multitudinous G-quadruplex/hemin complexes in the presence of K+, generating significantly enhanced current, enabling enzyme-free, label-free and highly sensitive detection of ACE, with a linear detection range of 100 fM to 50 nM and a detection limit of 68.36 fM (S/N = 3). The constructed aptasensor achieved the reliable detection of ACE in vegetable soil and cucumber samples, demonstrating its potential application prospects in environmental protection and food supervision.

A DNA machine-based magnetic resonance imaging nanoprobe for in vivo microRNA detection

In situ monitoring microRNA (miRNA) expression in vivo holds immense potential for directly visualizing the occurrence and progression of tumors. However, the significant barrier to developing a probe that can overcome the low abundance of miRNAs while providing an output signal with unlimited tissue penetration depth remains formidable. In this study, we developed a DNA machine-based magnetic resonance imaging nanoprobe (MRINP) for amplified detection of miR-21 in vivo. The MRINP was constructed with superparamagnetic Fe3O4 nanoparticles (NPs), paramagnetic Gd-DOTA complexes, and miR-21-activated DNA machines; the DNA machine was composed of hairpin DNAzyme (HD) strands serving as the DNAzyme walker and hairpin substrate (HS) strands serving as the track. Once uptake into tumor cells, the intracellular miR-21 specifically recognized and hybridized with the HD strand, restoring the activity of DNAzyme. Subsequently, the DNAzyme walker autonomously traveled on the surface of MRINP, and each step movement of the DNAzyme walker resulted in the cleavage of its substrate strands and the ensued release of the Gd-DOTA complex-labeled oligonucleotides, turning on the T1 signal of Gd-DOTA complexes for in situ imaging of miR-21 in tumor-bearing mice. This strategy would offer a promising approach for mapping tumor-specific biomarkers in vivo with unlimited penetration depth.

Site-specific RNA modification via initiation of in vitro transcription reactions with m6A and isomorphic emissive adenosine analogs.

The templated enzymatic incorporation of adenosine and its analogs, including m6A, thA and tzA into RNA transcripts, has been explored. Enforced transcription initiation with excess free nucleosides and the native triphosphates generates 5'-end modified transcripts, which can be 5'-phosphorylated and ligated to provide full length, singly modified RNA oligomers. To explore structural integrity, functionality and utility of the resulting non-canonical purine-containing RNA constructs, a MazF RNA hairpin substrate has been synthesized and analyzed for its susceptibility to this endonuclease. Additionally, RNA substrates, containing a singly incorporated isomorphic emissive nucleoside, can be used to monitor the enzymatic reactions in real-time by steady state fluorescence spectroscopy.

Dephosphorylation Switch DNAzyme-RCA Circuit: A Robust Strategy for the Homogeneous and Reliable Detection of FTO Demethylase.

Fat mass and obesity-associated protein (FTO) plays a crucial role in regulating the dynamic modification of N6-methyladenosine (m6A) in eukaryotic mRNA. Sensitive detection of the FTO level and efficient evaluation of the FTO demethylase activity are of great importance to early cancer diagnosis and anticancer drug discovery, which are currently challenged by limited sensitivity/precision and low throughput. Herein, a robust strategy based on the dephosphorylation switch DNAzyme-rolling circle amplification (RCA) circuit, termed DSD-RCA, is developed for highly sensitive detection of FTO and inhibitor screening. Initially, the catalytic activity of DNAzyme is silenced by engineering with an m6A modification in its catalytic core. Only in the presence of target FTO can the methyl group on DNAzyme be eliminated, resulting in the activation of the catalytic activity of DNAzyme and thus cleaving the hairpin substrate to release numerous primers. Different from the conventional methods that use the downstream cleavage primer with the original 3'-hydroxyl end directly as the RCA primer with the problem of high background signal, which should be compensated by additional separation and wash steps in heterogeneous format, our DSD-RCA assay uses the upstream cleavage primer with a 2',3'-cyclic phosphate terminus at the 3'-end serving as an intrinsically blocked 3' end. Only after a dephosphorylation reaction mediated by T4 polynucleotide kinase can the upstream cleavage primers with a resultant 3'-hydroxyl end be extended by RCA. With the high signal-to-noise ratio and homogeneous property, the proposed platform can sensitively detect FTO with a limit of detection of 31.4 pM, and the relative standard deviations (RSDs %) ranging from 0.8 to 2.0% were much lower than the heterogeneous methods. The DSD-RCA method was applied for analyzing FTO in cytoplasmic lysates from different cell lines and tissues of breast cancer patients and further used for screening FTO inhibitors without the need for separation or cleaning, providing an opportunity for achieving high throughput and demonstrating the potential applications of this strategy in disease diagnostics, drug discovery, and biological applications.

Movable toehold for leakless self-assembly circuits

Nonenzymatic self-assembly circuit utilizing hairpin substrates has been developed to be a powerful tool for information transduction, amplification and computation. However, the sensitivity, stability and application of this circuit are impeded by the presence of leakage which refers to undesired triggering in the absence of input. Herein, we proposed a movable toehold principle to suppress leakage and accelerate the catalytic reaction through removing partial hairpin toehold responsible for the leakage and transferring it to the catalyst. With movable toehold, catalytic hairpin assembly (called mtCHA) exhibited an excellent signal-to-background ratio of over 100, high robustness and improved specificity. In more complex circuit, including proximity recognition, signal amplification of small molecules (such as ATP), logic network, autocatalysis circuit and two-layer cascade circuit, mtCHA also demonstrated satisfactory performance. Our findings suggest that mtCHA holds great potential for broader applications, and the approach of repurposing harmful fragments into beneficial candidates can provide valuable insights for other chemical systems.

A novel RCA-based DNA sensor system for specific and quantitative detection of Klebsiella pneumonia

The timely detection of Klebsiella pneumoniae (K. pneumoniae) infection is of great importance for patient management and the prevention of nosocomial infections. In this study, we present the development of a novel detection assay that enables sensitive and specific identification of K. pneumoniae-encoded topoisomerase IV (K. pneumoniae Topo IV) using a DNA sensor system in combination with Rolling Circle Amplification (RCA) technology. The DNA sensor system was designed by immobilizing DNA hairpin substrates on RCA primers modified slide surface. This modular reacting mode enhances the adaptability and flexibility of the sensor, thereby improving its universality as a sensing platform. When K. pneumoniae is present, the active Topo IV triggers the cleavage-ligation of DNA hairpin substrates, followed by RCA and labeling with fluorescent probes. This results in the visualization of K. pneumoniae at the single-molecule level by the DNA sensor, compared to situations when there are no K. pneumoniae present. The reliability and sensitivity of the DNA sensor were verified using purified enzymes and bacterial extracts, which establishes the foundation for quantitative detection of clinical samples. Notably, the sensor's detection limit for K. pneumoniae extracts was found to be 70 colony-forming units (CFU)/µL, a range considered clinically relevant for infections. Moreover, the DNA sensor system exhibited high sensitivity and accuracy in detecting K. pneumoniae from clinical samples, suggesting its potential application in clinical settings for detecting pathogenic microbes beyond K. pneumoniae.

A sensitive and rapid electrochemical biosensor for sEV-miRNA detection based on domino-type localized catalytic hairpin assembly

Small extracellular–vesicule-associated microRNA (sEV-miRNA) is an important biomarker for cancer diagnosis. However, rapid and sensitive detection of low-abundance sEV-miRNA in clinical samples is challenging. Herein, a simple electrochemical biosensor that uses a DNA nanowire to localize catalytic hairpin assembly (CHA), also called domino-type localized catalytic hairpin assembly (DT-LCHA), has been proposed for sEV-miRNA1246 detection. The DT-LCHA offers triple amplification, (i). CHA system was localized in DNA nanowire, which shorten the distance between hairpin substrate, inducing the high collision efficiency of H1 and H2 and domino effect. Then, larger numbers of CHAs were triggered, capture probe bind DT-LCHA by exposed c sites. (ii) The DNA nanowire can load large number of electroactive substance RuHex as amplified electrochemical signal tags. (iii) multiple DT-LCHA was carried by the DNA nanowire, only one CHA was triggered, the DNA nanowire was trapped by the capture probe, which greatly improve the detection sensitivity, especially when the target concentration is extremely low. Owing to the triple signal amplification in this strategy, sEV-miRNA at a concentration of as low as 24.55 aM can be detected in 20 min with good specificity. The accuracy of the measurements was also confirmed using reverse transcription quantitative polymerase chain reaction. Furthermore, the platform showed good performance in discriminating healthy donors from patients with early gastric cancer (area under the curve [AUC]: 0.96) and was equally able to discriminate between benign gastric tumors and early cancers (AUC: 0.77). Thus, the platform has substantial potential in biosensing and clinical diagnosis.Graphical

Engineering DNA tetrahedron as a sensing surface of lateral flow test strips and ratiometric visual detection of exosomal microRNA-150–5p

We here report that DNA tetrahedral probe-based sensing surfaces are capable of efficiently capturing gold nanoparticles (AuNPs) on lateral flow test strips, and we have developed a ratiometric biosensor for exosomal microRNAs by integrating a tetrahedral probe and a catalytic hairpin assembly (CHA). DNA tetrahedrons were devised with barcodes and labeled with biotin as capture probes on the test and control lines of the strip. The CHA system consists of two hairpin substrates that can form stable duplexes via a programmable assembly reaction triggered by exosomal miRNAs. The duplexes hybridized with barcoded tetrahedra via complementary sequences and bound to streptavidin-modified AuNPs (SA-AuNPs) via biotin. Thus, a red line was observed on the test line owing to the immobilized SA-AuNPs. The remaining SA-AuNPs were captured by using a biotinylated tetrahedron on the control line. Because the barcoded and biotinylated tetrahedra consume SA-AuNPs simultaneously, a stronger signal in the test line results in a weaker signal in the control line, thus forming a ratiometric result. Tetrahedral probes avoid movement along with sample flow on sensing zones like single-stranded probes. The strip can have a detection limit of 58.90 fM with high selectivity due to the use of ratiometric color changing response, CHA signal amplification and tetrahedral capture probes. This system is able to rapidly detect exosomal microRNA-150–5p and may have a potential utility in point-of-care diagnosis of incipient diabetic nephropathy.

A highly sensitive and specific fluorescent strategy for the detection of Visfatin based on nonlinear hybridization chain reaction

This study established a nonlinear hybridization chain reaction system in which trigger DNA initiates self-sustained assembly of double-stranded hairpin substrates labelled with Sulfo-Cyanine3 (Cy-3) into fluorescent dendritic nanostructures for visfatin detection. The trigger DNA chain initially bound with the anti-visfatin aptamer will be freed with visfatin, which induces continuous chain branching growth and results in an exponential increase in fluorescence signals. Under optimal conditions, the fluorescence intensity increased linearly with visfatin concentrations ranging from 1 ng mL−1 to 100 ng mL−1 (the limit of detection dropped to 0.028 ng mL−1). Furthermore, our study showed that magnetic bead separation minimizes the nonspecific signal of the method. The specificity, stability, and sensitivity of this method were also tested in the study. The results revealed excellent specificity, stability and sensitivity. In the aspect of application evaluation, we tested visfatin in clinical serum samples and obtained credible and accordant results so we suppose that our detection method might have bright prospects in biological molecule technology and early clinical discovery of type 2 diabetes (T2DM).

Target-promoted specific activation of m6A-DNAzyme for SPEXPAR-amplified and highly sensitive non-label electrochemical assay of FTO demethylase.

The demethylase of fat mass and obesity related protein (FTO) is critical to regulate the dynamic N6-methyladenosine (m6A) modification of eukaryotic mRNAs, and its overexpression has found to be closely related to the initiation of several cancers. On the basis of a target-promoted specific activation of DNAzyme strategy coupled with self-primer exponential amplification reaction (SPEXPAR) cycles and DNA supersandwich assemblies, the highly sensitive and label-free electrochemical FTO assay approach is established. The modification of the catalytic core nucleobase of the DNAzyme probe by m6A can inhibit its cleavage activity. The presence of target FTO catalyzes the elimination of the methyl group to restore the DNAzyme activity, which cleaves the hairpin substrates to trigger the SPEXPAR for yielding many ssDNAs. The capture of these DNAs on the sensor electrode leads to the initiation of supersandwich assembly formation of long dsDNAs. Tremendous electrochemical signal probe of [Ru(NH3)6]Cl3 are then absorbed on these dsDNAs to produce highly amplified catalytic currents with the assistance of K3[Fe(CN)6] for detecting trace FTO with 63.1fM detection limit. Furthermore, the sensor can be employed for selective assay of FTO in cell lysates, revealing the great potential of this sensing strategy for biomedical and biological study applications.

Development of a selection assay for small guide RNAs that drive efficient site-directed RNA editing.

A major challenge confronting the clinical application of site-directed RNA editing (SDRE) is the design of small guide RNAs (gRNAs) that can drive efficient editing. Although many gRNA designs have effectively recruited endogenous Adenosine Deaminases that Act on RNA (ADARs), most of them exceed the size of currently FDA-approved antisense oligos. We developed an unbiased in vitro selection assay to identify short gRNAs that promote superior RNA editing of a premature termination codon. The selection assay relies on hairpin substrates in which the target sequence is linked to partially randomized gRNAs in the same molecule, so that gRNA sequences that promote editing can be identified by sequencing. These RNA substrates were incubated in vitro with ADAR2 and the edited products were selected using amplification refractory mutation system PCR and used to regenerate the substrates for a new round of selection. After nine repetitions, hairpins which drove superior editing were identified. When gRNAs of these hairpins were delivered in trans, eight of the top ten short gRNAs drove superior editing both in vitro and in cellula. These results show that efficient small gRNAs can be selected using our approach, an important advancement for the clinical application of SDRE.

Promiscuous splicing-derived hairpins are dominant substrates of tailing-mediated defense of miRNA biogenesis in mammals.

Canonical microRNA (miRNA) hairpins are processed by the RNase III enzymes Drosha and Dicer into ∼22 nt RNAs loaded into an Argonaute (Ago) effector. In addition, splicing generates numerous intronic hairpins that bypass Drosha (mirtrons) to yield mature miRNAs. Here, we identify hundreds of previously unannotated, splicing-derived hairpins in intermediate-length (∼50-100 nt) but not small (20-30 nt) RNA data. Since we originally defined mirtrons from small RNA duplexes, we term this larger set as structured splicing-derived RNAs (ssdRNAs). These associate with Dicer and/or Ago complexes, but generally accumulate modestly and are poorly conserved. We propose they contaminate the canonical miRNA pathway, which consequently requires defense against the siege of splicing-derived substrates. Accordingly, ssdRNAs/mirtrons comprise dominant hairpin substrates for 3' tailing by multiple terminal nucleotidyltransferases, notably TUT4/7 and TENT2. Overall, the rampant proliferation of young mammalian mirtrons/ssdRNAs, coupled with an inhibitory molecular defense, comprises a Red Queen's race of intragenomic conflict.

A sensitive “turn-on” fluorescent biosensor for the detection of biomarkers based on three-dimensional DNA walker

Three-dimensional (3D) DNA walker have been widely acknowledged as a promising strategy for improving the detection sensitivity of biosensors. In this work, a sensitive “turn-on” fluorescent biosensor for the detection of biomarkers based on three-dimensional DNA walker was developed. Herein, DNA walker strands, fluorophores (FAM and Cy5)-labeled hairpin substrate strands, HER2-specific aptamer, and gold nanoparticles (AuNPs) were involved to fabricate the nanoprobe1 (nanop1) for human epidermal growth factor receptor 2 (HER2) and nanoprobe2 (nanop2) for microRNA-21 (miRNA-21). The composite fluorescent probe comprising of nanop1 and nanop2 was employed for the multiplex determination of HER2 and miRNA-21 without cross interference. This fluorescent biosensor was sensitive with the detection limits of 0.01 ng mL−1 for HER2 and 0.018 pM for miRNA-21, which was mainly due to the signal amplification of Nb.BbvcI powered 3D DNA walker. Moreover, this biosensor was selective and reliable for the detection of HER2 and miRNA-21 spiked in the serum samples. Therefore, this present 3D DNA walker-based biosensor is not only highly sensitive and specific, but also is accurate because of the multiplex detection of biomarkers, which can be expected to be used for the application in clinical field.

NiCo2S4 nanoparticle-dispersed MoS2 nanosheets for catalytic and sensitive electrochemical aptamer sensing of ochratoxin A via cascaded amplifications

Highly sensitive monitoring of mycotoxins is of major importance for food safety and human health. In this work, on the basis of a new catalytic sensing interface of NiCo2S4 nanoparticle-dispersed MoS2 nanosheet (NiCo2S4 @MoS2)-modified electrode and the signal amplifications by 3D DNA walker and hybridization chain reaction (HCR), a sensitive electrochemical aptamer sensor for the detection of ochratoxin A (OTA), one of the most toxic mycotoxin widely distributed in food, is developed. The synthesized NiCo2S4@MoS2 shows enhanced catalytic current responses toward the methylene blue (MB) signal tag. The presence OTA results in the activation of the DNA walker to cyclically cleave the hairpin substrate DNAs to release many triggering sequences for the initiation of HCR between two hairpins, leading to the attachment of numerous MB-labeled strands on the sensor electrode. Subsequent electrocatalytic oxidation of MB thus yields drastically enhanced current responses for highly sensitive sensing of OTA with the detection limit of 0.42 pg mL−1 in the dynamic range of 0.5 pg mL−1-1 ng mL−1. Moreover, this sensing platform exhibits high selectivity and can be applied to monitor OTA spiked in real samples, showing its potential for the development of robust aptamer sensors for various mycotoxins.

Highly sensitive α-hemolysin nanopore detection of MUC1 based on 3D DNA walker

Mucin 1(MUC1) is an effective marker of breast cancer, so it is of great significance to develop a simple, sensitive and highly selective MUC1 detection sensor. Herein, we constructed a label-free nanopore biosensor for rapid and highly sensitive detection of MUC1. The presence of MUC1 triggered the modification of the DNAzyme walking chain on the surface of Fe3O4 nanoparticles and separation from the aptamer. In the presence of Zn2+, DNAzyme catalyzed hydrolytic cleavage of the hairpin substrate at the scissile rA. The DNAzyme was divided into two fragments and ssDNA was released. ssDNA products from the hairpin substrate can generate a current blocking signal during α-hemolysin nanopore testing. The frequency of signature events showed a linear response toward the concentration of MUC1 in the range of 0.01 nM–100 nM. The sensing system also exhibited high selectivity against other protein and can be used for the detection of real sample.

A highly sensitive and versatile fluorescent biosensor for pathogen nucleic acid detection based on toehold-mediated strand displacement initiated primer exchange reaction

Existing detection methods for pathogen nucleic acid detection, such as polymerase chain reaction (PCR), are complicated and expensive to perform. Here, we report a simple and versatile strategy for highly sensitive detection of pathogen nucleic acid based on toehold-mediated strand displacement initiated primer exchange amplification (t-PER). In the presence of the target, the blocked hairpin substrate is released by toehold-mediated strand displacement, which triggers the primer exchange reaction amplification. Then, multiple long tandem-repeat single-strands generated by PER open the molecular beacon to recover the fluorescence signal. The t-PER protocol also successfully directly detected human papilloma virus from clinical cervical swab samples, with consistent results compared to real time-polymerase chain reaction (RT-PCR). Moreover, the versatility and clinical feasibility of this method was further confirmed by measuring Epstein-Barr virus, hepatitis B virus, and Ureaplasma urealyticum from different clinical samples (serum samples and urine samples). This simple platform enabled specific and sensitive detection of pathogen nucleic acid in a format that might hold great potential for point-of-care infection diagnosis.

Dual DNAzyme-catalytic assembly of G-quadruplexes for inducing the aggregation of gold nanoparticles and developing a novel antibiotic assay method.

By utilizing a target biorecognition reaction to induce the self-assembly of G-quadruplexes and the aggregation of gold nanoparticles (Au NPs), this work develops a novel colorimetric biosensing method for kanamycin (Kana) antibiotic detection. The compact G-quadruplex structure was assembled from its two half-split sequences which were designed in two hairpin substrates of the Mg2+-dependent DNAzyme (MNAzyme). Besides hybridizing with the aptamer strand, the MNAzyme sequence was also split into two half fragments to be designed in the two substrates. Upon the aptamer-recognition reaction toward Kana, the MNAzyme strand could be quantitatively released to cause the exposure of the split G-quadruplex-sequences on two hairpin substrate-modified Au NPs and simultaneous release of two half fragments of the MNAzyme-sequence. Thus, the K+-assisted self-folding of G-quadruplexes causes the cross-linking of the two Au NPs to realize the Au NP aggregation-based colorimetric signal output (measured at the largest absorption peak near 520nm). Meanwhile, the self-assembled formation of the second MNAzyme drastically amplified the signal response. Under the optimal conditions, a wide linear range from 0.1 pg mL-1to 10ngmL-1 and an ultrahigh sensitivity with the detection limit of 76fgmL-1 were obtained. The dose-recovery experiments in real samples showed satisfactory results with recoveries from 98.4 to 105.4% and relative errors compared with the ELISA method less than 4.1%. Due to thehigh selectivity, excellent repeatability and stability, and simple manipulation, this method indicates a promising potential for practical applications. A novel homogeneous biosensing method was developed for the convenient detection of the kanamycin antibiotic. The target biorecognition-induced and dual DNAzyme-catalytic assembly of G-quadruplexes enabled the amplified aggregation of gold nanoparticles for the simple, cheap, stable, and ultrasensitive colorimetric signal transduction of the method.

Isomorphic Fluorescent Nucleosides Facilitate Real-Time Monitoring of RNA Depurination by Ribosome Inactivating Proteins.

Ribosome-inactivating proteins, a family of highly cytotoxic proteins, interfere with protein synthesis by depurinating a specific adenosine residue within the conserved α-sarcin/ricin loop of eukaryotic ribosomal RNA. Besides being biological warfare agents, certain RIPs have been promoted as potential therapeutic tools. Monitoring their deglycosylation activity and their inhibition in real time have remained, however, elusive. Herein, we describe the enzymatic preparation and utility of consensus RIP hairpin substrates in which specific G residues, next to the depurination site, are surgically replaced with tz G and th G, fluorescent G analogs. By strategically modifying key positions with responsive fluorescent surrogate nucleotides, RIP-mediated depurination can be monitored in real time by steady-state fluorescence spectroscopy. Subtle differences observed in preferential depurination sites provide insight into the RNA folding as well as RIPs' substrate recognition features.

Label-free detection of HPV mRNA with an artificial chaperone-enhanced MNAzyme (ACEzyme)-based electrochemical sensor

Nucleic acid biosensors for point-of-care (POC) diagnostic applications are highly desirable. The ability to detect DNA and RNA in a simple, rapid, affordable and portable format leads to a range of important applications for early screening in the field of disease monitoring and management. Herein, we report the development of an isothermal, label-free electrochemical biosensor that was designed on the basis of target-driven MNAzyme cleavage activity. Hybridization with HPV mRNA, a model nucleic acid target, activated MNAzyme and initiated the cleavage of immobilized hairpin substrates, leading to changes in the electrochemical signal. Under optimal conditions, a detection limit of 2.6 pM was obtained with an incubation time of 60 min. Furthermore, an artificial chaperone-enhanced MNAzyme (ACEzyme) system was integrated to an electrochemical biosensor for the first time. The analytical performance of the biosensor was enhanced, and the detection time was significantly reduced by the addition of PLL-g-Dex, which exhibits nucleic acid chaperone-like activity. A detection limit of 0.88 pM was obtained with a threefold decrease in incubation time without prior amplification. The proposed biosensing platform shows the advantages of simple fabrication and operation, good selectivity in the presence of single-base mismatch, and excellent versatility in a complex mixture of total RNA. We believe that this isothermal, label-free, and protein-free nucleic acid analysis platform could provide foundations for the further development of a universal nucleic acid biosensing platform for clinical application.

A Target-Feedback Rolling-Cleavage signal amplifier for ultrasensitive electrochemical detection of miRNA with Self-Assembled CeO2@Ag hybrid nanoflowers

Currently, developing an effective and easy-to-operate signal amplification assay to detect the trace-amount miRNAs in serum remains a significant challenge. Herein, an ultrasensitive CeO2@Ag hybrid nanoflower (CeO2@Ag HNF)-labeled electrochemical biosensor was developed for sensing miRNA, based on a target-feedback rolling-cleavage (TFRC) signal amplifier. CeO2@Ag HNFs possessing a unique three-dimensional layered structure were synthesized without any complex reaction conditions, such as heating and vacuum. The bimetallic nanoflowers provided a large surface area and excellent CAT-like activity, thereby enhancing electrochemical performance. Based on the principle of branched catalytic hairpin assembly, target miRNA could continuously trigger the assembly of branched junctions with ends composed of DNAzyme. The activated DNAzyme was able to cleave the hairpin substrate efficiently and release to capture more CeO2@Ag HNFs label. The process combining target-driven branched catalytic hairpin assembly and DNAzyme-assisted rolling cleavage were defined as TFRC signal amplification. This sensitive electrochemical biosensor exhibited good linear relationship of 1 fM − 1 nM with a detection limit down to 0.89 fM. The proposed method is expected to have a promising application in the miRNA-associated fundamental research and clinical diagnosis.