Dual Signal Amplification Research Articles

CRISPR-empowered electrochemical biosensor for target amplification-free and sensitive detection of miRNA

The clustered regularly interspaced short palindromic repeats (CRISPR) system provides a new molecular diagnostic tool for construction of biosensor platforms due to its high programmability and target specificity. Herein, we developed a CRISPR-empowered electrochemical biosensor by combining the advantages of CRISPR/Cas13a and primer exchange reaction (PER), named PER-E-CRISPR, for target amplification-free and sensitive detection of miR-21. Dual-signal amplification procedures involve the binding of target miR-21 induced by CRISPR-based amplification, along with the hybridization of multiple short single-stranded DNA strands with PER concatemers. When target miR-21 is present, CRISPR/Cas13a specifically recognizes the target miRNA, triggering the trans-cleavage activity of CRISPR/Cas13a. Then Cas13a/crRNA/miRNA cleaved the predesigned ribonucleotide site in hairpin 1 (HP1) and released trigger to open hairpin 2 (HP2) modified on the electrode surface. Then PER bridge sequence contained in HP2 is exposed and hybridized with PER concatemers, following multiple short single-stranded DNA tagged with methylene blue (ssDNA-MB) bond with the PER concatemers. Under optimized conditions, PER-E-CRISPR assay for detecting miR-21 exhibits linearity in dynamic range from 10−13 to 10−7 M, and we obtained a limit of detection (LOD) of 30.2 fM. The established PER-E-CRISPR biosensor shows perfect practical performance in actual plasma, which may have great promising prospects for miRNA detection in the field of molecular diagnosis.

Electrochemical DNA Biosensors with Dual-Signal Amplification Strategy for Highly Sensitive HPV 16 Detection.

Cervical cancer is an important topic in the study of global health issues, ranking fourth among women's cancer cases in the world. It is one of the nine major cancers that China is focusing on preventing and treating, and it is the only cancer that can be prevented through vaccination. Systematic and effective screening for human papilloma (HPV) infection, which is closely linked to the development of cervical cancer, can reduce cervical cancer incidence and mortality. In this paper, an electrochemical sensor was designed to detect HPV 16 using dual-signal amplification. An APTES-modified glassy carbon electrode was used for improved stability. Gold nanoparticles and a chain amplification reaction were combined for signal amplification. The limit of detection (LOD) of this electrochemical sensor was 1.731 × 10-16 mol/L, and the linear response of the target detector range was from 1.0 × 10-13 mol/L to 1.0 × 10-5 mol/L (R2 = 0.99232). The test of serum sample recovery showed that it has good anti-interference, and the performance of all aspects was improved to different degrees compared with the previous research from the team. The designed sensor is centered around the principles of low cost, high sensitivity and stability, which provides new ideas for the future development of cervical cancer prevention and electrochemical biosensors.

A dual signal amplification system with specific signal identification for rapid and sensitive detection of miRNA

False positive which is mostly caused by the nonspecific amplification has severely hindered the development of nucleic acid detection and it is hard to avoid. Therefore, specific signals recognition and output in nucleic acid amplification are crucial to reliability of clinical diagnosis. Herein, we proposed a one-step and rapid miRNA detection strategy with specific signal identification, dual amplification and output. And this strategy was named as high-temperature hybridization chain reaction coupled with strand displacement amplification (HSA). In HSA, we well designed a target signal recognition, replication, and output probe (RRO probe). If the target miRNA exists, RRO probe can initiate a strand displacement amplification and output a target-related special single-stranded DNA (trigger). And the trigger can be identified by a high-temperature hybridization chain reaction and initiate a secondary signal amplification. As a result, the quantitative determination of HSA for miRNA-21 was in the range of 100 fM to 100 pM in 30 min, and with a detection limit of 82 fM. Moreover, with high sensitivity and rapidity, HSA has been successfully used to detect miRNA-21 in real samples.

Programmable Clostridium perfringens Argonaute-Based, One-Pot Assay for the Multiplex Detection of miRNAs.

Assays for the molecular detection of miRNAs are typically constrained by the level of multiplexing, especially in a single tube. Here, we report a general and programmable diagnostic platform by combining mesophilic Clostridium perfringens Argonaute (CpAgo) with exponential isothermal amplification (EXPAR), which is a dual-signal amplification strategy, allowing for the rapid and sensitive detection of multiple miRNAs with single-nucleotide discrimination in one pot. The CpAgo-based One-Pot (COP) assay achieved a limit of detection of 1 zM miRNA within 30 min of turnaround time and a wide concentration range. This COP assay was applied to simultaneously detect four miRNAs in a single tube from clinical serum samples, showing superior analytical performance in distinguishing colorectal cancer patients from healthy individuals. This programmable, one-pot, multiplex, rapid, and specific strategy offers great promise in scientific research and clinical applications.

Electrochemical biosensor for highly sensitive detection of cTnI based on a dual signal amplification strategy of ARGET ATRP and ROP

Cardiac troponin I (cTnI), a gold biomarker for the diagnosis of acute myocardial infarction (AMI), plays a vital role in the early diagnosis, treatment and prognosis analysis of AMI. In this paper, an electrochemical biosensor for the highly sensitive determination of cTnI was fabricated based on the dual signal amplification strategy of electron transfer atom transfer radical polymerization (ARGET ATRP) and ring-opening polymerization (ROP) for the first time. Briefly, the thiolate cTnI-aptamer 1, which was bonded to the electrode via Au–S bonds, specifically captured cTnI to the electrode surface. Then, cTnI-aptamer 2 (Apt2) was successfully introduced to the electrode surface to form Apt-cTnI-Apt sandwich structure. Subsequently, the initiator BIBB was connected to Apt2 through bromination reaction, and then the resulting ATRP polymer was employed as a macromolecular initiator for the succeeding reaction. Next, the monomers, α-amino acid-N-carboxylic acid anhydride ferrocene derivatives (NCA-Fc), used for the ROP reaction produced numerous electroactive polymers on the electrode surface. The dual action of ARGET ATRP and ROP significantly improved sensitivity of cTnI biosensor assay, the prepared biosensor displayed a wide linear detection range from 100 fg mL−1 to 100 ng mL−1, with a detection limit of 32.24 fg mL−1. The method exhibited favorable selectivity, simple operation and excellent stability. Furthermore, the biosensor still rendered satisfactory analytical performance in the detection of clinical serum samples, indicating the application potential in clinical diagnosis.

Electrochemical Detection of Tumor Cell-Derived Exosomes Based on Cyclic Enzyme Scission and Hybridization Chain Reaction Dual-Signal Amplification

Tumor cell-derived exosomes are considered a potential source of cancer biomarkers. Here, we developed an electrochemical sensing platform for the rapid and simple detection of exosomes, using the CCRF-CEM exosome as a model. The platform utilizes cyclic nicking enzyme cleavage and a hybridization chain reaction (HCR) for dual-signal amplification. A hairpin aptamer probe (HAP) containing an aptamer was designed for the assay. The specific binding between the aptamer and PTK7, present on the exosome surface, causes a conformational change in the HAP. This facilitates hybridization between the HAP and the linker DNA, which subsequently triggers cyclic cleavage of the nicking endonuclease towards the linker DNA. Therefore, exosome detection is transformed into DNA detection. By combining this approach with HCR signal amplification, we achieved high-sensitivity electrochemical detection of CCRF-CEM exosomes, down to 1.1 × 104 particles/mL. Importantly, this assay effectively detected tumor exosomes in complex biological fluids, demonstrating the potential for clinical diagnosis.

Dual-amplification system based on CRISPR-Cas12a and horseradish peroxidase-tethered magnetic microspheres for colorimetric detection of microcystin-LR

A novel dual-amplification system based on CRISPR-Cas12a and horseradish peroxidase (HRP) was developed for colorimetric determination of MC-LR. This dual-amplification was accomplished by combining the nuclease activity of CRISPR-Cas12a with the redox activity of HRP. HRP linked to magnetic beads through an ssDNA (MB-ssDNA-HRP) was used to induce a color change of the 3,3′,5,5′-tetramethylbenzidine (TMB)-H2O2 chromogenic substrate solution. Specific binding of MC-LR with its aptamer initiated the release of a complementary DNA (cDNA), which was designed to activate the trans-cleavage activity of CRISPR-Cas12a. Upon activation, Cas12a cut the ssDNA linker in MB-ssDNA-HRP, causing a reduction of HRP on the magnetic beads. Consequently, the UV–Vis absorbance of the HRP-catalyzed reaction was decreased. The dual-signal amplification facilitated by CRISPR-Cas12a and HRP enabled the colorimetric detection of MC-LR in the range 0.01 to 50 ng·mL−1 with a limit of detection (LOD) of 4.53 pg·mL−1. The practicability of the developed colorimetric method was demonstrated by detecting different levels of MC-LR in spiked real water samples. The recoveries ranged from 86.2 to 118.5% and the relative standard deviation (RSD) was 8.4 to 17.6%. This work provides new inspiration for the construction of effective signal amplification platforms and demonstrates a simple and user-friendly colorimetric method for determination of trace MC-LR.Graphical

Dual Signal Amplification by Urease Catalysis and Silver Nanoparticles for Ultrasensitive Colorimetric Detection of Nucleic Acids.

Signal amplification techniques are highly desirable for the analysis of low-level targets that are closely related with diseases and the monitoring of important biological processes. However, it is still challenging to achieve this goal in a facile and economical way. Herein, we developed a novel dual signal amplification strategy by combining urease catalysis with the release of Ag+ from silver nanoparticles (AgNPs). This strategy was used for quantifying a DNA sequence (HIV-1) related with human immunodeficiency virus (HIV). DNA target HIV-1 hybridizes with the capture DNA probe on magnetic beads and the reporter DNA probe on AgNPs, forming a sandwich complex. The captured AgNPs are then transformed into numerous Ag+ ions that inactivate numerous ureases. Without catalytic production of ammonia from urea, the substrate solution shows a low pH 5.8 that will increase otherwise. The pH change is monitored by a pH indicator (phenol red), which allows for colorimetric detection. The proposed approach is sensitive, easy to use, economic, and universal, exhibiting a low detection limit of 9.7 fM (i.e., 1.94 attomoles) and a dynamic linear range of 4 orders for HIV-1 sequence detection.

Enzyme-free dual amplification biosensor based on functional nucleic acid and CDs/CoOOH for detection of leukemia fusion gene

Acute promyelocytic leukemia (APL) is an acute myeloid leukemia (AML) with a specific fusion gene target, PML/RARα fusion gene (PML/RARα), which is formed by the translocation of chromosomes 15 and 17. Detection of PML/RARα is the most reliable parameter for the diagnosis, treatment adjustment, efficacy evaluation, prognosis analysis and relapse prediction of APL. In this study, a novel biosensor was constructed for rapid enzyme-free detection of PML/RARα using DNAzyme and carbon dots/cobalt oxhydroxide nanosheet complexs (CDs/CoOOH). In the detection system, the separated DNAzymes could specifically recognize and bind together by the PML/RARα to form a complete DNAzyme for shearing hairpin probe (HP), then generated trigger, which was the first signal amplification. Then, trigger could hybridize with the capture probe (CP) anchored to streptavidin (SA) modified microplate as well as fluorescence quenching signal probe (SP@CDs/CoOOH). Finally, ascorbic acid (AA) was added to decompose CoOOH and the fluorescence of CDs was released, which was the second signal amplification. Through the dual signal amplification of DNAzyme and CDs/CoOOH, PML/RARα could be detected quickly and sensitively, which overcame the limitation of protein enzyme in traditional fluorescence methods, showing potential clinical application value in the diagnosis and treatment of leukemia.

DNAzyme@CuSNPs dual mimic enzyme probe-amplified chemiluminescent imaging array immunosensor for multiple chicken cytokines detection

The detection of cytokines plays an important role in clinical diagnosis and immune mechanism research of chicken diseases. In this work, a novel and ultrasensitive chemiluminescent (CL) imaging array immunosensor was proposed to detect multiple chicken cytokines based on DNAzyme@CuS nanoparticles (DNAzyme@CuSNPs) dual mimic enzyme signal amplification strategy. DNAzyme@CuSNPs owns excellent peroxidase property, which was modified with second antibody (Ab2) to prepare DNAzyme@CuSNPs detection probe, and demonstrated high catalysis CL imaging signal due to synergistic catalysis. Chicken interleukin-4 (ChIL-4) and chicken interferon-γ (ChIFN-γ) were used as model analysis samples, the DNAzyme@CuSNPs-based CL imaging immunosensor achieved simultaneous and high-throughput detection of ChIL-4 and ChIFN-γ with wide linear range of 10−3–102 ng/mL, and the detection limits are 0.41 pg/mL and 0.36 pg/mL, respectively. The multiplex chicken cytokines CL imaging array immunosensor shows a high sensitivity, wide linear range, excellent specificity and acceptable stability. This research opens dual mimic enzyme signal-amplified strategy to develop sensitive CL imaging immunoassay for chicken diseases detection application.

Immobilization-free electrochemical homogeneous aptasensor for highly sensitive detection of carcinoembryonic antigen by dual amplification strategy

Electrochemical aptasensor has been widely studied, while its practical application is limited by the unavoidable variations of aptamer loading densities and low signal amplification efficiency. To overcome these restrictions, an immobilization-free and label-free electrochemical homogeneous aptasensor was constructed for carcinoembryonic antigen (CEA) assay by combining RecJf exonuclease-mediated target cycling strategy and rolling circle amplification technology. In this system, the pre-immobilization of aptamers or other relevant signal elements on the electrode substrate is no longer necessary, thus the electrochemical homogeneous aptasensor shows good versatility on different transducers. Moreover, the whole recognition and signal amplification process are activated instantaneously by a non-professional operation of the solution mixture. This strategy can not only increase the stability (95.1% after 30 days of storage) and reproducibility (2.12% among five independent electrodes), but also further improve the sensitivity (detection limit of fg mL−1 level) due to the free target recognition and dual signal amplification in the homogeneous solution phase. The proposed immobilization-free electrochemical homogeneous aptasensors on different electrode substrates both achieve satisfactory results in actual sample tests, which has the potential for commercial applications and the establishment of other target platforms in the future.

The sensor platform combined with dual signal amplification and based on UCNPs and CRISPR/Cas12a for MiRNA-21 detection

Cancer has become the research target of researchers because of its serious threat to human life and health. MicroRNA levels can change as cancer progresses and become a biomarker for cancer detection. In this work, a novel sensor system was designed for the detection of miRNA-21 which utilizes the luminescence properties of upconverted nanoparticles (UCNPs) and the trans-cleavage of CRISPR/Cas12a, and also introduces a dual signal amplification mediated by Exonuclease III (Exo III) and phi29 DNA polymerase. The improved hairpin probe (HP) can not only be opened by the UCNPs photocontrollably, but also guide the target to cycle under the Exo III cut, achieving the first amplification of the signal. Subsequently, rolling circle amplification (RCA) realizes the signal secondary amplification. A large number of repeated sequences, which binds to Cas12a/crRNA and activates the trans-cutting activity of Cas12a, are generated in the process of RCA. Finally, the fluorescence signal can be recovered and highly sensitive detection for targets can be realized. The construction of one-to-many technology between target and signal is realized due to the dual signal amplification and the detection limit is 6.01 fM. In addition, the sensor system also has shown excellent performance in serum and cell lysates.

Electrochemical detection of isocarbophos in apple and cabbage with Ti3C2TX/arginine and serine-functionalized graphene quantum dot@gold nanostar aerogel and target cycle dual signal amplification

The paper reports one strategy for synthesis of Ti3C2TX/arginine and serine-functionalized graphene quantum dot@Au nanostar aerogel (Ti3C2TX/RS-GQD@Au). Positively charged RS-GQDs bound with negatively charged Ti3C2TX sheets to form Ti3C2TX aerogel. Then, HAuCl4 was in situ reduced with ascorbic acid in the presence of L-glutathione to form gold nanostars on Ti3C2TX sheets. The resulting Ti3C2TX/RS-GQD@Au shows one three-dimensional structure. The gold nanostars were homogeneously dispersed on Ti3C2TX sheets. Introduction of RS-GQD realizes construction of Schottky heterojunction. The presence of L-glutathione triggers an asymmetric structural evolution of gold nanocrystals and leads to more exposed high-index crystal facets. Unique structure achieves to high conductivity, structural stability and catalytic activity. Ti3C2TX/RS-GQD@Au was used for construction of electrochemical sensor for detection of isocarbophos coupled with target cycle amplification. The peak current at 0.27 V linearly increases with the increase of isocarbophos concentration between 1 × 10−11 and 8 × 10−16 M with 2.9 × 10−16 M of detection limit (S/N = 3). The analytical method provide advantage of sensitivity and specificity for detection of isocarbophos in apple and cabbage.

Hybridization chain reaction cascaded amplification platform for sensitive detection of pathogen

Rapid, sensitive, and accurate identification of pathogens is vital for preventing and controlling fish disease, reducing economic losses in aquaculture, and interrupting the spread of food-borne diseases in human populations. Herein, we proposed a hybridization chain reaction (HCR) cascaded dual-signal amplification platform for the ultrasensitive and specific detection of pathogenic microorganisms. A couple of specific primers for target bacterial 16S rRNAs were used to obtain amplified target single-stranded DNAs (AT-ssDNA). Then, AT-ssDNA initiated HCR amplification along with the opening of fluorophore (FAM) and a quencher (BHQ1) labeled hairpin reporter probe (H1), and the FAM fluorescence signal recovered. The proposed strategy could achieve a detection limit down to 0.31 CFU/mL for Staphylococcus aureus (S. aureus), 0.49 CFU/mL for Escherichia coli (E. coli) in buffer, and a linear range from 1 to 1 × 106 CFU/mL for S. aureus, 1 to 1 × 107 CFU/mL for E. coli. Furthermore, this platform enabled sensitive and precise detection of pathogenic microorganisms in complex samples such as fish blood and different organ tissues (large intestine, gallbladder, heart, liver, ren, gill, skin), which shows great potential in disease prevention and control in aquatic products.

Catalytic hairpin assembly-assisted dual-signal amplification platform for ultrasensitive detection of tumor markers and intelligent diagnosis of gastric cancer

Quantification of extracellular tumor markers has shown great promise for non-invasive cancer diagnosis. Combined detection of multiple tumor markers instead of a single one is valuable for accurate diagnosis. Here, we integrate CRISPR-Cas12a with DNA catalytic hairpin assembly (CHA) to doubly amplify the output signal for detecting microRNA-182 (miR-182), which is overexpressed by gastric cancer patients. Additionally, we develop a CHA system with self-replicating capacity (SRCHA) to realize dual-signal amplification for the detection of carcinoembryonic antigen (CEA), a broad-spectrum tumor marker. The proposed cascade amplification strategies enable ultrasensitive detection of miR-182 and CEA with low LODs of 0.063 fM and 4.8 pg mL−1, respectively. Moreover, we design a ternary “AND” logic gate using different concentrations of miR-182 and CEA as inputs, which demonstrates intelligent diagnosis of gastric cancer staging with a high accuracy of 93.3% in a clinical cohort of 30 individuals. Overall, our study expands the application of CRISPR-Cas12a in biosensing and provides a new diagnostic strategy for non-invasive liquid biopsy of gastric cancer before resorting to a traumatic tissue biopsy.

An Electrochemical Sensor for Trimethoprim Based on a Magnetic Molecularly Imprinted Carbon Paste Electrode

In order to achieve simple, rapid, and highly sensitive detection of trimethoprim (TMP), a magnetic molecularly imprinted carbon paste electrode (MCPE) was prepared by drop-coating magnetic molecularly imprinted polymer (MIP@Fe3O4@MWNTs) on the surface of reduction graphene oxide (rGO)/MCPE doped with Fe3O4@MWNTs. The introduction of multi-walled carbon nanotubes (MWNTs) and rGO served as dual signal-amplification materials, which can improve the response sensitivity of the sensor. In addition, the magnetic interaction between the substrate electrode and the molecularly imprinted material was beneficial to increasing the stability of the sensor. As expected, the electrochemical sensor not only showed sensitivity and selectivity for the detection of TMP, but it also possessed good stability. The detection range for TMP was 4.0 × 10−9~5.0 × 10−4 mol/L, and the detection limit was 1.2 × 10−9 mol/L. The response performance varied within 10% when the sensor was placed for more than 2 months and used more than 60 times. The spiked recoveries of TMP in environmental water samples, urine samples, and pharmaceuticals (drugs) were between 91% and 110%, and the relative standard deviation (RSD) was within 5%.

Simple and Ultrasensitive Detection of Glioma-Related ctDNAs in Mice Serum by SERS-Based Catalytic Hairpin Assembly Signal Amplification Coupled with Magnetic Aggregation.

Circulating tumor DNA (ctDNA) is more representative and accurate than biopsy and is also conducive to dynamic monitoring, facilitating accurate diagnosis and prognosis of glioma. Therefore, the present study aimed to establish and validate a novel amplified method for the detection of IDH1 R132H and BRAF V600E, which were associated with the genetic diagnosis of glioma. A dual-signal amplification method based on magnetic aggregation and catalytic hairpin assembly (CHA) was constructed for the simultaneous detection of ctDNAs. When target ctDNAs are present, the CHA reaction is initiated and leads to the assembly of Au-Ag nanoshuttles (Au-Ag NSs) onto magnetic beads (MBs). Further enrichment of MBs under an external magnetic field facilitated the dual-signal amplification of SERS. The limit of detection (LOD) for IDH1 R132H and BRAF V600E in serum was as low as 6.01 aM and 5.48 aM. The reproducibility and selectivity of the proposed SERS analysis platform was satisfactory. Finally, the platform was applied to quantify IDH1 R132H and BRAF V600E in the serum of subcutaneous-tumor‑bearing nude mice, and the results obtained by SERS were consistent with those from quantitative real-time polymerase chain reaction (qRT-PCR). The present study showed that the dual-signal amplification method is a simple and ultrasensitive strategy for gliomas-associated ctDNAs detection, which is crucial for early diagnosis and dynamic monitoring.

Vanillin-Catalyzed highly sensitive luminol chemiluminescence and its application in food detection.

Among various chemiluminescence (CL) systems, luminol-H2O2 system is used extensively due to its cheapness and sensitivity. Herein, 4-hydroxy-3-methoxybenzaldehyde, known as vanillin, was firstly found to be able to catalyze H2O2 very efficiently to produce •OH and O2•-, which can be used to enhance the CL of luminol-H2O2 as Co+. In alkaline aqueous solution, vanillin catalyzed the dissociation of H2O2 into active •OH and O2•- radicals and accelerated luminol-H2O2 reaction to emit strong CL signal. Combining the stabilizing function of β-CD, CL intensity of luminol-H2O2 was enhanced further. Thus, dual-signal amplification of luminol-H2O2 chemiluminescence based on the catalyzing function of vanillin and the stabilizing function of β-CD was proposed and its mechanism was explored deeply in the manuscript. Interestingly, vanillin is a highly prized flavor compound broadly used as food additive, however, the excessive intake of vanillin is harmful to human and thus the determination of vanillin is very important. On the basis of the luminol-β-CD-H2O2/vanillin reaction, a low-cost, rapid and simple CL sensor has been established to detect vanillin. The sensor was able to detect vanillin in the range of 1.0μM∼75μM with a detection limit of 0.89μM (S/N=3). It can also be used for CL imaging detection with satisfactory results.

A Novel SPR Immunosensor Based on Dual Signal Amplification Strategy for Detection of SARS-CoV-2 Nucleocapsid Protein

Since the global outbreak of coronavirus disease 2019 (COVID-19), it has spread rapidly around the world. The nucleocapsid (N) protein is one of the most abundant SARS-CoV-2 proteins. Therefore, a sensitive and effective detection method for SARS-CoV-2 N protein is the focus of research. Here, we developed a surface plasmon resonance (SPR) biosensor based on the dual signal-amplification strategy of Au@Ag@Au nanoparticles (NPs) and graphene oxide (GO). Additionally, a sandwich immunoassay was utilized to sensitively and efficiently detect SARS-CoV-2 N protein. On the one hand, Au@Ag@Au NPs have a high refractive index and the capability to electromagnetically couple with the plasma waves propagating on the surface of gold film, which are harnessed for amplifying the SPR response signal. On the other hand, GO, which has the large specific surface area and the abundant oxygen-containing functional groups, could provide unique light absorption bands that can enhance plasmonic coupling to further amplify the SPR response signal. The proposed biosensor could efficiently detect SARS-CoV-2 N protein for 15 min and the detection limit for SARS-CoV-2 N protein was 0.083 ng/mL, with a linear range of 0.1 ng/mL~1000 ng/mL. This novel method can meet the analytical requirements of artificial saliva simulated samples, and the developed biosensor had a good anti-interference capability.

Elegant biosensor based on magnetic beads and target-directed enzyme-free dual-amplification for the detection of miRNA-let-7a

Research on the use of microRNAs (miRNAs) for cancer diagnosis is a hot topic because miRNAs are a key indicator of gene expression and hopeful biomarkers. The high-performance detection of miRNAs in early disease diagnosis has attracted attention from the research community. Herein, we construct a robust fluorescence biosensor for miRNA-let-7a detection that uses an enzyme-free dual-signal amplification strategy that combines magnetic bead-combined entropy-driven catalysis (MB-EDC) and catalytic hairpin assembly (CHA). MB-EDC uses a multifunctional DNA probes of M1-MB that contain a three-stranded substrate M1 and magnetic beads. By using the EDC reaction and magnet recovery technology, the reversibility of target recirculation process is effectively reduced. When miRNA-let-7a is introduced, MB-EDC occurs, recirculating the target and eliminating the interference of other strands by using a magnet. The trigger released in the MB-EDC then activates the CHA reaction, giving rise to an amplified fluorescence signal. This isothermal enzyme-free MB-EDC and CHA scheme can provide double signal amplification for precise analysis of miRNA-let-7a. The biosensor system for miRNA-let-7a detection has a low detection limit of 8.3 pM. Moreover, it has high specificity and relatively good detection capabilities under challenging conditions. Therefore, this sensor can promote the exploration of miRNAs in cancer diagnostics.