A Novel CRISPR/Cas12a-Mediated Ratiometric Dual-Signal Electrochemical Biosensor for Ultrasensitive and Reliable Detection of Circulating Tumor Deoxyribonucleic Acid.

Circulating tumor DNA (ctDNA) holds great promise as a noninvasive biomarker for cancer diagnosis, treatment, and prognosis. However, the accurate and specific quantification of low-abundance ctDNA in serum remains a significant challenge. This study introduced, for the first time, a novel exponential amplification reaction (EXPAR)-assisted CRISPR/Cas12a-mediated ratiometric dual-signal electrochemical biosensor for ultrasensitive and reliable detection of ctDNA. To implement the dual-signal strategy, a signal unit (ssDNA-MB@Fc/UiO-66-NH2) was prepared, consisting of methylene blue-modified ssDNA as the biogate to encapsulate ferrocene signal molecules within UiO-66-NH2 nanocarriers. The presence of target ctDNA KRAS triggered EXPAR amplification, generating numerous activators for Cas12a activation, resulting in the cleavage of ssDNA-P fully complementary to the ssDNA-MB biogate. Due to the inability to form a rigid structure dsDNA (ssDNA-MB/ssDNA-P), the separation of ssDNA-MB biogate from the UiO-66-NH2 surface was hindered by electrostatic interactions. Consequently, the supernatant collected after centrifugation exhibited either no or only a weak presence of Fc and MB signal molecules. Conversely, in the absence of the target ctDNA, the ssDNA-MB biogate was open, leading to the leakage of Fc signal molecules. This clever ratiometric strategy with Cas12a as the "connector", reflecting the concentration of ctDNA KRAS based on the ratio of the current intensities of the two electroactive signal molecules, enhanced detection sensitivity by at least 60-300 times compared to single-signal strategies. Moreover, this strategy demonstrated satisfactory performance in ctDNA detection in complex human serum, highlighting its potential for cancer diagnosis.

CRISPR/Cas12a-coupled multiplexed strand displacement amplification for miRNA155 one-tube detection: via a dual-cavity PCR tube.

A CRISPR/Cas12a-coupled multiplexed strand displacement amplification (CMSDA) for the detection of miR155 has been developed. Non-specific amplification was avoided by designing a single-stranded DNA template with a hairpin structure. The detection target miR155 was used as a primer to initiate a multiple-strand displacement reaction to produce abundant ssDNA. ssDNA was recognized by the Cas12a/CrRNA binary complex, activating the trans-cleaving activity of Cas12a. The multiple-strand displacement reaction is more efficiently detected compared with a single-strand displacement reaction. The detection range is from 250 pM to 1 nM, and the limit of the detection is 6.5 pM. The proposed method showed a good applicability in complex serum environments, indicating that the method has a broad prospect for disease detection and clinical application. In addition, we designed a dual-cavity PCR tube, which realized one-tube detection of miRNA155 and avoided open-cap contamination.

CRISPR/Cas12a-Powered EC/FL Dual-Mode Controlled-Release Homogeneous Biosensor for Ultrasensitive and Cross-Validated Detection of Messenger Ribonucleic Acid.

Accurate detection of cancer-associated mRNAs is beneficial to early diagnosis and potential treatment of cancer. Herein, for the first time, we developed a novel CRISPR/Cas12a-powered electrochemical/fluorescent (EC/FL) dual-mode controlled-release homogeneous biosensor for mRNA detection. A functionalized ssDNA P2-capped Fe3O4-NH2 loaded with methylene blue (P2@MB-Fe3O4-NH2) was synthesized as the signal probe, while survivin mRNA was chosen as the target RNA. In the presence of the target mRNA, the nicking endonuclease-mediated rolling circle amplification (NEM-RCA) was triggered to produce significant amounts of ssDNA, activating the collateral activity of Cas12a toward the surrounding single-stranded DNA. Thus, the ssDNA P1 completely complementary to ssDNA P2 was cleaved, resulting in that the ssDNA P2 bio-gate on Fe3O4-NH2 could not be opened due to electrostatic interactions. As a result, there was no or only a little MB in the supernatant after magnetic separation, and the measured EC/FL signal was exceedingly weak. On the contrary, the ssDNA P2 bio-gate was opened, enabling MB to be released into the supernatant, and generating an obvious EC/FL signal. Benefiting from the accuracy of EC/FL dual-mode cross-verification, high amplification efficiency, high specificity of NEM-RCA and CRISPR/Cas12a, and high loading of mesoporous Fe3O4-NH2 on signal molecules, the strategy shows aM-level sensitivity and single-base mismatch specificity. More importantly, the practical applicability of this dual-mode strategy was confirmed by mRNA quantification in complex serum environments and tumor cell lysates, providing a new way for developing a powerful disease diagnosis tool.

Ultrathin Co-N-C Layer Modified Pt-Co Intermetallic Nanoparticles Leading to a High-Performance Electrocatalyst toward Oxygen Reduction and Methanol Oxidation.

The development of low platinum-based alloy electrocatalysts is crucial to accelerate the commercialization of fuel cells, yet remains a synthetic challenge and an incompatibility between activity and stability. Herein, a facile procedure to fabricate a high-performance composite that comprises Pt-Co intermetallic nanoparticles (IMNs) and Co, N co-doped carbon (Co-N-C) electrocatalyst is proposed. It is prepared by direct annealing of homemade carbon black-supported Pt nanoparticles (Pt/KB) covered with a Co-phenanthroline complex. During this process, most of Co atoms in the complex are alloyed with Pt to form ordered Pt-Co IMNs, while some Co atoms are atomically dispersed and doped in the framework of superthin carbon layer derived from phenanthroline, which is coordinated with N to form Co-Nx moieties. Moreover, the Co-N-C film obtained from complex is observed to cover the surface of Pt-Co IMNs, which prevent the dissolution and agglomeration of nanoparticles. The composite catalyst exhibits high activity and stability toward oxygen reduction reactions (ORR) and methanol oxidation reactions (MOR), delivering outstanding mass activities of 1.96 and 2.92 A mgPt -1 for ORR and MOR respectively, owing to the synergistic effect of Pt-Co IMNs and Co-N-C film. This study may provide a promising strategy to improve the electrocatalytic performance of Pt-based catalysts.

Electrochemical detection of the p53 gene using exponential amplification reaction (EXPAR) and CRISPR/Cas12a reactions.

An improved electrochemical sensor has been developed for sensitive detection of the p53 gene based on exponential amplification reaction (EXPAR) and CRISPR/Cas12a. Restriction endonuclease BstNI is introduced to specifically identify and cleave the p53 gene, generating primers to trigger the EXPAR cascade amplification. A large number of amplified products are then obtained to enable the lateral cleavage activity of CRISPR/Cas12a. For electrochemical detection, the amplified product activates Cas12a to digest the designed block probe, which allows the signal probe to be captured by the reduced graphene oxide-modified electrode (GCE/RGO), resulting in an enhanced electrochemical signal. Notably, the signal probe is labeled with large amounts of methylene blue (MB). Compared with traditional endpoint decoration, the special signal probe effectively amplifies the electrochemical signals by a factor of about 15. Experimental results show that the electrochemical sensor exhibits wide ranges from 500 aM to 10 pM and 10 pM to 1 nM, as well as a relatively low limit detection of 0.39 fM, which is about an order of magnitude lower than that of fluorescence detection. Moreover, the proposed sensor shows reliable application capability in real human serum, indicating that this work has great prospects for the construction of a CRISPR-based ultra-sensitive detection platform.

Catalytic Hairpin Assembly-Driven Ratiometric Dual-Signal Electrochemical Biosensor for Ultrasensitive Detection of MicroRNA Based on the Ratios of Fe-MOFs and MB-GA-UiO-66-NH2.

In this work, a novel ratio electrochemical biosensing platform based on catalytic hairpin assembly target recovery to trigger dual-signal output was developed for ultrasensitive detection of microRNA (miRNA). To achieve the ratiometric dual-signal strategy, methylene blue (MB), an electrochemical indicator, was ingeniously loaded into the pores of graphene aerogel (GA) and metal-organic framework (MOF) composites with high porosity and large specific surface area, and another electrochemical indicator Fe-MOFs with distinct separation of redox potential was selected as a signal probe. Concretely, with the presence of the target miRNA, the CHA process was initiated and the signal probe was introduced to the electrode surface, producing abundant double-stranded H1-H2@Fe-MOFs-NH2. Then, the measurement and analysis of the prepared ratiometric electrochemical biosensor by differential pulse voltammetry (DPV) showed that the introduction of the target miRNA led to an increase in the oxidation peak signal of Fe-MOFs (+0.8 V) and a decrease in the oxidation peak signal of MB (-0.23 V). Therefore, the peak current ratio of IFe-MOFs/IMB could be employed to accurately reflect the actual concentration of miRNA. Under optimal conditions, the detection limit of the proposed biosensor was down to 50 aM. It was worth noting that the proposed biosensor exhibited excellent detection performance in a complex serum environment and tumor cell lysates, showing great potential in biosensing and clinical diagnosis.

A test strip electrochemical disposable by 3D MXA/AuNPs DNA-circuit for the detection of miRNAs.

The simple and reliable detection of microRNAs is of great significance for studying the biological functions, molecular diagnosis, disease treatment and targeted drug therapy of microRNA. In this study, we introduced a novel Ti3C2Tx (MXene) aerogels (denoted as MXA) composite gold nano-particles (AuNPs)-modified disposable carbon fiber paper (CFP) electrode for the label-free and sensitive detection of miRNA-155. Firstly, in the presence of MXene, graphene oxide (GO) and ethylenediamine (EDA), the 3D MXene hydrogel was formed by self-assembly method, and then adding the freeze-dried 3D MXA dropwise to CFP. Subsequently, electrodepositing AuNPs on the CFP/MXA was done to construct a 3D disposable DNA-circuit test strip with excellent interface. Under the optimum experimental conditions, the detection limit of 3D disposable DNA circuit strip for miRNA-155 was 136 aM (S/N = 3). The CFP/MXA/AuNPs (CMA) electrode also has a wide dynamic range (20 fM to 0.4 µM), with a span of 4 orders of magnitude. Notably, we also tested the practicality of the sensor in 8 clinical samples. The technological innovations in the detection and quantification of microRNA in this work may be helpful to the study new aspects of microRNA biology and the development of diagnosis.

Sandwich-type microRNA biosensor based on graphene oxide incorporated 3D-flower-like MoS2 and AuNPs coupling with HRP enzyme signal amplification.

A sandwich electrochemical biosensing strategy for ultrasensitive detection of miRNA-21 was developed by using graphene oxide incorporated 3D-flower-like MoS2 (3D MoS2-rGO) nanocomposites as the substrate and horseradish peroxidase (HRP)-functionalized DNA strand 1 (S1)-gold nanoparticles (S1-AuNPs-HRP) as signal amplification probes. Herein, 3D MoS2-rGO nanocomposites not only had a large specific surface area and excellent conductivity, but also provided more attachment sites for electrodepositing AuNPs. In the presence of target miRNA, a sandwich structure was formed, and the determination of the miRNA-21 was carried out by measuring the DPV response of H2O2 mediated by hydroquinone (HQ) at a potential of + 0.052 V (vs AgCl reference electrode). Under the optimal experimental conditions, the as-prepared biosensor enabled the ultrasensitive detection of miRNA-21 from 5 fM to 0.5 µM with the low detection limit of 0.54 fM (S/N = 3), comparable or lower than previous reported methods for miRNA-21 detection, which benefited from the synergistic amplification of 3D MoS2-rGO and AuNPs-HRP. The prepared biosensor showed satisfactory selectivity, reproducibility, and stability towards miRNA-21 detection. The biosensor was feasible for accurate and quantitative detection of miRNA-21 in normal human serum samples with RSD below 5.86%, which showed a great potential in clinical analysis and disease diagnosis.

A universal electrochemical biosensor based on CRISPR/Cas12a and a DNA tetrahedron for ultrasensitive nucleic acid detection.

Herein, a universal nucleic acid analysis platform was constructed for sensitive and accurate detection of miRNA-155 and ctDNA using isothermal amplification-assisted CRISPR/Cas12a and a tetrahedral DNA nanostructure (TDN) supported sensing interface. Under the optimal experimental conditions, the prepared sensor achieved specific detection of miRNA-155 and ctDNA at as low as aM levels in 2.6 h. Furthermore, the platform was also successfully applied to human serum sample recovery experiments and cancer cell lysates, demonstrating outstanding reliability and accuracy. We firmly believe that this work provides a universal, sensitive, and practical tool for early clinical diagnosis.

Simultaneous detection of dihydroxybenzene isomers in the environment by a free-standing flexible ZnCo2O4 nanoplate arrays/carbon fiber cloth electrode.

The simultaneous determination of dihydroxybenzene isomers is highly valuable for early environmental monitoring, but it is still a challenge. In this work, a free-standing flexible electrode was prepared for the simultaneous detection of hydroquinone (HQ), catechol (CC), and resorcinol (RC). The bimetallic zinc/cobalt zeolitic imidazolate frameworks nanoplate arrays (Zn/Co-ZIF NPAs) grown in situ on the carbon fiber cloth (CFC) was fabricated by a facile static synthesis method, and the porous ternary ZnCo2O4 NPAs derived from Zn/Co-ZIF NPAs were formed by annealing in air. Due to the fast electron transmission, abundant active sites and excellent electrocatalytic properties with enzyme-like kinetic performance of the ZnCo2O4/CFC electrode, the as-proposed sensor showed a wilder linear response (2-500 µM), a lower detection limits (0.03 µM HQ, 0.06 µM CC and 0.15 µM RC) and a higher sensitivity (23.58 µA µM-1 cm-2 HQ, 17.72 µA µM-1 cm-2 CC, and 15.18 µA µM-1 cm-2 RC), respectively. More importantly, the proposed electrochemical sensor exhibited excellent detection performance in complex water samples, providing a strategy for the detection of other toxic substances in the ecological environment.

An ultrasensitive electrochemical biosensor for microRNA-21 detection via AuNPs/GAs and Y-shaped DNA dual-signal amplification strategy.

Herein, a gold nanoparticles/graphene aerogels (AuNPs/GAs) modified electrochemical biosensor with catalytic hairpin assembly (CHA) and Y-shaped DNA nanostructure dual-signal amplification approaches for ultrasensitive microRNA-21 (miR-21) detection was successfully constructed, which displayed an ultra-wide detection linear range from 5 fM to 50 nM, as well as a relatively low detection limit (LOD) of 14.70 aM (S/N = 3). Furthermore, the sensing strategy had excellent specificity among highly homologous miRNA family members and exhibited satisfactory analytical performance for miRNA detection.

Highly sensitive and facile microRNA detection based on target triggered exponential rolling-circle amplification coupling with CRISPR/Cas12a.

MicroRNAs (miRNAs) play a crucial role in the regulation of gene expression and have been implicated in many diseases. Herein, we develop a target triggered exponential rolling-circle amplification coupling with CRISPR/Cas12a (T-ERCA/Cas12a) system, which can achieve the ultrasensitive detection with simple operation and no annealing procedure. In this assay, T-ERCA combines the exponential amplification with rolling-circle amplification by introducing a dumb-bell probe with two enzyme recognition sites. miRNA-155 targets are activators that trigger exponential rolling circle amplification to produce large amounts of ssDNA, which is then recognized by CRISPR/Cas12a for further amplification. Compared with single EXPAR or RCA combined with CRISPR/Cas12a, this assay shows higher amplification efficiency. Therefore, benefiting from the excellent amplification effect of T-ERCA and the high recognition specificity of CRISPR/Cas12a, the proposed strategy shows a wide detection range from 1 fM to 5 nM with a LOD (limit of detection) down to 0.31 fM. Moreover, it shows good application ability for assessing miRNA levels in different cells, indicating that the T-ERCA/Cas12a may provide a new guidance for molecular diagnosis and clinical practical application.

Duplex-specific nuclease powered 3D DNA walker and quantum dots barcodes for homogeneous electrochemical detection of microRNAs.

Multiplex microRNAs (miRNAs) detection is beneficial for early diagnosis and prognosis of cancer. Herein, duplex-specific nuclease (DSN) powered 3D DNA walker and quantum dots (QDs) barcodes were designed for the simultaneous detection of miRNAs in a homogeneous electrochemical sensor. In the proof-of-concept experiment, the effective active area of the as-prepared graphene aerogel-modified carbon paper (CP-GAs) electrode was ∼14.30 times larger than that of the traditional glassy carbon electrode (GCE), endowing the enhanced capability of loading more metal ions for ultrasensitive detection of miRNAs. In addition, DSN-powered target recycling and DNA walking strategy assured the sensitive detection of miRNAs. After the introduction of magnetic beads (MNs) and electrochemical double enrichment strategies, the integration of triple signal amplification methods yielded good detection results. Under optimal conditions, towards simultaneous detection of microRNA-21 (miR-21) and miRNA-155 (miR-155), a linear range of 10-16-10-7 M and a sensitivity of 10 aM (miR-21) and 2.18 aM (miR-155) were achieved, respectively. It was worth mentioning that the prepared sensor can detect miR-155 down to 0.17 aM, which was also extremely advantageous among the sensors reported so far. What's more, through verification, the prepared sensor had good selectivity and reproducibility, and exhibited good detection ability in complex serum environments, showing great potential in early clinical diagnosis and screening.

Flexible carbon fiber cloth supports decorated with cerium metal- organic frameworks and multi-walled carbon nanotubes for simultaneous on-site detection of Cd2+ and Pb2+ in food and water samples.

The widespread heavy metal pollution endangers human health; hence, accurate on-site detection and quantification of heavy metal content in the surroundings is a vital step in reversing the harmful effect. Herein, an electrochemical sensor based on flexible cerium metal-organic framework@multi-walled carbon nanotubes/carbon cloth (CeMOF@MWCNTs/CC) was constructed for simultaneous on-site detection of Cd2+ and Pb2+ in food and water samples. The rich carboxyl groups of MWCNTs provided abundant sites for the adsorption of Cd2+ and Pb2+, and the mutual conversion of Ce3+ and Ce4+ in CeMOF facilitated the reduction and reoxidation of metal ions. The prepared electrode showed excellent performance in the simultaneous measurement of Cd2+ and Pb2+, with detection limits of 2.2 ppb and 0.64 ppb, respectively. More importantly, the sensing platform has been successfully used to detect simultaneously Cd2+ and Pb2+ in grain and water samples, and the detection results were consistent with the standard methods, showing great potential in environmental monitoring and food safety.

CRISPR/Cas12a and primer-assisted rolling circle amplification integrated ultra-sensitive dual-signal sensing platform for EGFR 19 detection.

Herein, we integrated CRISPR/Cas12a with primer-assisted rolling circle amplification (PARCA) to specifically detect EGFR 19 from the genome. We fused the method into fluorescent and electrochemical detection systems forming a stable and sensitive dual-signal sensing platform. The fluorescent detection system stably detected EGFR 19 in a linear range from 500 fM to 10 nM with an ultra-low background signal. The electrochemical detection system possessed a detection limit as low as 42 aM due to the introduction of nanomaterial UIO-66-NH2. The dual-signal sensing platform showed superior performance in complex serum samples and real cell genomes and provided a flexible and dynamic approach for the ultra-sensitive detection of EGFR 19.

Endonuclease-Assisted PAM-free Recombinase Polymerase Amplification Coupling with CRISPR/Cas12a (E-PfRPA/Cas) for Sensitive Detection of DNA Methylation.

DNA methylation is considered as a potential cancer biomarker. The evaluation of DNA methylation level will contribute to the prognosis and diagnosis of cancer. Herein, we propose a novel assay based on endonuclease-assisted protospacer adjacent motif (PAM)-free recombinase polymerase amplification coupling with CRISPR/Cas12a (E-PfRPA/Cas) for sensitive detection of DNA methylation. The methylation-sensitive restriction enzyme (MSRE) is first used to selectively digest unmethylated DNA, while the methylated target remains structurally intact. Therefore, the methylated target can initiate the RPA reaction to generate a large amount of double-stranded DNA (dsDNA). To avoid the dependence of PAM site of CRISPR/Cas12a, one of the RPA primers is designed with 5'-phosphate terminuses. After treating with Lambda, the sequence with 5'-phosphate modification will be degraded, leaving the single-stranded DNA (ssDNA). The CRISPR/Cas12a can accurately locate ssDNA without PAM, then initiating its trans-cleavage activity for further signal amplification. Meanwhile, non-specific amplification can be also avoided under Lambda, effectively filtering the detection background. Benefiting from the specificity of MSRE, the high amplification efficiency of Lambda-assisted RPA, and the self-amplification effect of CRISPR/Cas, the E-PfRPA/Cas assay shows outstanding sensitivity and selectivity, and as low as 0.05% of methylated DNA can be distinguished. Moreover, the lateral flow assay is also introduced to exploit the point-of-care diagnostic platform. Most importantly, the proposed method shows high sensitivity for determination of genomic DNA methylation from cancer cells, indicating its great potential for tumor-specific gene analysis.

Ultrasensitive fluorescent biosensor for detecting CaMV 35S promoter with proximity extension mediated multiple cascade strand displacement amplification and CRISPR/Cpf 1.

A novel fluorescent biosensor was proposed for detecting the CaMV 35S promoter in genetically modified organisms (GMOs). It was based on a proximity extension mediated multiple cascade strand displacement amplification connected with CRISPR/Cpf 1 (termed PE-MC/SDA-CRISPR/Cpf1). In this protocol, the CaMV 35S was recognized by proximity reaction in the presence of two adjacent primer probes. The proximity extension further triggered the multiple cascade strand displacement amplification (MC/SDA), generating a mass of ssDNA. The products compelled the trans-cleavage activity of CRISPR/Cpf 1, so as to cleave nearby ssDNA-FQ reporters and generate a strong fluorescent signal. The ingenious three-link combination design allowed the CaMV 35S a low background interference. And the MC/SDA combined with CRISPR/Cpf 1 dramatically improved the detection sensitivity. Under optimized conditions, the detection linear range of ultrasensitive fluorescent biosensor for CaMV 35S was from 50 fM to10 pM and 10 pM-500 pM, along with the limit of detection (LOD) down to 14.4 fM. The sensing platform also had excellent performance in the assay of selectivity and real samples. Therefore, the method earned great application potential for transgenic crops.

Simultaneous detection of CaMV35S and T-nos utilizing CRISPR/Cas12a and Cas13a with multiplex-PCR (MPT-Cas12a/13a).

Here, we established a strategy (MPT-Cas12a/13a) that combined CRISPR/Cas12a and Cas13a for simultaneously detecting CaMV35S and T-nos based on multiplex PCR (M-PCR) and transcription. It realized a simultaneous detection mode with different signals in the same space. The MPT-Cas12a/13a had excellent sensitivity with the limit of detection as low as 11 copies of T-nos and 13 copies of CaMV35S and it had outstanding specificity and anti-interference ability in actual sample analysis. Therefore, it is a potential candidate in the detection of GM crops.

Metal-organic framework-derived porous ternary ZnCo2O4 nanoplate arrays grown on carbon cloth for simultaneous electrochemical determination of ascorbic acid, dopamine, and uric acid.

Metal-organic frameworks derived from ternary metal oxide directly grown on the conductive substrate have attracted great interest in electrochemical sensing. In this work, metal-organic framework-derived ternary ZnCo2O4 nanoplate arrays that were grown on carbon cloth (ZnCo2O4 NA/CC) are fabricated and applied for the electrochemical determination of ascorbic acid (AA), dopamine (DA), and uric acid (UA). Field emission scanning electron microscope (FESEM) reveals that a network-like CC substrate is covered with considerable nanoplate arrays, presenting a large specific area. X-ray photoelectron spectroscopy (XPS) demonstrates the nanoplate arrays to be composed of ZnCo2O4. Benefiting from the unique array morphology and ternary element composition, the ZnCo2O4 NA/CC shows desirable performances for simultaneous detection of AA, DA, and UA. The individual detection limits are 7.14 µM for AA, 0.25 µM for DA, and 0.33 µM for UA. Additionally, the ZnCo2O4 NA/CC is successfully applied for the quantitative determination of AA, DA, and UA in spiked serum samples, showing its great application potential.

MXene-MoS2 heterostructure collaborated with catalyzed hairpin assembly for label-free electrochemical detection of microRNA-21.

Abnormal expression of microRNAs is greatly associated with the occurrence of various cancer types, revealing great potential of microRNA as biomarkers for cancer diagnosis and prognosis. Herein, a MXene-MoS2 heterostructure enhancing electrochemical biosensor coupled with catalytic hairpin assembly (CHA) amplification approach for label-free determination of microRNA-21 (miR-21) was successfully assembled. In particular, the unique micro-nano heterostructure with large specific area and favorable electroconductivity exhibited the ability of excellent confinement effect. Thus, rendered the MXene-MoS2 heterostructure the ability to trigger more target recycling reaction, giving new vitality to the traditional CHA amplification method. Meanwhile, thionine (Thi) and gold nanoparticles (AuNPs) were anchoring at the surface of MXene-MoS2 heterostructure, respectively, empowered the sensor the capability of capture probes fixation and miR-21 label-free determination. When numerous electronegative double-stranded DNA generated, the electron transfer was greatly hindered, resulting in signal decrease. Accordingly, the design denoted a broad dynamic range from 100 fM to 100 nM and a detection limit of about 26 fM, comparable or lower than previous reported methods for miR-21 detection. Furthermore, the sensing platform supplied satisfactory selectivity, reproducibility and stability towards the miR-21 detection. The real sample determination also showed a promising performance under clinical circumstance. Finally, from the clinical standpoint, the proposed biosensor is a considerable platform toward early disease detection and monitoring.