MiRNA detection kit based on Cas12a self-circulation and CHA double cascade amplification

Abstract

The invention discloses a miRNA detection kit and method based on Cas12a self-circulation and CHA double cascade amplification, and belongs to the technical field of biosensing. The method comprises the following steps: firstly, converting target miRNA into a double-stranded DNA activator by utilizing self-assembly of a catalytic hairpin; furthermore, a CRISPR-Cas12a positive feedback self-circulation system based on specially designed scgRNA is triggered, so that exponential amplification of a signal is realized; finally, the Au-coated CuO core-shell nano-enzyme with high catalytic activity is released by cutting a connecting chain, and TMB-HO chromogenic reaction is catalyzed, so that colorimetric detection is realized. According to the present invention, the linear amplification mode and the index amplification mode are ingeniously fused, the heterojunction nano-enzyme is used to enhance the signal, and the kit has advantages of high sensitivity (the detection limit can achieve 1.39 fM), strong specificity, simple operation, no complex instrument, visual detection realization and the like, and has wide application prospects in the fields of early cancer screening and immediate cancer diagnosis.

Description

Technical Field

[0001] This invention discloses a miRNA detection kit and method based on Cas12a self-circulation and CHA dual-cascade amplification, belonging to the field of biosensing technology. The method first utilizes catalytic hairpin self-assembly to convert the target miRNA into a double-stranded DNA activator; then, it triggers a CRISPR-Cas12a positive feedback self-circulation system based on specially designed scgRNA, achieving exponential signal amplification; finally, it releases a highly catalytically active Au@Cu2O core-shell nanozyme by cleaving the linker strand, catalyzing the TMB-H2O2 colorimetric reaction for colorimetric detection. This invention cleverly integrates linear and exponential amplification modes and utilizes heterojunction nanozymes to enhance the signal, offering advantages such as high sensitivity (detection limit down to 1.39 fM), strong specificity, simple operation, no need for complex instruments, and the ability to perform visual detection. It has broad application prospects in the field of early cancer screening and point-of-care diagnosis. Background Technology

[0002] MicroRNAs are a class of non-coding single-stranded RNA molecules, approximately 19-24 nucleotides in length, that play crucial regulatory roles in life processes such as cell differentiation, proliferation, and apoptosis. Numerous studies have shown that many miRNAs are abnormally expressed in the peripheral blood (e.g., serum, plasma) of cancer patients, making them highly promising non-invasive biomarkers for early cancer diagnosis and prognostic monitoring. For example, miRNA-21 has been widely confirmed to be closely related to the development and progression of various malignant tumors, including lung cancer, breast cancer, and colorectal cancer. Therefore, achieving accurate and rapid detection of specific miRNAs in serum (especially at concentrations as low as fM) is of great significance for early cancer screening and personalized treatment.

[0003] Currently, the "gold standard" for miRNA detection is real-time quantitative polymerase chain reaction (qRT-PCR). Although this method has high sensitivity and specificity, it heavily relies on expensive precision thermal cyclers, skilled operators, and complex RNA extraction and reverse transcription steps, resulting in a long detection cycle and making it difficult to meet the needs of point-of-care testing in resource-limited areas or at the bedside.

[0004] To overcome the above limitations, researchers have developed a variety of new detection technologies, but significant shortcomings still exist:

[0005] 1. CRISPR-Cas System-Based Detection Technologies: Represented by the CRISPR-Cas12a system, these technologies rely on the non-specific trans-cleavage activity of the Cas12a protein activated by the target nucleic acid to degrade reporter molecules (such as fluorescently labeled single-stranded DNA), thereby generating a signal. Related patents (such as CN116790720A) disclose such methods. However, these methods typically follow a linear reaction pattern of "one target activating one Cas12a protein," resulting in limited signal amplification and insufficient sensitivity for ultra-low abundance targets. More importantly, the direct activation efficiency of the Cas12a protein on RNA targets is far lower than its activation efficiency on DNA targets, limiting its application in the direct detection of miRNAs.

[0006] 2. Isothermal amplification technology based on catalytic hairpin self-assembly (CHA): CHA is an enzyme-free nucleic acid self-assembly technology that amplifies signals through a strand displacement reaction triggered by the target analyte. Related patents (such as CN112649484B) utilize CHA for signal amplification. However, CHA itself is primarily a linear amplification process, and its amplification efficiency (typically 10^2-10^3 times) is still inferior to exponential amplification technologies (such as PCR). Therefore, using it alone is insufficient to achieve the sensitivity required for detecting trace amounts of miRNA in clinical samples.

[0007] 3. Nanozyme-based colorimetric sensing technology: Nanomaterial-based enzyme mimics (nanozymes) are used to replace natural horseradish peroxidase (HRP) for colorimetric detection due to their low cost and high stability. Related patents (such as CN110849953B) involve the application of copper-based oxide nanomaterials. However, single nanomaterials (such as pure Cu2O or AuNRs) often have limited catalytic activity, resulting in slow colorimetric reactions and low signal contrast. Furthermore, the synthesis process of some high-performance nanomaterials (such as gold nanorods) often introduces toxic surfactants (such as CTAB), leading to poor biocompatibility and hindering clinical testing applications.

[0008] In summary, existing technologies struggle to simultaneously meet the core requirements of point-of-care testing (POCT): high sensitivity (fM level), high specificity (single-base resolution), ease of operation (isothermal, no complex instruments required), and easy signal interpretation (visualization). Therefore, there is an urgent need to develop a novel integrated sensing strategy. Summary of the Invention

[0009] (a) Purpose of the invention The primary objective of this invention is to provide a novel miRNA detection method, aiming to effectively integrate and overcome the shortcomings of existing single technologies. Specific objectives include:

[0010] 1. Construct a cascade reaction system that can generate exponential signal amplification for trace amounts of miRNA.

[0011] 2. It solves the problem of low activation efficiency when the Cas12a system directly detects RNA targets, without relying on the reverse transcription step.

[0012] 3. Develop a high-performance nanozyme signal probe with catalytic activity far exceeding that of a single component and good biocompatibility.

[0013] 4. The entire testing process is carried out at a constant temperature (e.g., 37°C), and the final results can be semi-quantitatively judged by directly observing color changes with the naked eye, thus forming a complete POCT solution suitable for primary healthcare scenarios.

[0014] 5. A dedicated reagent kit for implementing the above method is provided.

[0015] (II) Technical Solution To achieve the aforementioned objectives, the core of this invention lies in the creative design of a three-tiered, interconnected "signal translation-exponential amplification-enhanced output" biosensing platform. This platform is not a simple aggregation of existing technologies, but rather achieves functional synergy and a leap in performance through ingenious molecular-level design.

[0016] 1. Overall Technical Solution This invention provides a method for detecting miRNAs based on Cas12a self-circulation and CHA dual-cascade amplification, characterized in that the method comprises the following three core steps performed sequentially:

[0017] Stage 1: CHA-mediated signal "translation" and pre-amplification. Using two carefully designed hairpin DNA probes (H1 and H2), under the catalysis of the target miRNA, each miRNA molecule is converted into a stable double-stranded DNA (dsDNA) complex (H1-H2) through an entropy-driven strand displacement reaction. Simultaneously, the miRNA is cyclically released to participate in the next conversion. The key role of this step is to isothermally and enzymatically convert RNA signals, which are difficult for Cas12a to efficiently recognize, into DNA signals that are highly sensitive to Cas12a, achieving a shift in detection dimension and initial linear amplification of the signal.

[0018] The second stage: Cas12a positive feedback loop (CONAN) exponential amplification based on scgRNA. This is one of the core innovations of this invention. The H1-H2 dsDNA generated in the first stage acts as an activator, binding to the pre-assembled Cas12a-crRNA complex and activating the trans-cleavage activity of Cas12a. The activated Cas12a does not only cleave traditional fluorescent reporter probes, but specifically cleaves a specially designed "locked" single-stranded guide RNA precursor (scgRNA). When scgRNA is not cleaved, its guide sequence is "locked" by its own hairpin structure and is inactive; once cleaved, it releases mature gRNA with full function. The newly released gRNA rapidly binds to free Cas12a protein in the reaction system, forming a new Cas12a-gRNA complex. This new complex can then cleave more scgRNA, releasing more gRNA, thus forming a positive feedback loop of "activation → scgRNA cleavage → gRNA release → new complex formation → reactivation". This process achieves exponential explosive growth of the signal, breaking through the linear amplification bottleneck of traditional CRISPR detection.

[0019] The third stage: High-contrast colorimetric signal output mediated by Au@Cu2O heterojunction nanozymes. This is another core innovation of this invention. Au@Cu2O core-shell nanozymes are immobilized on the surface of magnetic beads via a single-stranded DNA linker that can be cleaved by Cas12a, forming a "magnetic bead-linker-nanozyme" composite probe. When the Cas12a in the second stage is activated and exhibits trans-cleavage activity, it indiscriminately cleaves this linker, causing a large amount of Au@Cu2O nanozymes to be released from the magnetic bead surface into the solution. After solid-liquid separation using an external magnetic field, the supernatant is added to develop chromogenic substrates (TMB and H2O2). The released Au@Cu2O nanozymes exhibit extremely high peroxidase-like activity, rapidly catalyzing the oxidation of TMB to blue oxTMB. The intensity of the blue color is directly proportional to the amount of nanozyme released, and consequently, to the concentration of the initial target miRNA. Quantitative detection can be performed using a microplate reader (absorbance at 650 nm) or by direct visual qualitative / semi-quantitative interpretation.

[0020] 2. Kit Components The present invention also provides a kit for carrying out the above-described method, characterized in that the kit comprises at least the following components:

[0021] CHA reaction module: contains hairpin probes H1 and H2 in lyophilized or solution form.

[0022] The Cas12a self-circulating reaction module contains the LbaCas12a protein, crRNA targeting the H1-H2 complex, and the key scgRNA. These components can be premixed or aliquoted.

[0023] Signal output module: contains a pre-constructed "magnetic bead-connecting chain-Au@Cu2O nanozyme" composite probe.

[0024] Chromogenic substrate module: contains TMB chromogenic solution and hydrogen peroxide (H2O2) solution.

[0025] Auxiliary reagents include reaction buffers (such as NEBuffer 2.1 or similar systems), washing buffers (such as PBS), etc.

[0026] Negative and positive controls: solutions without the target and target miRNA standards at known concentrations.

[0027] 3. Specific preparation process of Au@Cu2O nanozymes To ensure high sensitivity of the signal output, the synthesis process of the nanozyme has been specifically optimized in this invention:

[0028] Synthesis of gold nanorod (AuNR) cores: The classic seed-mediated growth method was adopted, with CTAB as the structure directing agent and ascorbic acid as the reducing agent. The aspect ratio of gold nanorods was controlled by adjusting the amount of silver ions (Ag⁺), so that they have strong absorption in the near-infrared region and provide templates for subsequent heterojunction growth.

[0029] Epitaxial growth of the Cu2O shell: Purified AuNRs were redispersed, and copper salt (CuCl2) was added as a precursor. Sodium dodecyl sulfate (SDS) was used as a mild surfactant, and reduction was performed with hydroxylamine hydrochloride (NH2OH·HCl) under alkaline conditions. By precisely controlling the reaction temperature, pH, and reagent concentration, Cu2O was uniformly epitaxially grown on the AuNR surface, forming a core-shell structure with a clear interface (Au@Cu2O). This structure was confirmed by transmission electron microscopy (TEM), X-ray diffraction (XRD), and X-ray photoelectron spectroscopy (XPS).

[0030] (III) Beneficial Effects Through the implementation of the above technical solution, the present invention has achieved the following significant beneficial effects, all of which are supported by data from specific embodiments:

[0031] 1. Order-of-magnitude breakthrough in sensitivity: Due to the effective coupling of "linear CHA amplification" and "exponential CONAN amplification," the detection limit (LOD) of this invention for the model target miRNA-21 reaches 1.39 fM (femtomolar), and maintains excellent linearity (R² = 0.996) over a wide dynamic range (spanning four orders of magnitude) from 1 pM to 10 nM. This sensitivity is 100-1000 times higher than that of using CHA alone or traditional Cas12a detection methods, meeting the needs for detecting trace amounts of miRNA in clinical serum.

[0032] 2. Perfectly solves the problem of Cas12a's "RNA insensitivity": This invention cleverly utilizes CHA as a "molecular translator," shifting the detection target from "RNA with low Cas12a activation efficiency" to "dsDNA with extremely high Cas12a activation efficiency." This not only bypasses the inherent defects of Cas12a but also eliminates the reverse transcription step, which is prone to introducing errors and increases costs and time, making the detection process more robust and faster.

[0033] 3. Significantly Improved Signal Output Intensity and Contrast: Benefiting from the electronic coupling effect at the Au-Cu2O heterojunction interface, the peroxidase-like activity (measured by the Michaelis constant Km and maximum reaction rate Vmax) of the prepared Au@Cu2O core-shell nanozyme is significantly superior to that of single AuNRs or commercial HRPs. In actual detection, this manifests as a deeper blue color development in positive samples within the same reaction time, while the background color of negative samples is lighter, resulting in an extremely high signal-to-noise ratio, making the visual judgment results highly reliable.

[0034] 4. Excellent Specificity and Anti-interference Capability: The specificity of this invention is guaranteed by two "checkpoints." The first checkpoint is the CHA reaction-dependent "toe-point substitution" mechanism, which is sensitive to single-base mismatches; the second checkpoint is the strict recognition of PAM sequences by the Cas12a-crRNA complex. This "double-checking" mechanism enables this invention to clearly distinguish the target miRNA-21 from its single-base, double-base mismatches, and other family members (such as miRNA-122). Even in complex matrices containing 10% human serum, the spiked recovery rate remains at 93.6%–102.6%, demonstrating its strong resistance to matrix interference.

[0035] 5. Truly Practical for POCT: The entire detection process can be completed in a 37°C constant temperature water bath or metal bath, eliminating the need for expensive equipment such as thermal cyclers, fluorescence microscopes, or electrochemical workstations. The operation is simple, involving sample addition, incubation, magnetic separation, and color development, with a total time controllable within 2 hours. Final result interpretation requires no instruments; a preliminary assessment of miRNA levels can be made by observing the change in solution color from colorless to blue, significantly lowering the barrier to entry and making it highly suitable for widespread application in community hospitals, clinics, and even home health monitoring.

[0036] 6. Preliminary clinical validation of effectiveness: In a blinded test using clinical serum samples, this invention successfully distinguished the serum from 8 lung cancer patients and 8 healthy volunteers. The difference in miRNA-21 expression levels detected was statistically significant (P < 0.001), and highly consistent with the conclusions of the standard qRT-PCR method. This preliminarily demonstrates the feasibility and reliability of this invention in practical clinical applications. Attached Figure Description

[0037] Figure 1 The overall principle and core component construction diagram of the detection method of this invention are as follows: ① Schematic diagram of the synthesis process of Au@Cu2O core-shell nanozyme, showing the step-by-step construction from gold nanorods to Au@Cu2O heterostructure; ② Schematic diagram of signal probe assembly, showing the structure of the composite probe of "carboxylated magnetic beads - single-stranded DNA adapter - Au@Cu2O nanozyme"; ③ Schematic diagram of the complete detection principle of the biosensor, clearly showing the entire process of three-stage signal amplification and transduction triggered by target miRNA in the form of flowchart: "catalytic hairpin self-assembly → Cas12a positive feedback self-circulation → nanozyme release → colorimetric signal output".

[0038] Figure 2Morphology, structure, and composition characterization of Au@Cu2O nanomaterials. (A) Transmission electron microscopy image of the gold nanorod core, showing its uniform rod-like morphology and size. (B) Transmission electron microscopy image of the Au@Cu2O core-shell structure, clearly showing that the surface of the gold nanorod is covered by a uniform Cu2O shell, forming a distinct core-shell heterojunction. (C) Elemental distribution map of Au@Cu2O nanoparticles, visually confirming the spatial relationship between the Au core and the Cu2O shell by superimposing the distribution of Au (red) and Cu (green) elements. (D) X-ray diffraction pattern of Au@Cu2O, showing characteristic diffraction peaks from gold (face-centered cubic structure) and cuprous oxide (cubic phase), proving the coexistence of the two phases. (E) Full X-ray photoelectron spectroscopy spectrum of Au@Cu2O. (FI) High-resolution XPS fitted spectra, namely: (F) C 1s spectrum, (G) Cu 2p spectrum, showing characteristic peaks of Cu⁺, confirming the Cu₂O phase; (H) O 1s spectrum; (I) Au 4f spectrum, whose binding energy shifts confirm the electronic interaction between Au and Cu₂O interface.

[0039] Figure 3 Characterization of the peroxidase-like activity of Au@Cu2O nanozymes. (A) Schematic diagram of the simulated colorimetric reaction of Au@Cu2O catalyzing the oxidation of TMB by hydrogen peroxide. (B) Bar chart of absorbance quantitative analysis after the colorimetric reaction of TMB catalyzed by different nanomaterials. Comparison of Au@Cu2O, pure AuNRs, pure Cu2O and blank control without nanozymes demonstrates that Au@Cu2O has the highest catalytic activity. (C) Typical kinetic curves of the TMB colorimetric reaction monitored at 650 nm wavelength. The reaction rates catalyzed by different nanozymes are compared, and the slope of the curves intuitively reflects the catalytic efficiency advantage of Au@Cu2O.

[0040] Figure 4: Feasibility verification diagram of catalytic hairpin self-assembly and Cas12a self-circulation system. (A) Non-denaturing polyacrylamide gel electrophoresis diagram verifying the CHA reaction. Each lane shows: marker (Lane 1), free target miRNA (Lane 2), hairpin probe H1 (Lane 3), hairpin probe H2 (Lane 4), mixture of H1 and H2 (Lane 5, no reaction), mixture of target and H1 (Lane 6), and the complete reaction system of target, H1, H2 and reporter probe H3 (Lane 7, a new band representing the product appears), proving that the CHA reaction was successfully triggered by the target miRNA. (B) Non-denaturing polyacrylamide gel electrophoresis diagram verifying the Cas12a / scgRNA component. Each lane shows: crRNA (Lane 1), scgRNA (Lane 2), premixed Cas12a, crRNA and scgRNA (Lane 3, showing complex formation). (C) Schematic diagram of the fluorescence detection principle used to characterize the Cas12a self-circulating system: After Cas12a is activated, it cleaves the fluorescence-quenched reporter probe, generating a fluorescence signal. (D) Real-time fluorescence kinetic curves of the system after adding different concentrations of target analytes. The rapid increase in fluorescence signal confirms the effective occurrence of scgRNA-based positive feedback self-circulating amplification of Cas12a.

[0041] Figure 5 Characterization of biosensor analytical performance (sensitivity and linearity). (A) Absorbance spectra measured at 650 nm for detecting different concentrations of target miRNA-21 (from blank to 10 μM). Color or absorbance values ​​show a gradient change with increasing concentration. (B) Calibration curves plotted based on data from (A): A plot of absorbance at 650 nm against the logarithm of target concentration shows good linearity in the range of 1 pM to 10 nM, and the linear regression equation and correlation coefficient (R²) are given. (C) The method detection limit is 1.39 fM, calculated based on detection data in the low concentration region according to the 3-fold blank standard deviation (3σ) criterion. The graph shows the signal and error bars at the low concentration points.

[0042] Figure 6: Evaluation charts of the specificity, stability, and reproducibility of the biosensor. (A) Specificity evaluation bar chart: Detection of perfectly matched miRNA-21, three-base mismatched sequences, two-base mismatched sequences, one-base mismatched sequences, and other non-target miRNAs (miRNA-144, miRNA-122) under the same conditions. The results show that only perfectly matched targets produce strong signals. (B) Storage stability test chart: Shows the signal retention rate of the newly prepared sensing material for the same concentration of miRNA-21 after storage for different times (e.g., 0, 7, 15, 30 days). (C) Reproducibility test chart: Multiple parallel detections (n≥3) of the same concentration of miRNA-21 samples were performed, and the relative standard deviation of the absorbance values ​​was calculated and displayed in the form of error bars, demonstrating that the method has good reproducibility.

[0043] Figure 7 : Validation chart for complex real-world sample analysis. (A, B) Bar chart showing the quantitative analysis results of miR-21 levels in serum samples from lung cancer patients and healthy controls (n=8 each), with significant differences in expression levels between the two groups. (C) Results of recovery tests using plasma samples with different concentrations of standard miR-21, validating the accuracy and reliability of this method in complex matrices.

[0044] Figure 8 High-resolution structural characterization of Au@Cu2O nanomaterials. (A) High-resolution transmission electron microscopy image of Au@Cu2O, showing clear lattice fringes in local areas, which can be used to analyze the interplanar spacing and crystal structure matching relationship between Au and Cu2O. (B) Energy dispersive spectroscopy (EDS) elemental analysis results of Au@Cu2O, listing the atomic percentage of each element to further verify its elemental composition and core-shell structure.

[0045] Figure 9 Characterization of the biosensor construction process. (A) Comparison of UV-Vis absorption spectra of different components (Au@Cu2O modified DNA, naked Au@Cu2O, and pure DNA) to verify the successful assembly of the signal probe. (B) UV-Vis absorption spectra of magnetic beads, crRNA, and their coupled complex to demonstrate the effective immobilization of crRNA on the surface of magnetic beads. (C) Difference in absorbance at 650 nm between the sensor system and the presence or absence of target miR-21 to demonstrate the functional response of the detection system.

[0046] Figure 10Results of experimental condition optimization. This includes optimization of key parameters for the catalytic hairpin self-assembly and Cas12a reaction: (A) Optimization of incubation temperature for the amplification reaction; (B) Optimization of Cas12a reaction time; (C) Optimization of the molar ratio of hairpin probes H1 to H2; (D) Optimization of the pH of the amplification reaction buffer; (E, F) Optimization of the reaction volume of Cas12a and crRNA. The optimal experimental conditions were determined by comparing signal intensity in each figure.

[0047] Figure 11 Comparative analysis with qRT-PCR: The detection results of this sensor are compared with those of the classic quantitative reverse transcription polymerase chain reaction (qRT-PCR) in detecting miR-21 expression levels. The comparison results of the two methods in terms of detection consistency, sensitivity, and correlation are presented, verifying the reliability and practicality of this method. Detailed Implementation

[0048] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. These embodiments are only for explaining the present invention and do not constitute a limitation on the scope of protection of the present invention. Unless otherwise specified, the reagents and materials used in the present invention are commercially available.

[0049] Example 1: Preparation and characterization of Au@Cu2O core-shell nanozymes Preparation steps: Gold nanorods (AuNRs) were synthesized using a seed-mediated growth method, and then a Cu2O shell was deposited on their surface using an in-situ reduction method to form an Au@Cu2O core-shell structure.

[0050] Characterization results: TEM images clearly show that gold nanorods with an average length of approximately 50 nm and a diameter of approximately 10 nm are encapsulated by a uniformly thick Cu₂O shell (approximately 8-10 nm), forming a distinct core-shell structure. XRD patterns show characteristic diffraction peaks of gold at 38.2°, 44.4°, 64.6°, and 77.5°, while characteristic diffraction peaks of Cu₂O appear at 29.6°, 36.5°, 42.4°, and 61.5°. XPS analysis shows a negative shift in the binding energy of the Au 4f orbitals and a positive shift in the binding energy of the Cu 2p orbitals, confirming electron transfer from Cu₂O to Au, i.e., the existence of interfacial electronic coupling effects.

[0051] Example 2: Assembly of a biosensing probe (Mag-S-Au@Cu2O) Mag-S preparation: After activation by EDC / NHS, carboxylated magnetic beads are covalently coupled with carboxyl-modified S2 probes and crRNA to prepare magnetic bead-crRNA complex (Mag-S).

[0052] Au@Cu2O-S preparation: Thiol-modified single-stranded DNA (S) self-assembles on the Au@Cu2O surface through Au-S bonds, and forms a stable signal probe after salt aging.

[0053] Example 3: Optimization of Operational Steps and Conditions in the Detection Process Standard procedure: 1. CHA reaction: The target miRNA-21 was reacted with H1 and H2 probes at 37℃ for 30 minutes. 2. Cas12a activation: Add Cas12a, Mag-S, and scgRNA, and react at 37°C for 30 minutes. 3. Signal release: After magnetic separation, Au@Cu2O-S was added and incubated at 37°C for 60 minutes. 4. Colorimetric detection: Add TMB / H2O2 and develop color for 10 minutes, then measure the absorbance at 650 nm. Optimization conditions: Temperature: 37℃; Time: CHA 30 min, Cas12a 30 min, probe incubation 60 min; pH: 7.0; Probe ratio: H1:H2 = 1:1; Cas12a volume: 2 μL (100 nM); crRNA concentration: 2.2 μL (10 μM) Performance: Linear range 1 pM-10 nM, detection limit 1.39 fM (R²=0.996).

[0054] Example 4: System Evaluation of Analytical Performance

[0055] 4.1 Sensitivity and Linearity: miRNA-21 standards with gradient concentrations from 10 fM to 10 nM were prepared and detected according to the steps in Example 3. A linear regression equation was obtained by plotting absorbance values ​​(A650) against the logarithm (logC) of the target concentration: Y = 0.088 logC + 1.199 (R² = 0.996). The limit of detection was calculated to be 1.39 fM, based on three times (3σ) of the standard deviation (σ) of the blank sample signal.

[0056] 4.2 Specificity test: 100 pM of perfectly matched miRNA-21, single-base mismatched sequences (SM), double-base mismatched sequences (DM), and irrelevant miRNA-122 and miRNA-141 were used for detection. Results showed that only perfectly matched miRNA-21 produced a strong signal response (A650 > 2.0), while the signals produced by all mismatched and irrelevant sequences (A650 < 0.15) were not significantly different from the blank control (A650 = 0.12).

[0057] 4.3 Repeatability and Stability: For the same concentration (1 pM) of miRNA-21 sample, six parallel assays were performed within the same batch of kits, and the intra-day relative standard deviation (RSD) was 4.2%; for assays performed on different dates and in different batches of kits, the inter-day RSD was 6.8%. After being stored in PBS at 4°C for 30 days, the prepared Mag-S-Au@Cu2O probe maintained more than 95% of its initial signal response capability.

[0058] Example 5: Detection of actual serum samples Serum samples were collected from eight lung cancer patients and eight healthy individuals, with the approval of the ethics committee and informed consent from the patients. All serum samples were diluted three-fold with PBS, heat-treated at 65°C for 10 minutes, and then 2 μL was directly used as the test sample, and the test was performed according to the steps in Example 3.

[0059] Results: The detection signal in the serum of the healthy group (mean A650 = 0.25 ± 0.08) was significantly lower than that in the lung cancer patient group (mean A650 = 1.89 ± 0.31). The difference between the two groups was statistically significant (P < 0.001) by t-test. Comparison of the detection results of this invention with the results of standard qRT-PCR tests performed in parallel on these samples showed a high positive correlation (Pearson correlation coefficient r = 0.972), confirming the accuracy and reliability of this invention in real, complex samples.

[0060] Conclusion: This invention successfully constructed a novel triple-cascade biosensing method integrating CHA, CRISPR-Cas12a positive feedback self-circulation, and heterojunction nanozyme catalytic amplification, and developed a corresponding detection kit based on this method. This method innovatively solves key problems in existing technologies, such as insufficient sensitivity, weak response to RNA targets, and unsatisfactory signal output, ultimately achieving ultrasensitive, highly specific, visual, and isothermal rapid detection of miRNAs. Preliminary clinical sample validation results demonstrate its significant application potential in the field of non-invasive early cancer screening. This invention is ingeniously designed, highly effective, easy to promote, and has good industrialization and market prospects.

Claims

1. A method for detecting miRNAs based on Cas12a self-circulation and CHA dual-cascade amplification, characterized in that, The method comprises the following three core reaction stages in sequence: (1) CHA-mediated signal translation and linear pre-amplification stage: The target miRNA is mixed with two pre-designed hairpin probes H1 and H2 under isothermal conditions; the target miRNA first hybridizes with the recognition domain of the H1 probe, triggering a conformational change of H1 and exposing its originally hidden fulcrum sequence; the exposed fulcrum sequence further binds complementary to the H2 probe, forming a thermodynamically more stable H1-H2 double-stranded DNA complex through an entropy-driven strand displacement reaction, while the target miRNA is cyclically released so that it can continue to catalyze the next round of reaction; this stage realizes the conversion of each target miRNA molecule into a corresponding H1-H2 dsDNA molecule, completing the translation from RNA signal to DNA signal and preliminary linear amplification; (2) Cas12a positive feedback self-circulating exponential amplification stage based on scgRNA: The H1-H2 dsDNA complex generated in step (1) is added to a reaction system containing Cas12a protein, crRNA and lock nucleic acid guide RNA precursor scgRNA; the H1-H2 complex acts as an activator, binding to the Cas12a-crRNA ribonucleoprotein complex to activate the trans-cleavage activity of Cas12a protein; the activated Cas12a protein specifically cleaves scgRNA molecules in the system; when the scgRNA is not cleaved, its guide sequence is locked by its own hairpin secondary structure and cannot function; after being cleaved by Cas12a, mature gRNA with complete activity is released; the newly released mature gRNA quickly binds to the free Cas12a protein in the reaction system to form a new Cas12a-gRNA complex; the new complex continues to cleave the remaining scgRNA, releasing more gRNA, thereby forming a positive feedback closed loop of "activation → cleavage of scgRNA → release of gRNA → formation of new complex → reactivation", realizing exponential amplification of the initial signal; (3) Au@Cu2O nanozyme release and visualization colorimetric signal output stage: In step (2), a signal output probe is added in advance or simultaneously. The probe is formed by magnetic microspheres covalently linked to Au@Cu2O core-shell nanozyme through a single-stranded DNA adapter. When Cas12a is activated and generates trans-cleavage activity in step (2), its non-specific cleavage activity will simultaneously degrade the DNA adapter connecting the magnetic beads and the nanozyme. This causes a large amount of Au@Cu2O nanozyme to be released from the surface of the magnetic beads into the reaction supernatant. Solid-liquid separation is performed by applying an external magnetic field, the magnetic beads are removed, and the supernatant containing free nanozyme is collected. The colorimetric substrate 3,3',5,5'-tetramethylbenzidine and hydrogen peroxide are added to the supernatant. The released Au@Cu2O nanozyme exerts peroxidase-like activity and catalyzes the oxidation of TMB to generate the blue product oxTMB. The quantitative or qualitative detection of the target miRNA is achieved by measuring the absorbance value of the solution at a wavelength of 650 nm or by directly observing the change in the blue depth of the solution.

2. The detection method according to claim 1, characterized in that, The hairpin probes H1 and H2 described in step (1) are designed as follows: the H1 probe is a single-stranded DNA that is partially complementary to form a hairpin structure. Its sequence contains a recognition domain that is partially or completely complementary to the target miRNA and is located in the loop region or stem-loop junction of the hairpin structure; and a fulcrum sequence that is masked by the initial hairpin structure. This fulcrum sequence is exposed by a strand displacement reaction after the miRNA binds to the recognition domain. The H2 probe is also a single-stranded DNA that forms a hairpin structure. Its sequence contains a sequence domain that is completely complementary to the fulcrum sequence exposed by H1 and is used to trigger hybridization with H1.

3. The detection method according to claim 1, characterized in that, The scgRNA mentioned in step (2) is an engineered RNA molecule whose sequence includes a scaffold sequence that binds to the Cas12a protein; a spacer sequence that is complementary to a specific region in the H1-H2 dsDNA complex; and an additional sequence for forming a self-locking hairpin structure. This additional sequence wraps the key part of the spacer sequence in a double-stranded stem, preventing it from effectively interacting with the Cas12a-crRNA complex. The scgRNA releases a mature gRNA with complete guiding function only after the self-locking structure is broken when the Cas12a protein cleaves it at a specific site.

4. The detection method according to claim 1, characterized in that, The Cas12a protein mentioned in step (2) is selected from LbaCas12a derived from Lachnospiraceae bacterium ND2006, and its amino acid sequence is shown in SEQ ID NO: 1, or is a functional variant of it with equivalent trans-cleavage activity.

5. The detection method according to claim 1, characterized in that, The Au@Cu2O core-shell nanozyme described in step (3) has the following structural features: The gold nanorods have an aspect ratio between 3.0 and 5.0 as their core and a cuprous oxide shell uniformly coats the surface of the gold nanorods with a shell thickness of 5-15 nm. A clear heterogeneous interface is formed between the gold core and the Cu2O shell, and there is electron transfer from Au to Cu2O, which generates an electronic coupling effect, thereby significantly enhancing its peroxidase-like catalytic activity.

6. The detection method according to claim 5, characterized in that, The preparation method of the Au@Cu2O core-shell nanozyme includes the following specific steps: (a) preparing gold nanorods using a seed-mediated growth method: in the presence of hexadecyltrimethylammonium bromide, chloroauric acid is reduced with sodium borohydride to obtain gold seeds, and then ascorbic acid is used as a reducing agent. By adjusting the amount of silver nitrate, gold is directed to grow on the seed surface to form nanorods; (b) purifying the gold nanorods to remove excess surfactant; (c) constructing a Cu2O shell by epitaxial growth: the purified gold nanorods are dispersed in water, and copper chloride, sodium dodecyl sulfate, sodium hydroxide and hydroxylamine hydrochloride are added sequentially, and the reaction is allowed to stand at room temperature; the hydroxylamine hydrochloride reduces Cu²⁺ to Cu⁺ and combines with OH⁻ to form a Cu2O shell by in-situ crystallization growth on the surface of the gold nanorods.

7. A kit for implementing the detection method according to any one of claims 1 to 6, characterized in that, The kit contains the following separately aliquoted components: (1) CHA reaction module: containing hairpin probes H1 and H2 dissolved in buffer or lyophilized form at concentrations of 10 nM–200 nM; (2) Cas12a self-circulating reaction module: containing purified LbaCas12a protein solution, crRNA solution targeting the H1-H2 complex, and scgRNA solution at concentrations of 50 nM–500 nM; (3) Signal output module: containing a suspension of the assembled "carboxylated magnetic beads-single-stranded DNA adapter-Au@Cu2O nanozyme" composite probe; (4) Chromogenic substrate module: containing TMB chromogenic solution and hydrogen peroxide solution, or a premix of both; (5) Reaction buffer: providing a buffer with optimal pH and ionic strength suitable for CHA reaction and Cas12a enzyme activity; (6) Controls: containing a negative control and at least one positive control standard of the target miRNA at a known concentration.

8. The reagent kit according to claim 7, characterized in that, The components in the kit are in the following forms: the core components of the CHA reaction module and the Cas12a self-circulating reaction module are pre-embedded in the cap or bottom of the reaction tube in the form of lyophilized microspheres or microdiscs; the composite probe of the signal output module is stored separately in a centrifuge tube in the form of a suspension; the chromogenic substrate is a ready-to-use solution, packaged in an opaque dropper bottle or ampoule.

9. The use of a detection method as described in any one of claims 1 to 6 or a kit as described in any one of claims 7 to 8 in the preparation of an in vitro diagnostic product, wherein the in vitro diagnostic product is used for: early screening or auxiliary diagnosis of cancer; patient prognosis monitoring or treatment effect evaluation; scientific research and quantitative analysis of microRNA biomarkers.

10. The application according to claim 9, characterized in that, The cancers include lung cancer, breast cancer, colorectal cancer, prostate cancer, stomach cancer, or liver cancer; the microRNAs to be detected include at least one of miRNA-21, miRNA-155, miRNA-10b, and let-7 family members.

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