Biosensor for detecting miRNA
By combining the CRISPR/Cas13a system and the dual signal amplification strategy of electrochemiluminescent hydrogel, a biosensor was constructed, which solved the problems of complexity and low sensitivity of existing miRNA detection methods, and realized simple, rapid miRNA recognition and high-sensitivity detection.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- GUANGAN PEOPLES HOSPITAL
- Filing Date
- 2026-03-03
- Publication Date
- 2026-05-15
AI Technical Summary
Existing miRNA detection methods are cumbersome, complex, time-consuming, and require expensive instruments. They also have low sensitivity and selectivity, making it difficult to achieve simple and rapid serum miRNA identification and nucleic acid signal amplification.
A biosensor was constructed using a transcription-coupled positive feedback CRISPR/Cas13a system and an electrochemiluminescent hydrogel, combined with a dual signal amplification strategy of Cas13a signal amplification and ECL hydrogel. The target miRNA was identified and its signal amplified using an aminocarbazole-sodium alginate-polyethylene glycol hydrogel modified with gold nanoparticles.
It significantly improves the sensitivity and specificity of miRNA detection, enables efficient recognition of low-concentration miRNAs and amplification of nucleic acid signals, reduces detection difficulty and cost, and lowers the detection limit to the aM level.
Smart Images
Figure CN122038575A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of miRNA detection technology, specifically relating to a biosensor for detecting miRNA. Background Technology
[0002] MicroRNAs (miRNAs) are a class of short, single-stranded non-coding RNAs that primarily regulate gene expression by pairing with target messenger RNAs (mRNAs) at the post-transcriptional level, inducing translational repression and / or promoting mRNA destabilization and degradation. As an important component of tumor-associated molecular regulatory networks, miRNAs can influence multiple key signaling pathways and target gene axes, playing a role in promoting or suppressing cancer development in processes such as cell proliferation, programmed cell death (e.g., apoptosis), and invasion and metastasis. Abnormal miRNA expression is closely related to patient clinical outcomes and prognostic assessment. Current research further demonstrates that different tissue origins and pathological states exhibit relatively specific miRNA expression profiles. miRNA lineages show high information content and application potential in distinguishing tumor from non-tumor tissues and in tumor subtyping and stratification, thus they are widely explored as candidate molecules for tumor biomarkers. It is worth noting that miRNAs not only exist inside cells, but can also be stably distributed in various body fluids such as plasma, serum, and urine. Their stability is related to the transport / protection methods such as encapsulation by exosomes or complexation with proteins, and can reflect the biological state of the source cells to a certain extent. These characteristics together lay the foundation for their advantages as non-invasive biomarkers.
[0003] Currently, methods for miRNA detection include electrochemical detection, Northern blotting, reverse transcription polymerase chain reaction (RT-PCR), and microarray technology. These traditional methods are cumbersome, complex, time-consuming, require expensive equipment, and have low sensitivity and selectivity. While RT-PCR is robust and reliable for miRNA detection, the entire process is complex, involving the isolation of miRNA from exosomes in the blood, miRNA elongation, reverse transcription to form cDNA, and finally amplification of the cDNA at different temperatures, thus requiring expensive equipment. These limitations have hindered miRNA detection. Summary of the Invention
[0004] To address the problems in the prior art, this invention provides a biosensor for detecting miRNA, achieving simple and rapid serum miRNA identification and nucleic acid signal amplification, thereby improving the sensitivity and specificity of target miRNA detection.
[0005] The technical problem solved by this invention is achieved by the following technical solution:
[0006] The present invention aims to provide a biosensor for detecting miRNA, comprising a transcription-coupled positive feedback CRISPR / Cas13a system and an electrochemiluminescent hydrogel, wherein the electrochemiluminescent hydrogel is an aminocarbazole-sodium alginate-polyethylene glycol hydrogel with gold nanoparticles modified on its surface.
[0007] Furthermore, the transcription-coupled positive feedback CRISPR / Cas13a system includes a Cas13a / crRNA complex, a single-stranded RNA fluorescent reporter probe, and a template strand.
[0008] Furthermore, the nucleotide sequence of the single-stranded RNA fluorescent reporter probe is as shown in SEQ ID NO.3; the nucleotide sequence of the template strand is as shown in SEQ ID NO.2.
[0009] Furthermore, the biosensor construction method includes: polishing the glassy carbon electrode surface on chamois leather with alumina powder; coating the treated glassy carbon electrode surface with an aminocarbazole-sodium alginate-polyethylene glycol hydrogel with gold nanoparticles on the treated glassy carbon electrode surface; drying to form a modified film; mixing tris(2-carboxyethyl)phosphine with single chain 1 (S1) and incubating it, then dropping it onto the hydrogel-modified glassy carbon electrode surface; subsequently blocking it with mercaptohexanol; capturing the pre-prepared dopamine-single chain 2 (DA-S2); reacting to construct the S1-DA-S2 structure; and then dropping the transcriptionally coupled positive feedback CRISPR / Cas13a system reaction mixture onto the glassy carbon electrode surface and incubating it.
[0010] Furthermore, an aminocarbazole-sodium alginate-polyethylene glycol hydrogel with gold nanoparticles was coated onto the treated glassy carbon electrode surface and dried at 35-40°C for 1-2 hours to form a modified film. Tris(2-carboxyethyl)phosphine was mixed with S1 and incubated at room temperature for 1 hour. The mixture was then blocked with mercaptohexanol at room temperature for 20-40 minutes to capture the pre-prepared DA-S2 at room temperature. The reaction was carried out for 1-2 hours to construct the S1-DA-S2 structure. The reaction mixture of the transcription-coupled positive feedback CRISPR / Cas13a system was dropped onto the glassy carbon electrode surface and incubated at 35-40°C for 50-70 minutes.
[0011] Furthermore, the construction method of the transcription-coupled positive feedback CRISPR / Cas13a system includes: preparing a reaction system containing Cas13a buffer, T7 RNA polymerase buffer, target miR-203b, single-stranded RNA fluorescent reporter probe, NTPs mixture, RNase inhibitor, T7 RNA polymerase, Cas13a / crRNA, template strand and DEPC-treated water, and incubating the above components at 35-40°C for 50-70 minutes.
[0012] Furthermore, in a reaction system with a total volume of 50 μL, the components included 10×Cas13a buffer (5 μL), T7 RNA polymerase buffer (5 μL), target miR-203b (2 μL), 100 nM single-stranded RNA (ssRNA) fluorescent reporter probe (5 μL), 10 mM NTPs mixture (10 μL), RNase inhibitor (1 μL, 50 U), 1 μM T7 RNA polymerase (4 μL), 1 μM Cas13a / crRNA (4 μL), 10 μM template strand (1.6 μL), and DEPC-treated water (12.4 μL).
[0013] Furthermore, the preparation method of the electrochemiluminescent hydrogel includes: adding aminocarbazole solution and calcium chloride to sodium alginate and polyethylene glycol aqueous solution, stirring and letting stand, and washing after hydrogel formation to obtain aminocarbazole-sodium alginate-polyethylene glycol hydrogel.
[0014] Furthermore, aminocarbazole solution and calcium chloride were added to sodium alginate and polyethylene glycol aqueous solution, respectively, and stirred at 300-700 rpm for 1-2 hours at room temperature, and then allowed to stand for 3-7 hours.
[0015] Furthermore, the method for modifying the surface of aminocarbazole-sodium alginate-polyethylene glycol hydrogel with gold nanoparticles includes: adding tetrachloroauric acid hexahydrate to the aminocarbazole-sodium alginate-polyethylene glycol hydrogel solution, stirring the mixture for 20-40 minutes, adding sodium borohydride to the solution, stirring at room temperature for 1-2 hours, and centrifuging, washing and resuspending the product for later use.
[0016] Electrochemiluminescence (ECL), an analytical technique combining the advantages of electrochemistry and spectroscopy, boasts characteristics such as low background, high sensitivity, high selectivity, rapid response, and wide dynamic range. These properties enable its integration with nucleic acid self-assembly and the CRISPR / Cas13a system, overcoming the shortcomings of high background signals and expensive signal readout equipment in nucleic acid detection, thus providing a wider linear range and lower detection limits. Porous hydrogels, due to their excellent biocompatibility, can effectively maintain the enzymatic activity of the CRISPR / Cas13a system. Furthermore, their high porosity structure promotes sufficient contact between the luminescent material, catalytic material, and hydrogen peroxide substrate, thereby shortening the electron transport path and significantly improving the detection efficiency of ECL.
[0017] This invention designs a CRISPR-based method for recognizing and initiating self-amplifying RNA analysis. Utilizing a dual amplification strategy between the template and the CRISPR / Cas13a system through synergistic positive feedback, it achieves simple and rapid serum miRNA recognition and nucleic acid signal amplification. This method provides a novel design concept and technical approach for constructing efficient and stable miRNA detection methods, and is expected to meet the clinical demand for high-sensitivity miRNA detection.
[0018] Compared with the prior art, the beneficial technical effects of the present invention are as follows:
[0019] (1) The miRNA detection platform constructed in this invention can specifically bind to target miRNA (such as miR-203b), and through the dual signal amplification effect of ECL hydrogel self-enhancement and Cas13a signal amplification strategy, the ECL signal amplification effect is significantly enhanced, thereby improving the sensitivity and specificity of target miRNA detection.
[0020] (2) In existing miRNA detection technologies, the detection of low-concentration miRNAs is limited due to the low abundance of miRNAs. However, this invention can easily detect low-concentration miRNAs by using the positive feedback Cas13a system signal coupled with ECL hydrogel dual signal amplification. It has a lower detection limit than traditional miRNA detection methods, and the detection limit is as low as aM.
[0021] (3) This invention utilizes the positive feedback Cas13a system of transcriptional coupling to specifically recognize target miRNA. Compared with traditional methods, this method only requires the preparation of porous hydrogels and simple design of target-specific recognition crRNA and template strands. With the help of T7 RNA transcriptase, Cas13a protein and NTPs, the entire detection system can be successfully established, which greatly reduces the difficulty and cost of detection.
[0022] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention, it can be implemented according to the contents of the specification. Furthermore, in order to make the above contents, objectives, features and advantages of the present invention more obvious and understandable, specific embodiments of the present invention are described below. Attached Figure Description
[0023] Figure 1 This is a schematic diagram of the transcription-coupled positive feedback CRISPR / Cas13a signal amplification strategy of the present invention.
[0024] Figure 2 This is a schematic diagram of the preparation process of the porous hydrogel of the present invention.
[0025] Figure 3This is a schematic diagram illustrating the principle of the ECL biosensor for detecting serum miRNA in this invention.
[0026] Figure 4 The figure shows the verification results of the transcription-coupled positive feedback CRISPR / Cas13a signal amplification strategy of the present invention.
[0027] Figure 5 SEM images and elemental distribution diagrams of the Au@ACZ-SA-PEG hydrogel prepared in this invention.
[0028] Figure 6 This is a graph showing the performance analysis results of the ECL biosensor of the present invention.
[0029] Figure 7 This is a graph showing the stability performance analysis results of the ECL biosensor of the present invention.
[0030] Figure 8 This is a diagram showing the clinical detection results of the ECL biosensor of the present invention. Detailed Implementation
[0031] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.
[0032] In addition, unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be obtained by purchasing from the market or prepared by existing methods.
[0033] Example 1: Construction of a transcription-coupled positive feedback CRISPR / Cas13a (TCPFC) signal amplification system
[0034] The TCPFC strategy proposed in this invention organically combines CRISPR-mediated target recognition and RNA transcription amplification reactions in a single system using a "one-pot" approach. This reaction system consists of a Cas13a / crRNA complex, T7 RNA polymerase, a template strand, and NTPs. The cascade reaction is initiated upon the introduction of the target (using miR-203b as an example). The core design of this strategy lies in the use of an RNA-DNA chimeric molecule (template strand), whose structure includes an RNA sequence that can be specifically cleaved by Cas13a / crRNA and a DNA sequence that serves as a transcription template.
[0035] Table 1: Nucleic acid sequences used in this invention
[0036]
[0037] The specific experimental steps are as follows: A 50 μL reaction system was prepared, comprising: 10×Cas13a buffer (5 μL), T7 RNA polymerase buffer (5 μL), different concentrations of target miR-203b (2 μL), 100 nM single-stranded RNA (ssRNA) fluorescent reporter probe (5 μL), 10 mM NTPs mixture (10 μL), RNase inhibitor (1 μL, 50 U), 1 μM T7 RNA polymerase (4 μL), 1 μM Cas13a / crRNA (4 μL), 10 μM template strand (1.6 μL), and DEPC-treated water (12.4 μL). The mixture was placed in a 200 μL reaction tube and incubated at 37°C for 60 minutes. Changes in fluorescence signal were monitored using a real-time quantitative PCR system.
[0038] Example 2: Construction of efficient and stable self-reinforcing ECL hydrogel
[0039] First, an aminocarbazole-sodium alginate-polyethylene glycol (ACZ-SA-PEG) hydrogel was synthesized via physical crosslinking. 100 μL of 40 mM aminocarbazole (ACZ) solution and 250 μL of 0.1 M calcium chloride (CaCl2) were added to 4 mL of 2% sodium alginate (SA) and 0.04% polyethylene glycol (PEG) aqueous solutions, respectively, and the mixture was stirred at 500 rpm for 1 hour at room temperature, then allowed to stand for 5 hours. After complete hydrogel formation, the mixture was thoroughly washed three times with deionized water to remove any ungelled components. Subsequently, gold nanoparticles (AuNPs) were synthesized on the surface of the hydrogel by adding 288 μL of tetrachloroauric acid hexahydrate (HAuCl4·6H2O) (1.0 wt%) to the synthesized hydrogel, which was prepared by dissolving 0.35 g of ACZ-SA-PEG hydrogel in 10 mL of deionized water. After rapidly stirring the mixture for 30 minutes, 10 μL of freshly prepared 1.9 M sodium borohydride (NaBH4) was added to the solution, and the mixture was stirred at room temperature for 1.5 hours. The final product was centrifuged with deionized water and washed three times, then resuspended in 0.1% chitosan solution and stored at 4°C until use.
[0040] Example 3: Construction of ECL Biosensor
[0041] First, the glassy carbon electrode (GCE) surface was polished on chamois leather using alumina (Al2O3) powder. Then, Au@ACZ-SA-PEG hydrogel was drop-coated onto the pretreated GCE surface and dried at 37°C for 1 hour to form a modified film on the electrode surface. Simultaneously, 160 μL of tris(2-carboxyethyl)phosphine (30 mM) was mixed with 40 μL of S1 (10 μM) and incubated at room temperature for 1 hour to activate the thiol groups at the ends of the S1 sequence. Next, 10 μL of the activated S1 was drop-added to the hydrogel-modified electrode surface, and incubation was performed overnight using Au-S bond interactions to achieve probe anchoring. Subsequently, the electrode was blocked with 1 mM mercaptohexanol (MCH) at room temperature for 30 minutes to eliminate non-specific adsorption sites. Then, using the base complementarity pairing principle, the pre-prepared DA-S2 was captured at room temperature and reacted for 1.5 hours to construct the S1-DA-S2 structure. Then, 10 μL of the TCPFC strategy reaction mixture was added dropwise to the electrode surface and incubated at 37°C for 60 minutes. After each modification step, the electrode was washed with phosphate-buffered saline (0.01 M PBS, pH 7.4). Finally, the constructed biosensor was placed in PBS detection buffer (0.1 M, pH 7.4) containing 30 mM potassium persulfate (K2S2O8) for ECL signal acquisition. The ECL measurement parameters were set as follows: photomultiplier tube voltage 700 V, potential scan range -2 V to 0 V, and scan rate 0.5 V / s.
[0042] Experiment 1: TCPFC Strategy Construction and Verification
[0043] To verify the feasibility of the TCPFC strategy, we first performed 12% PAGE gel electrophoresis analysis. For example... Figure 4 As shown in lane A, lane 5 exhibits four nucleic acid bands with different electrophoretic migration rates, indicating that under the control condition lacking Cas13a, the RNA fragment in the template substrate remains intact and does not undergo non-specific degradation. In lane 6, characteristic cleavage products from the template strand were observed, confirming that the formation of the crRNA-target double-stranded complex successfully activated Cas13a, thereby mediating specific cleavage of the chimeric template strand. Notably, lane 7 simultaneously displays the Cas13a cleavage product and a high abundance of transcripts generated by T7 RNA polymerase (T7 RNAP). This result indicates that the crRNA-target double-stranded complex not only triggers Cas13a-mediated cleavage but also synergistically promotes the T7 RNAP-driven transcription process, resulting in a large amount of RNA product and signal amplification.
[0044] Furthermore, fluorescence spectroscopy kinetic analysis further validated the effectiveness of the TCPFC strategy. Figure 4B). Comparative results showed that the synergistic system containing both Cas13a / crRNA and T7 RNAP (curve f) exhibited significantly enhanced fluorescence intensity compared to the Cas13a / crRNA system alone (curve g). Subsequently, we examined the linear detection range and limit of detection (LOD) of this strategy for miRNA-203b using fluorescence spectroscopy. The results showed that the fluorescence signal intensity continuously increased with increasing target concentration. Within the concentration range of 10 fM to 10 nM (… Figure 4 The detection system exhibited good linear response; the fluorescence intensity (I) was linearly correlated with the logarithm of the target concentration (lgC). Figure 4 D), the linear regression equation is I = 613.2lgC (miR-203b) +362.1 (R = 0.994).
[0045] Figure 4 The results of the validation of the transcription-coupled positive feedback CRISPR / Cas13a signal amplification strategy are shown in the figure. (A) is a PAGE image of the signal amplification system. (B) is a fluorescence kinetic diagram of the signal amplification system. a: Target; b: Template; c: Cas13a / crRNA; d: Reporter; e: ssRNA+Template+Cas13a / crRNA+T7 RNA polymerase; f: ssRNA+Target+Template+Cas13a / crRNA; g: ssRNA+Target+Template+Cas13a / crRNA+T7 RNA polymerase. (C) shows the fluorescence intensity of different concentrations of miRNA-203b based on the TCPFC strategy. (D) shows the linearity analysis of miRNA-203b using the TCPFC strategy.
[0046] Experimental Example 2: Verification of Hydrogel Synthesis
[0047] The morphology of the synthesized porous hydrogel was characterized using scanning electron microscopy (SEM). Figure 5 These results demonstrate that the ACZ-SA-PEG hydrogel is a three-dimensional microstructure with multiple pores, providing a large surface area and diverse active sites. Furthermore, the elemental distribution of the Au@ACZ-SA-PEG hydrogel was verified using characteristic K-line (EDX) elemental mapping. Figure 5 As shown in DH, most Au NPs are uniformly distributed on the ACZ-SA-PEG hydrogel, and Au NPs account for only a small portion of the total elemental composition.
[0048] Figure 5The images show the SEM images and elemental distribution maps of the prepared Au@ACZ-SA-PEG hydrogel, where AC represents the SEM image of the Au@ACZ-SA-PEG hydrogel, and DH represents the combined EDS elemental mapping images of C, N, O, Au, and their respective components.
[0049] Experimental Example 3: Performance Analysis of ECL Biosensors
[0050] The analytical performance of the prepared biosensor on three miRNA characteristics was verified. Figure 6 A shows the ECL intensity-time curve of the ECL biosensor as the miR-203b concentration increases; the signal gradually increases with the improvement of analytical level. Figure 6 As shown in B and C, a strong linear relationship was observed between the logarithm of miR-203b concentration (ranging from 100 aM to 1 nM) and ECL intensity, with the linear equation: I = 1785.8 log C(miR-203b) - 1941.2 (R = 0.991), and the detection limit was 68.82 aM. Figure 6 D). ECL and PCR test results also showed good consistency. Figure 6 E).
[0051] Figure 6 Performance analysis of the ECL biosensor. (A) ECL intensity-time curves corresponding to different concentrations of miRNA-203b (10 aM to 10 nM). (B) Calibration plot of the ECL biosensor for miRNA-203b detection. (C) Specificity of the ECL biosensor for miRNA-203b. (D) Correlation between parallel detections (n=20) of the constructed ECL biosensor and RT-qPCR. (E) Box plot of the detection limit of the ECL biosensor.
[0052] We also evaluated the stability performance of ECL, such as Figure 7 As shown in Figure A, the calculated relative standard deviations (RSDs) were 1.10%, 1.01%, 1.13%, and 3.06%, respectively, indicating that the prepared ECL biosensor exhibits excellent stability. Similarly, the stability performance of the six electrodes at 1 pM was also investigated. Figure 7 B) The calculated RSD of 2.73% demonstrates the feasibility of technical reproducibility in the measurement. A batch of biosensors was stored at 4°C, and their stability was evaluated at different time intervals. Figure 7 As shown in Figure C, even after 8 days, the ECL signal did not change significantly (RSD = 1.78%), indicating satisfactory storage stability.
[0053] Figure 7The graph shows the ECL stability analysis, where (A) represents the stability of miRNA-203b measured by ECL. (B) shows the reproducibility of the ECL biosensor measuring miRNA-203b (1pM) on six different electrodes. (C) shows the storage stability of miRNA-203b (10pM) in the ECL biosensor after 8 days.
[0054] Experimental Example 4: Clinical Application of ECL Biosensors
[0055] In addition, we simultaneously detected the expression level of miR-203b in serum samples from 220 individuals (80 healthy individuals, 40 early-stage patients, and 100 late-stage patients) to validate the application of the biosensor in clinical testing. The results showed that miRNA-203b expression increased progressively in the serum of healthy individuals, early-stage patients, and late-stage patients, with significant differences between the two groups. Figure 8 A). Furthermore, miRNA-203b can distinguish between the three population groups with high precision. Figure 8 (B and C).
[0056] Figure 8 The images show the clinical test results of ECL biosensor. A is a violin plot of serum miRNA-203b expression in normal individuals, early-stage patients, and late-stage patients. B and C are ROC curves and confusion matrices for the diagnosis of serum miRNA-203b in normal individuals, early-stage patients, and late-stage patients.
[0057] The sequence numbers of the above embodiments of the present invention are for descriptive purposes only and do not represent the superiority or inferiority of the embodiments.
[0058] The embodiments of the present invention have been described above with reference to the accompanying drawings. However, the present invention is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of the present invention without departing from the spirit and scope of the claims. All of these forms are within the protection scope of the present invention.
Claims
1. A biosensor for detecting miRNA, characterized in that, It includes a transcription-coupled positive feedback CRISPR / Cas13a system and an electrochemiluminescent hydrogel, which is an aminocarbazole-sodium alginate-polyethylene glycol hydrogel with gold nanoparticles on its surface.
2. The biosensor for detecting miRNA as described in claim 1, characterized in that: The transcription-coupled positive feedback CRISPR / Cas13a system includes a Cas13a / crRNA complex, a single-stranded RNA fluorescent reporter probe, and a template strand.
3. The biosensor for detecting miRNA as described in claim 2, characterized in that: The nucleotide sequence of the single-stranded RNA fluorescent reporter probe is shown in SEQ ID NO.3; the nucleotide sequence of the template strand is shown in SEQ ID NO.
2.
4. The biosensor for detecting miRNA as described in claim 1, characterized in that: The biosensor construction method includes: polishing the glassy carbon electrode surface on chamois leather with alumina powder; coating the treated glassy carbon electrode surface with an aminocarbazole-sodium alginate-polyethylene glycol hydrogel with gold nanoparticles on the treated glassy carbon electrode surface; drying to form a modified film; mixing tris(2-carboxyethyl)phosphine with single chain 1 and incubating it; then dropping it onto the hydrogel-modified glassy carbon electrode surface; subsequently blocking it with mercaptohexanol; capturing the pre-prepared dopamine-single chain 2; reacting it; and then dropping the transcription-coupled positive feedback CRISPR / Cas13a system reaction mixture onto the glassy carbon electrode surface and incubating it.
5. A biosensor for detecting miRNA as described in claim 4, characterized in that: The nucleotide sequence of single-strand 1 is shown in SEQ ID NO.4; the nucleotide sequence of single-strand 2 is shown in SEQ ID NO.
5.
6. The biosensor for detecting miRNA as described in claim 4, characterized in that: An aminocarbazole-sodium alginate-polyethylene glycol hydrogel with gold nanoparticles was coated onto the treated glassy carbon electrode surface and dried at 35-40°C for 1-2 hours to form a modified film. Tris(2-carboxyethyl)phosphine was mixed with single chain 1 and incubated at room temperature for 1 hour. The mixture was then blocked with mercaptohexanol at room temperature for 20-40 minutes. The pre-prepared dopamine-single chain 2 was captured at room temperature and reacted for 1-2 hours. The reaction mixture of the transcription-coupled positive feedback CRISPR / Cas13a system was dropped onto the glassy carbon electrode surface and incubated at 35-40°C for 50-70 minutes.
7. A biosensor for detecting miRNA as described in claim 4, characterized in that: The method for constructing a transcription-coupled positive feedback CRISPR / Cas13a system includes: preparing a reaction system containing Cas13a buffer, T7 RNA polymerase buffer, target, single-stranded RNA fluorescent reporter probe, NTPs mixture, RNase inhibitor, T7 RNA polymerase, Cas13a / crRNA, template strand, and DEPC-treated water. After mixing the above components, incubate at 35-40°C for 50-70 minutes.
8. A biosensor for detecting miRNA as described in claim 4, characterized in that: The preparation method of electrochemiluminescent hydrogel includes: adding aminocarbazole solution and calcium chloride to sodium alginate and polyethylene glycol aqueous solution, stirring and letting stand, and washing after hydrogel formation to obtain aminocarbazole-sodium alginate-polyethylene glycol hydrogel.
9. A biosensor for detecting miRNA as described in claim 8, characterized in that: Add aminocarbazole solution and calcium chloride to sodium alginate and polyethylene glycol aqueous solution, respectively, and stir at 300-700 rpm for 1-2 hours at room temperature, then let stand for 3-7 hours.
10. A biosensor for detecting miRNA as described in claim 8, characterized in that: The method for modifying gold nanoparticles on the surface of aminocarbazole-sodium alginate-polyethylene glycol hydrogel includes: adding tetrachloroauric acid hexahydrate to an aminocarbazole-sodium alginate-polyethylene glycol hydrogel solution, stirring the mixture for 20-40 minutes, adding sodium borohydride to the solution, stirring at room temperature for 1-2 hours, and centrifuging, washing and resuspending the product for later use.