Synchronous real-time detection method for multiple nucleic acid markers based on electrochemiluminescence and hybridization chain reaction of metal framework material

Abstract

The invention discloses a method for synchronously and instantly detecting multiple nucleic acid markers based on electrochemiluminescence and hybridization chain reaction of a metal framework material, which comprises the following steps: sequentially modifying a RuMOF nano material and a nucleic acid capture probe CP-NH2 on the surface of an ITO (Indium Tin Oxide) electrode to construct a biological sensing interface with a strong ECL (Electrochemiluminescence) signal; according to the present invention, the concentration of the to-be-detected miRNA is determined by combining the clustering regular interval short palindromic repeat / Cas13a (CRISPR / Cas13a) and the hybrid chain reaction (HCR) technology, and the detection method has characteristics of good specificity and good sensitivity, and can achieve the synchronous instant detection of various miRNAs.

Description

Technical Field

[0001] The present invention relates to the field of biomedical engineering, and in particular to a method for simultaneous instant detection of multiple nucleic acid markers based on electrochemiluminescence of metal frame materials and hybridization chain reaction. Background Art

[0002] MiRNA in urine has become a research hotspot for non-invasive diagnosis and prognosis monitoring of chronic kidney disease (CKD) due to its advantages of non-invasive collection and early detection. In recent years, the CRISPR-Cas system has shown great potential in gene editing and other research fields. The CRISPR / Cas system can be used as a molecular scissors, with strong nucleic acid targeting and high specificity in target-induced gene / nucleic acid cleavage, good biocompatibility, and programmability. It has been successfully used for high-sensitivity nucleic acid detection. However, in order to improve the amplification efficiency, most CRISPR / Cas technologies require pre-amplification of the target nucleic acid (reverse transcription is required for RNA detection). However, the reaction conditions of pre-amplification are usually incompatible with CRISPR / Cas, which can easily cause false positive results. Therefore, it is necessary to combine other signal amplification technologies to further improve the detection sensitivity and specificity. In addition to the above problems, the traditional electrochemiluminescence (ECL) assay usually adopts a three-electrode system, which requires an electrochemical workstation to power the system and relies on PMT for signal acquisition. Only one sample can be measured at a time, which is difficult to meet the needs of high-throughput on-site rapid detection. Summary of the invention

[0003] The present invention aims to solve the above technical problems. To this end, the present invention proposes a method for simultaneous and instant detection of multiple nucleic acid markers.

[0004] Before elaborating on the scheme of the present invention, the following English abbreviations are explained: MOF: metal organic framework, ECL: electrochemiluminescence, DMF: N,N-dimethylformamide, CRISPR / Cas13a: regularly interspaced short palindromic repeats / Cas13a, HCR: hybridization chain reaction, PEI: polyethyleneimine, CS: chitosan, BSA: bovine serum albumin, ssRNA: single-stranded RNA.

[0005] The technical solution of the present invention is as follows:

[0006] A method for simultaneous instant detection of multiple nucleic acid markers, comprising:

[0007] S1. Preparation of metal organic framework material RuMOF;

[0008] S2, add RuMOF solution to each hole on the surface of ITO electrode, let it stand at room temperature to dry, then add PEI and CS mixed solution, let it stand at room temperature to dry, then add glutaraldehyde aqueous solution, incubate at room temperature, then add CP-NH2, incubate at 25℃-37℃, then use BSA solution to incubate at 25℃-37℃ to block nonspecific adsorption sites, and obtain a bioelectrode with surface modified with nano ECL luminescent material RuMOF and nucleic acid capture probe CP-NH2;

[0009] S3, dropping the miRNA to be tested and the CRISPR / Cas13a reaction solution onto the surface of the bioelectrode, reacting at 25°C-37°C, and then dropping the HCR reaction solution onto the surface of the bioelectrode and incubating;

[0010] S4. Using an ECL detection device in combination with the bioelectrode to determine the concentration of the miRNA to be tested.

[0011] In step S2, RuMOF is modified onto the surface of the ITO electrode, and the ECL luminophore is fixed using a mixed solution of PEI and CS. In some embodiments, in step S2, the nucleic acid capture probe CP-NH2 is modified with an amino group at the 5' end, and the CP modified with an amino group at the 5' end is connected to the amino groups of PEI and CS on the surface of the ITO electrode through glutaraldehyde, thereby chemically fixing the CP to the surface of the ITO electrode.

[0012] In some embodiments, the ITO electrode described in step S2 is cleaned by ultrasonic cleaning with detergent, anhydrous ethanol, and deionized water in sequence before use, and then blown dry with nitrogen.

[0013] In some embodiments, the initial concentrations of RuMOF, PEI, CS, glutaraldehyde aqueous solution, CP-NH2, and BSA solution described in step S2 are 10 mg / mL, 0.1%, 0.1%, 2.5%, 1 μM, and 1%, respectively, and the corresponding dropwise added volume ratio is 2:1:1:1:1:1:1.

[0014] In some embodiments, the CRISPR / Cas13a reaction solution components described in step S3 include: 1 μM LWCas13a, 0.2 μM crRNA, 10X Reaction buffer, 4000U / mL RNase inhibitor, and enzyme-free water, wherein the crRNA has its spacer sequence adjusted according to different miRNA targets.

[0015] In some embodiments, the HCR reaction solution components of step S3 include: 10 μM H1, 10 μM H2, 30 mM Mg 2+ and 300 mM Na + 1X TE.

[0016] H1 and H2 are hairpin probes, and the 3' ends of both are labeled with the fluorescent dye Cy5 that can undergo energy resonance transfer with RuMOF, wherein H2 can recognize and bind to the nucleic acid capture probe CP-NH2 on the bioelectrode.

[0017] The detection principle of the technical solution of the present invention is: when the target miRNA does not exist, Cas13 has no side cutting function, so the CP remains intact; when the hairpin probe (H1 and H2) marked with Cy5 is added, the HCR reaction is triggered, and a large amount of Cy5 fluorescent dyes H1 and H2 are introduced into the electrode surface, and energy resonance transfer can occur between RuMOF and Cy5, resulting in the quenching of the ECL signal of RuMOF. However, in the presence of target miRNA, miRNA specifically binds to the guide sequence (crRNA) of Cas13a, activates the trans-cutting activity of Cas13a, cuts the CP on the electrode surface, and causes it to be unable to trigger the HCR reaction, thereby maintaining the ECL signal of RuMOF.

[0018] The technical solution of the present invention can combine ECL, CRISPR / Cas13a and HCR to improve the detection sensitivity, and can be used for the detection of chronic kidney disease-related miRNA in urine samples.

[0019] In some specific embodiments, the preparation process of the metal organic framework material RuMOF in step S1 includes: dissolving Co(No3)2·6H2O and Ru(bpy)3Cl2·6H2O in N,N-dimethylformamide (DMF) organic solvent by a one-step solvothermal method to obtain a mixed solution, and the mixed solution is reacted to obtain in-situ encapsulated Ru(bpy)3 2+ The metal organic framework material RuMOF, the reaction temperature is 22°C-25°C; wherein the final concentration of Co(No3)2·6H2O in the mixed solution is 0.35 mmol, and the final concentration of Ru(bpy)3Cl2·6H2O in the mixed solution is 0.08 mmol.

[0020] In these specific embodiments, the reaction temperature of 22°C-25°C is different from the traditional 60°C condition, and the low temperature slows down the growth rate of MOF. The preparation method can obtain a RuMOF with a hydrated particle size of about 346nm and a regular octahedron (complete lattice), which is about 1 / 10 of the size (4.1μM) of the RuMOF synthesized at 60°C, while ensuring that the crystal structure and functional groups of RuMOF remain unchanged, while improving the uniformity and specific surface area of ​​the electrode surface modification.

[0021] In these specific embodiments, the RuMOF preparation process can convert Ru(bpy)3 2+In situ encapsulation in the MOF framework as the luminophore of the ECL reaction. Tested at the same voltage, it has a more uniform and stronger ECL signal than the traditional RuMOF synthesized at 60°C.

[0022] In a further embodiment, the step of dissolving Co(No3)2·6H2O and Ru(bpy)3Cl2·6H2O in N,N-dimethylformamide organic solvent to obtain a mixed solution comprises: ultrasonically dissolving Co(No3)2·6H2O and Ru(bpy)3Cl2·6H2O in N,N-dimethylformamide (DMF) organic solvent, and then adding oxalic acid and hydrochloric acid solutions, wherein the final concentration of oxalic acid in the mixed solution is 5 mmol, the final concentration of hydrochloric acid in the mixed solution is 0.2 mol, and the N,N-dimethylformamide organic solvent is a solvent with a ratio of N,N-dimethylformamide: deionized water of 2:1.

[0023] In a further embodiment, the mixed solution is allowed to react in a polytetrafluoroethylene reactor.

[0024] In a further embodiment, step S1 further comprises: after the reaction of the mixed solution is completed, the reaction solution is collected and centrifuged, the precipitate is collected, and the precipitate is washed multiple times with N,N-dimethylformamide (DMF), anhydrous ethanol, and deionized water, and finally the precipitate is placed in a 60°C vacuum drying oven and dried overnight. Preferably, the centrifugal speed is 10000 rpm and the centrifugal time is 10 min. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 TEM, EDS, and particle size images corresponding to the RuMOF synthesized at room temperature (22-25°C) and the traditional 60°C in Example 1 of the present invention;

[0026] Figure 2 The XPS, XRD, FTIR and ECL corresponding to the RuMOF synthesized at room temperature (22-25°C) and the traditional 60°C in Example 1 of the present invention;

[0027] Figure 3 This is a detection principle diagram of the method for simultaneous and immediate detection of multiple miRNAs related to chronic kidney disease in Example 2 of the present invention;

[0028] Figure 4It is a feasibility verification of the detection principle of the detection method in Example 2 of the present invention, wherein (A) ECL emission spectra of Ru(bpy)2+ and RuMOF, Cy5 fluorescence excitation and emission spectra; (B) fluorescence detection of CRISPR / Cas13a trans-cleavage activity; (C) PAGE electrophoresis diagram verifies HCR reaction, 1. CP (1 μM), 2. H1 (1 μM), 3. H2 (1 μM), 4. H1 (1 μM) + H2 (1 μM), 5. H1 (1 μM) + CP (0.2 μM), 6. H1 (1 μM) + H2 (1 μM) M) + CP (0.2 μM), 7. H2 (1 μM) + CP (0.2 μM), (D) Verification of CRISPR / Cas13a and HCR cascade reaction, 1. H1 + H2 + CP, 2. H1 + H2 + CP (Cas13a + CP), 3. H1 + H2 + CP (Cas13a + crRNA + CP), 4. H1 + H2 + CP (Cas13a + crRNA + miR-21 + CP), 5. H1 + H2 + CP (Cas13a + crRNA + NC + CP), 6. H1 + H2;

[0029] Figure 5 This is the electrode modification verification in Example 2 of the present invention; wherein (A) SEM image of ITO bare electrode; (B) SEM image of ITO electrode surface after modification with RuMOF;

[0030] a.ITO,

[0031] b.ITO / RuMOF / PEI / CS,

[0032] c.ITO / RuMOF / PEI / CS / CP,

[0033] d.ITO / RuMOF / PEI / CS / CP / BSA,

[0034] EIS spectrum of ITO / RuMOF / PEI / CS / CP / BSA / HCR (C); CV curve (D); (E)

[0035] a.ITO / RuMOF / PEI / CS,

[0036] b.ITO / RuMOF / PEI / CS / CP / BSA,

[0037] c.ITO / RuMOF / PEI / CS / CP / BSA / CRISPR(miR-21) / HCR,

[0038] ECL spectrum of edITO / RuMOF / PEI / CS / CP / BSA / CRISPR(NC) / HCR;

[0039] Figure 6 The portable ECL detection results of the three chronic kidney disease miRNAs in the embodiments of the present invention: miRNA21, miRNA192, and miRNA29 were analyzed, including (AC) miR-21, miR-192, and miR-29 detection standard curves; (DF) miR-21, miR-192, and miR-29 (100 pM) repeatability test; (GI) the effects of different types of interference sequences (10 μM) and targets (0.1 μM) miR-21, miR-192, and miR-29 on ECL signals;

[0040] Figure 7 Linear correlation between the detection results of three chronic kidney disease miRNAs in the embodiments of the present invention: miRNA21, miRNA192, and miRNA29 using portable ECL and the concentrations spiked in artificial urine (N=15), including (A) miR-21, (B) miR-192, and (C) miR-29; Bland-Altman analysis of the consistency between the detection results of the embodiments of the present invention and the spiked concentrations, (D) miR-21, (E) miR-192, and (F) miR-29. DETAILED DESCRIPTION

[0041] The following embodiments of the present invention are described in detail. The following embodiments are only intended to illustrate the present invention, but are not intended to limit the scope of the present invention in any way.

[0042] Example 1: Preparation of metal organic framework material RuMOF

[0043] The specific preparation process of RuMOF is as follows: weigh Co(No3)2·6H2O (weighing amount is 102.8mg, 0.35mmol) and Ru(bpy)3Cl2·6H2O (weighing amount is 60mg, 0.08mmol) in a 50mL centrifuge tube, add 20mL DMF solution and 10mL deionized water, add 200μL oxalic acid solution (0.75M, DMF as solvent) and 2mL hydrochloric acid solution (3M) after ultrasonic dissolution, mix well and transfer to a polytetrafluoroethylene reactor, react at room temperature (22℃-25℃), and the reaction time is 3 days. After the reaction is completed, the reaction solution is collected and centrifuged (the centrifugal speed is 10000rpm, and the centrifugal time is 10 minutes), and the precipitate is collected and washed with DMF, anhydrous ethanol, and deionized water for multiple times. Finally, the precipitate is placed in a 60℃ vacuum drying oven and dried overnight to obtain Ru(bpy)3 2+ Metal-organic framework material RuMOF.

[0044] like Figure 1The TEM, EDS, and particle size diagrams of RuMOF at 22°C-25°C and the traditional 60°C reaction temperature are shown, indicating that the RuMOF size decreases as the reaction temperature is lowered. This preparation method can obtain a RuMOF with a hydrated particle size of about 346nm and a regular octahedron (complete lattice), which is about 1 / 10 of the size of the RuMOF synthesized at 60°C. While ensuring that the RuMOF crystal structure and functional groups remain unchanged, the uniformity and specific surface area of ​​the electrode surface modification are improved.

[0045] It can be seen from TEM that the RuMOF synthesized at room temperature (22-25°C) is still a regular octahedron with a size of about 343nm and a hydrated particle size of about 346nm, which is about 1 / 10 of the size (4.1μM) of the RuMOF synthesized at 60°C. EDS experimental results show that the RuMOF synthesized at room temperature (22-25°C) also contains C, N, O, Co and Ru elements.

[0046] like Figure 2 Shown are the XPS, XRD, and ECL of RuMOF at room temperature (22-25°C) and the traditional reaction temperature of 60°C, indicating that while ensuring that the crystal structure and functional groups of RuMOF remain unchanged, when tested at the same voltage, the small-sized RuMOF synthesized at room temperature (22-25°C) is modified more uniformly and has a stronger ECL signal.

[0047] Example 2: Method for simultaneous and immediate detection of multiple miRNAs related to chronic kidney disease

[0048] like Figure 3 The diagram shows the detection principle of the method for simultaneous and immediate detection of multiple miRNAs related to chronic kidney disease in Example 2. The detection method comprises the following steps:

[0049] Step 201. Use Example 1 to prepare the metal organic framework material RuMOF.

[0050] Step 202. The ITO electrode was ultrasonically cleaned with detergent, anhydrous ethanol, and deionized water for 10 minutes in sequence, then dried with nitrogen, and placed in a 60°C oven for several hours for further drying. A 10 mg / mL RuMOF solution (20 μL) was added to each hole on the surface of the ITO electrode, and after standing and drying at room temperature, a mixed solution of 0.1% PEI and 0.1% CS (10 μL) was added, and then stood and dried at room temperature. Then, a 2.5% glutaraldehyde aqueous solution (10 μL) was added, incubated at room temperature for 30 minutes, and then 1 μM CP-NH (10 μL) was added, and incubated at 37°C for 1 hour. Subsequently, a 1% BSA solution (10 μL) was incubated at 37°C for 30 minutes to block nonspecific adsorption sites, and a bioelectrode with a surface modified with nano-ECL luminescent material RuMOF and a nucleic acid capture probe CP-NH2 was obtained, so that a biosensing interface with a strong ECL signal was constructed.

[0051] Step 203. Rinse the electrode with deionized water several times, dry it naturally, and then drop the CRISPR / Cas13a reaction solution (20 μL) containing the miRNA sample to be tested onto the surface of the bioelectrode. After reacting at 37°C for 30 min, drop the HCR reaction solution (10 μL) onto the surface of the bioelectrode and incubate for 2 hours.

[0052] Table 1 shows the nucleic acid sequences used in Example 2.

[0053] Table 1

[0054]

[0055] Table 2 shows the CRISPR / Cas13a reaction system used in Example 2.

[0056] Table 2

[0057] Components Add volume Final concentration 1 μM Cas13a 1μL 20nM 0.2 μM crRNA 2.5μL 10nM 0.1μMtarget 2.5μL 5nM 2μM FAM-report 2.5μL 100nM 10×Reactionbuffer 5μL 1× 40000U / mL RNase inhibitor 1μL 0.8 U / μL Enzyme-free water 35.5μL /

[0058] Table 3 shows the HCR reaction system used in Example 2.

[0059] Table 3

[0060]

[0061] Step 204. Using ECL detection device, the concentration of miRNA to be tested is determined in combination with the bioelectrode. The detection principle is: first, RuMOF is modified to the surface of the ITO electrode, and then the ECL luminophore is fixed using a mixed solution of PEI and CS. Subsequently, the capture probe (CP) of the RNA single strand is modified on the electrode surface, and the CP modified with an amino group at the 5' end is connected to the amino group of PEI and CS on the electrode surface by glutaraldehyde, thereby fixing the CP to the electrode surface. In the presence of target miRNA, miRNA binds to crRNA, activates the side cutting activity of Cas13a, and thus cuts the CP modified on the electrode surface. Different miRNA sequences are specifically distinguished by the crRNA of Cas13a. CP serves as both a side cutting substrate of CRISPR / Cas13a and a trigger chain for subsequent HCR reactions. The 3' ends of the hairpin H1 and hairpin H2 in the HCR reaction solution components are modified with fluorescent dye Cy5, so the HCR reaction will introduce a large amount of Cy5 on the ITO electrode surface, quenching the ECL signal of RuMOF by energy resonance transfer. If CP is cleaved, the HCR reaction cannot be triggered, allowing RuMOF to maintain a high ECL signal. Therefore, the relationship between miRNA concentration and ECL signal intensity can be established.

[0062] Figure 4 To verify the feasibility of the detection principle of the detection method in this embodiment 2, Figure 5 For the electrode modification verification in this Example 2, Figure 4 and Figure 5 The feasibility of detecting miRNA using electrodes combined with ECL, CRISPR, and HCR technology was confirmed.

[0063] Example 3: Electrochemiluminescent biosensor detection for simultaneous and immediate detection of multiple miRNAs associated with chronic kidney disease

[0064] In Example 3, the miRNA samples to be tested include miR21, miR192, miR29 related to chronic kidney disease, RNA sequences with 3 base mismatches with the target sequence, and other miRNAs related to CKD for detection.

[0065] Before the test, 25 μL of 100 mM DBEA (containing 10 mM PBS, pH=7.4) solution was added to each detection well of the ITO electrode, and the ECL signal was extracted using a commercial desktop machine or a self-developed portable device.

[0066] like Figure 6As shown, with the increase of miRNA concentration, the number of Cas13a activated trans-cutting activity increases, resulting in an increase in the number of CP cuts, thereby reducing the triggered HCR reaction, reducing the amount of Cy5 introduced to the electrode surface, and the ECL signal becomes stronger and stronger. The calibration curve between the normalized ECL intensity and the logarithm of the miRNA concentration was calculated by formula 1. MiR-21, miR-192 and miR-29 showed a good linear relationship in the concentration range of 2.0fM-2.0μM, 2.0fM-0.20μM and 2.0fM-2.0μM, respectively (R=0.98, R=0.98, R=0.98), and the LODs were 0.15fM, 0.096fM and 0.45fM (S / N=3), respectively.

[0067] ΔE=|E-E0| / E0 (Formula 1)

[0068] Subsequently, the reproducibility test was carried out with miRNA at a concentration of 100 pM. The RSDs of five repeated measurements of miR-21, miR-192 and miR-29 were 1.35%, 1.44% and 0.44%, respectively, indicating that the reproducibility of this research scheme was good.

[0069] At the same time, only the corresponding target miRNA (0.1 μM) can enhance the ECL signal, while other similar miRNA sequences (10 μM) fail to cause the enhancement of the ECL signal. This is because the guide sequence crRNA of the Cas13a protein is specifically designed for each target miRNA, and only the target miRNA can bind to it in a completely complementary pair, thereby activating the trans-cleavage activity of Cas13a.

[0070] Example 4: Portable electrochemiluminescent biosensor for simultaneous and immediate detection of multiple miRNAs associated with chronic kidney disease

[0071] In Example 4, the miRNA samples to be tested were artificial urine containing chronic kidney disease-related miR21, miR192, and miR29 for standard addition experiments and recovery analysis. Three different concentrations of miRNA (10fM, 10pM, 10nM) were mixed to prepare 15 artificial urine samples of different concentrations for recovery analysis. ECL signals were extracted using a commercial desktop or a self-developed portable device.

[0072] As shown in Table 4 and Figure 7As shown, the spiked recovery rates of the three miRNAs were all between 90-115%, showing good recovery rates, indicating that this research scheme can accurately quantify the target miRNA in the presence of the other two miRNAs. The spiked concentration was linearly fitted with the detection results of the portable ECL detection device. The fitting slopes of miR-21, miR-192 and miR-29 detection were 1.001, 0.999, and 1.000, respectively, and the correlation coefficients were all 0.99, indicating that the detection results of this research scheme in different samples were consistent with the spiked concentrations. Bland-Altman analysis showed that almost all the scattered points were distributed within the consistency interval with a confidence level of 95%, further indicating that the detection results of this method had a good level of consistency with the spiked concentrations. The developed multiple miRNA detection schemes can achieve simultaneous and instant detection of three miRNAs for chronic kidney disease in clinical urine samples.

[0073] Table 4 Recovery results of three miRNA artificial urine spike detection

[0074]

[0075]

[0076]

[0077] The embodiments of the present invention are described in detail above with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge scope of ordinary technicians in the technical field without departing from the purpose of the present invention.

Claims

1. A method for simultaneous and real-time detection of multiple nucleic acid markers, characterized in that: include: S1. Preparation of metal organic framework material RuMOF; S2, dropping RuMOF solution into each hole on the surface of ITO electrode, standing at room temperature to dry, then dropping polyethyleneimine and chitosan mixed solution, standing at room temperature to dry, then adding glutaraldehyde aqueous solution, incubating at room temperature, then dropping CP-NH2, incubating at 25℃-37℃, then, using bovine serum albumin solution to incubate at 25℃-37℃ to block nonspecific adsorption sites, to obtain a bioelectrode with surface modified with nano ECL luminescent material RuMOF and nucleic acid capture probe CP-NH2; S3, dropping the miRNA to be tested and the CRISPR / Cas13a reaction solution onto the surface of the bioelectrode, reacting at 25°C-37°C, and then dropping the HCR reaction solution onto the surface of the bioelectrode and incubating; S4. Using an ECL detection device in combination with the bioelectrode to determine the concentration of the miRNA to be tested.

2. The method for simultaneous and instant detection of multiple nucleic acid markers according to claim 1, characterized in that: The nucleic acid capture probe CP-NH2 described in step S2 is modified with an amino group at the 5' end. The CP modified with an amino group at the 5' end is connected to the amino groups of PEI and CS on the surface of the ITO electrode by glutaraldehyde, thereby chemically fixing the CP to the surface of the ITO electrode.

3. The method for simultaneous and instant detection of multiple nucleic acid markers according to claim 1, characterized in that: Before use, the ITO electrode was cleaned with detergent, anhydrous ethanol, and deionized water by ultrasonic cleaning, and then dried with nitrogen.

4. The method for simultaneous and instant detection of multiple nucleic acid markers according to claim 1, characterized in that: The initial concentrations of RuMOF, PEI, CS, glutaraldehyde aqueous solution, CP-NH2, and BSA solution described in step S2 are 10 mg / mL, 0.1%, 0.1%, 2.5%, 1 μM, and 1%, respectively, and the corresponding dropwise addition volume ratio is 2:1:1:1:1:1:

1.

5. The method for simultaneous and instant detection of multiple nucleic acid markers according to claim 1, characterized in that: The components of the CRISPR / Cas13a reaction solution described in step S3 are: 1 μM LWCas13a, 0.2 μM crRNA, 10X Reaction buffer, 4000U / mL RNase inhibitor, and enzyme-free water, wherein the crRNA has its spacer sequence adjusted according to different miRNA targets.

6. The method for simultaneous and instant detection of multiple nucleic acid markers according to claim 1, characterized in that: The HCR reaction solution described in step S3 is composed of 10 μM H1, 10 μM H2, 30 mM Mg 2+ and 300 mM Na + 1X TE; H1 and H2 are hairpin probes, and the 3' ends of both are labeled with the fluorescent dye Cy5 that can undergo energy resonance transfer with RuMOF, wherein H2 can recognize and bind to the nucleic acid capture probe CP-NH2 on the bioelectrode.

7. The method for simultaneous and instant detection of multiple nucleic acid markers according to claim 1, characterized in that: Step S1 includes: By a one-step solvothermal method, Co(No3)2·6H2O and Ru(bpy)3Cl2·6H2O were dissolved in N,N-dimethylformamide organic solvent to obtain a mixed solution, and the mixed solution was reacted to obtain in-situ encapsulated Ru(bpy3 2+ Metal organic framework material RuMOF, reaction temperature: 22℃-25℃; The final concentration of Co(No3)2·6H2O in the mixed solution is 0.35 mmol, and the final concentration of Ru(bpy)3Cl2·6H2O in the mixed solution is 0.08 mmol.

8. The method for simultaneous and instant detection of multiple nucleic acid markers according to claim 7, characterized in that: The step of dissolving Co(No3)2·6H2O and Ru(bpy)3Cl2·6H2O in an N,N-dimethylformamide organic solvent to obtain a mixed solution, include: Co(No3)2·6H2O and Ru(bpy)3Cl2·6H2O were ultrasonically dissolved in N,N-dimethylformamide organic solvent, and then oxalic acid and hydrochloric acid solutions were added, wherein the final concentration of oxalic acid in the mixed solution was 5 mmol, the final concentration of hydrochloric acid in the mixed solution was 0.2 mol, and the N,N-dimethylformamide organic solvent was a solvent with a ratio of N,N-dimethylformamide: deionized water of 2:

1.

9. The method for simultaneous and instant detection of multiple nucleic acid markers according to claim 7, characterized in that: The mixed solution is allowed to stand in a polytetrafluoroethylene reactor for reaction.

10. The method for simultaneous and instant detection of multiple nucleic acid markers according to claim 7, characterized in that: Also includes: After the reaction of the mixed solution is completed, the reaction solution is collected and centrifuged to collect the precipitate, and the precipitate is washed multiple times with N,N-dimethylformamide, anhydrous ethanol and deionized water respectively.

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