Electrochemical detection method of miRNA-141

Abstract

The invention relates to a miRNA-141 electrochemical sensor, which is based on a bispecific nuclease (DSN) mediated cyclic amplification signal amplification strategy (RCA), adopts basic group specific complementary pairing, and synthesizes a copper nano-cluster (Cu NCs) as an electrochemical signal through an in-situ DNA template, and is used for ultra-sensitive analysis of miRNA-141. The detectable linear range of the sensor is 1 fM to 100 nM, and the detection limit is 0.35 fM. The detection performance, selectivity and applicability of the method have great application prospects in the aspects of gene detection, clinical research and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, and particularly relates to an electrochemical detection method for miRNA-141. Background Art

[0002] In order to support the Healthy China strategy, continuous innovation and development of biosensor detection technology, analysis efficiency and application strategy have vital scientific significance and use value. As a potential biomarker for early clinical cancer diagnosis, the abnormal expression of miRNA-141 is closely related to a variety of cancers, such as lung cancer, liver cancer, prostate cancer and gastric cancer. However, miRNA-141 has the characteristics of short sequence, low abundance and high homologous sequence similarity, which puts higher requirements on the highly sensitive and specific analysis and detection technology of miRNA-141.

[0003] However, the sensitivity and specificity of the methods for detecting miRNA-141 in the prior art cannot meet the detection requirements. Summary of the invention

[0004] The purpose of the present invention is to provide an electrochemical detection method for miRNA-141, which has high sensitivity and specificity.

[0005] The purpose of the present invention is achieved by the following technical solutions:

[0006] The electrochemical detection method of miRNA-141 comprises the following steps:

[0007] (1) Testing with different concentrations of miRNA-141 solution to prepare a standard curve;

[0008] (2) Detecting the sample to be tested and calculating the concentration of miRNA-141 in the sample to be tested according to the standard curve.

[0009] In the present invention, the method for detecting a sample to be tested or a miRNA-141 solution of different concentrations comprises the following steps:

[0010] (1) Carboxyl-modified Fe3O4 nanoparticles react with primer P1 modified with 5'-terminal amino group to obtain P1-Fe3O4 complex; DNA1, 2 and 3 are bound to the P1-Fe3O4 complex by the base complementary pairing principle to obtain a hybrid complex; the hybrid complex and double-stranded specific nuclease are reacted with a sample to be tested or a miRNA-141 solution of different concentrations at 60-70° C. for 20-40 min to perform a DSN-mediated target cyclic amplification reaction to obtain ssDNA released after the action of DSN;

[0011] (2) The 5'-end carboxyl-modified primer P2 is combined with the surface amino-modified mesoporous silica nanospheres and reacts with the ssDNA released after the DSN in step (1) to obtain a dsDNA-MSNs complex rich in A and T bases;

[0012] (3) The capture probe CP modified with a thiol group at the 5' end is covalently assembled onto an indium tin electrode modified with three-dimensional gold nanoclusters to obtain a CP / GNC / ITO electrode; the CP / GNC / ITO is immersed in a solution containing an auxiliary probe HP and a dsDNA-MSNs complex, and the HP and dsDNA-MSNs complex are assembled onto the surface of the CP / GNC / ITO to obtain a dsDNA-MSNs / HP / CP / GNC / ITO electrode; then, ascorbic acid solution and anhydrous copper sulfate solution are sequentially added to the surface of the dsDNA-MSNs / HP / CP / GNC / ITO at 35-40°C for 40-60 minutes to promote the formation of copper nanoclusters; then, the dsDNA-MSNs / HP / CP / GNC / ITO after the reaction is detected by differential pulse voltammetry using a CHI650E workstation.

[0013] In the present invention, the sequence of the primer P1 modified with an amino group at the 5' end is as follows: 5'-NH2-C6-CCATCTTTACCAGACAGTGTTA-3'; the sequence of DNA1 is as follows: 5'-ACACATATATAT-3'; the sequence of DNA2 is as follows: 5'-CTGGATATATAT-3'; the sequence of DNA3 is as follows: 5'-AGATATATATAT-3'; the sequence of the primer P2 modified with a carboxyl group at the 5' end is as follows: 5'-COOH-C6-ATATATAT-3'; the sequence of HP is as follows: 5'-GTGTATATATAT-3'; the sequence of the capture probe CP modified with a thiol group at the 5' end is as follows: 5'-SH-C6-ATATATAT-3'.

[0014] In the present invention, the abscissa of the standard curve is 1gC, C is the concentration of miRNA-141, and the ordinate is the peak current difference between different concentrations of miRNA-141 and a blank sample; the blank sample is an ultrapure aqueous solution without miRNA-141.

[0015] The principle of the detection method of the present invention ( Figure 1) is as follows: With miRNA-141 as the detection object, a new type of label-free electrochemical detection method for miRNA was established through a double-stranded specific nuclease (DSN)-mediated target cyclic amplification strategy (RCA), and copper nanoclusters (Cu NCs) were synthesized in situ with DNA templates as electrochemical detection signals. First, three single-stranded DNAs rich in A and T bases (ssDNA, i.e., DNA1 / 2 / 3) were paired with primer P1 (single-strand) modified on magnetic mesoporous Fe3O4 nanospheres through partial base complementarity. When the target miRNA-141 is present, P1 specifically recognizes and binds to miRNA-141 with high affinity to form a double strand, thereby releasing ssDNA. In the RCA strategy with DSN, DSN efficiently recognizes and cleaves the DNA strand in the fully complementary paired DNA double strand or DNA / RNA hybrid double strand, while it has no enzyme activity against single-stranded DNA and single / double-stranded RNA. Therefore, the target miRNA is released from the double strand and hybridizes with the unreacted P1 to undergo a cyclic reaction of cleavage, release, and hybridization. After magnetic separation using magnetic Fe3O4, the released ssDNA hybridizes with single-stranded DNA (P2) rich in A and T bases loaded on mesoporous silica nanospheres (MSNs) to obtain double-stranded DNA silica nanospheres (dsDNA-MSNs) complexes rich in A and T bases. Subsequently, it is layered with auxiliary probes (HP) and capture probes (CP) on indium tin electrodes (ITO) electrochemically deposited with three-dimensional gold nanoclusters (GNCs) to form a large amount of double-stranded DNA rich in AT bases and used as a template to generate a large amount of double-stranded DNA rich in AT bases in the presence of reducing agents and Cu. 2+ Under the action of Cu NCs synthesized in situ by DNA template, high sensitivity and selective detection of miRNA-141 were achieved by three-electrode system differential pulse voltammetry (DPV).

[0016] The electrochemical detection method of miRNA-141 provided by the present invention has the following beneficial effects:

[0017] (1) High sensitivity. The method of the present invention uses a double-stranded specific nuclease (DSN)-mediated target cyclic amplification strategy (RCA) and a DNA template to in situ synthesize copper nanoclusters (Cu NCs) as electrochemical detection signals, which not only amplifies the readout of the electrochemical signal, but also greatly accelerates the transfer of electrons by the nanomaterial, significantly improving the sensitivity and stability of the detection. The electrochemical detection method provided by the present invention has a linear detection range of 1fM-100nM for miRNA-141 and a detection limit of 0.35fM.

[0018] (2) High specificity. A variety of different sequences have no interference in the detection of miRNA-141 by the method of the present invention.

[0019] (3) Accurate results: The recovery rate of miRNA-141 by the method of the present invention is between 95% and 105%.

[0020] (4) High versatility. The method of the present invention is simple to operate, low in cost, and can detect low-concentration miRNA-141, thus having good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Detection principle of the method of the present invention

[0022] Figure 2 Standard curve preparation results. Figure 2 (A) DPV spectrum of the method of the present invention for detecting different concentrations (0, 1fM, 10fM, 100fM, 1pM, 10pM, 100pM, 1nM, 10nM, 100nM) of miRNA-141, the horizontal axis is the potential, the unit is V, the vertical axis is the current, the unit is μA; Figure 2 (B) shows the linear relationship between different concentrations of miRNA-141 and DPV response value. The ordinate is the peak current difference between different concentrations of miRNA-141 and blank sample, in μA; the abscissa is lgC, C is the concentration of miRNA-141, in molar (M); Figure 2 (C) Specificity of the method of the present invention, Blank is a blank sample, and Mixture is a mixture of miRNA-21, let-7a, miRNA-155, and miRNA-141.

[0023] Figure 3 (A) EIS characterization of the biosensor assembly process: (a) ITO; (b) GNC / ITO; (c) CP / GNC / ITO; (d) HP / CP / GNC / ITO; (e) dsDNA-MSNs / HP / CP / GNC / ITO. The abscissa is the real part of the impedance in ohms, and the ordinate is the imaginary part of the impedance in ohms. Figure 3 (B) Gel electrophoresis analysis, lane 1-DNA1, lane 2-DNA2, lane 3-DNA3, lane 4-complex of DNA1, 2 and 3 with P1, lane 5-ssDNA after DSN action. DETAILED DESCRIPTION

[0024] The Duplex-Specific Nuclease Kit was purchased from Shenzhen Newbond Biotechnology Co., Ltd., with the product number EA003, and includes DSN (double-strand specific nuclease, DSN) and 2×DSN annealing solution.

[0025] In the present invention, all nucleic acid fragments (Table 1) were dissolved in ultrapure water to the required concentration and stored at -4°C for future use. All experimental water was ultrapure water (≥18 MΩ, Milli-Q, Millipore).

[0026] Example 1 Electrochemical Detection Method of miRNA-141

[0027] The electrochemical detection method of miRNA-141 comprises the following steps:

[0028] 1. Make a standard curve

[0029] (1)DSN-mediated target cyclic amplification reaction

[0030] Fe3O4 nanoparticles were prepared according to the method in the literature (the preparation method was disclosed in Zhu, M., & Diao, G. (2011). Synthesis of porous Fe3O4 nanospheres and its application for the catalytic degradation of xylenol orange. The Journal of Physical Chemistry C, 115 (39), 18923-18934): 1.5 g FeCl3·6H2O, 1 g polyvinyl pyrrolidone and 2 g sodium acetate were added to 30 mL of ethylene glycol. After mechanical stirring for 2 h, the mixture was transferred to a Teflon-lined stainless steel reactor and heated at 200°C for 8 h. The black product in the precipitate was collected by magnetic separation, then washed with ethanol 5 times, and dried in a vacuum drying oven at 60°C for 24 h to obtain magnetic Fe3O4 nanoparticles. Carboxyl modification of Fe3O4 nanoparticles: 30 mg of Fe3O4 nanoparticles were dispersed in 40 mL of deionized water, ultrasonicated for 30 min, and then 126 mg of oxalic acid was added to the above solution under mechanical stirring. After reacting at room temperature for 1.5 h, magnetic separation was performed and washed with distilled water for 5 times. Finally, it was dried in a vacuum drying oven at 60 ° C for 24 h to obtain carboxyl-modified Fe3O4 nanoparticles.

[0031] The DSN-mediated target cyclic amplification reaction includes the following steps: taking 100 μL of carboxyl-modified Fe3O4 nanoparticles with a concentration of 1 mg / mL, dispersing them in a PBS buffer (0.1 M, pH 7.4) containing 10 mM NHS (N-hydroxysuccinimide) and 20 mM EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide), and gently shaking them at room temperature for 1 hour; then, adding 5 μL of 5'-terminal amino-modified primer P1 with a concentration of 100 μM, and shaking overnight, so that the carboxyl-modified Fe3O4 nanoparticles and the 5'-terminal amino-modified primer P1 are covalently bonded to form a P1-Fe3O4 complex. After magnetic separation, the P1-Fe3O4 complex was washed with PBS buffer and dispersed in 200 μL of PBS buffer (0.1M, pH 7.4), and 5 μL of a 100 μM mixture of single-stranded DNA 1, 2 and 3 rich in A and T bases (abbreviated as ssDNA mixture) was added and placed at 37°C for 2 hours. Through the principle of base complementary pairing, DNA 1, 2 and 3 were respectively bound to the P1-Fe3O4 complex to obtain a hybrid complex. The hybrid complex was magnetically separated and washed with PBS buffer to remove unreacted DNA chains. The hybrid complex was redispersed in 200 μL of PBS buffer (0.1M, pH7.4) to obtain a hybrid complex suspension. 4 μL of the above hybrid complex suspension and 1 μL of DSN (0.1 unit) were mixed with 5 μL of different concentrations of miRNA-141 aqueous solution (concentrations were 0, 1fM, 10fM, 100fM, 1pM, 10pM, 100pM, 1nM, 10nM, 100nM; when the concentration was 0, it was a blank sample), and incubated at 65°C for 30 minutes to perform DSN-mediated target cyclic amplification reaction. Subsequently, 5 μL of 2×DSN annealing solution was added to each reaction solution, and the reaction was carried out at 50°C for 5 minutes to terminate the cyclic amplification reaction, and then heated at 90°C for 20 minutes to completely inactivate DSN and cooled to room temperature to obtain ssDNA (i.e., DNA1, 2, and 3) released after DSN action, which contained a large number of A and T bases.

[0032] The structure of the primer P1 with 5'-terminal amino group modification is as follows: 5'-NH2-C6-CCATCTTTACCAGACAGTGTTA-3'; the mixture of single-stranded DNA 1, 2 and 3 rich in A and T bases: the mixture contains single-stranded DNA 1, 2 and 3 at a molar ratio of 1:1:1, wherein the sequence of DNA 1 is: 5'-ACACATATATAT-3'; the sequence of DNA 2 is: 5'-CTGGATATATAT-3'; the sequence of DNA 3 is: 5'-AGATATATATAT-3'. The concentration of the mixture of single-stranded DNA rich in A and T bases = the concentration of DNA 1 = the concentration of DNA 2 = the concentration of DNA 3.

[0033] (2) MSNs loaded with double-stranded DNA rich in A and T bases (dsDNA-MSNs)

[0034] The synthesis of surface amino-modified mesoporous silica nanospheres (MSNs) was carried out according to a previously reported method (Wei, J., Chang, W., Qileng, A., Liu, W., Zhang, Y., Rong, S., Liu, Y. (2018). Dual-modal split-type immunosensor for sensitive detection of microcystin-LR: Enzyme-induced photoelectrochemistry and colorimetry. Analytical chemistry, 90 (15), 9606-9613.): 0.1 g of hexadecyltrimethylammonium bromide (CTAB) was dissolved in 7 mL of water, and then 1.8 mL of ethanol, 3 mL of ether, 1 mL of tetraethyl orthosilicate (TEOS) and 0.1 mL of 25% ammonia water were added in sequence. After stirring for 4 h, the mixture was centrifuged, the precipitate was collected, washed with ultrapure water, and calcined at 550°C to obtain a calcined product. Then, 10 mg of the calcined product was dispersed in 7 mL of ethanol, and 0.1 mL of 3-aminopropyltriethoxysilane (APTES) was added for amination treatment. The mixture was reacted at 30 °C with stirring for 24 h, and finally centrifuged. The precipitate was washed, dried, and then prepared into a 1 mg / mL MSNs suspension using 0.1 M, pH 7.4 PBS buffer.

[0035] The dsDNA-MSNs complex was prepared as follows: 5 μL of 100 μM 5'-end carboxyl-modified primer P2 was added to PBS buffer (0.1 M, pH 7.4) containing 10 mM NHS and 20 mM EDC, activated at room temperature for 1 h, and then added to 100 μL of 1 mg / mL MSNs suspension (solvent is 0.1 M, pH 7.4 PBS buffer), shaken overnight, so that the surface of the MSNs containing amino functional groups and the 5'-end carboxyl-modified primer P2 are covalently bonded to form a P2-MSNs complex. Then, the ssDNA released after the DSN action in step (1) was added to the P2-MSNs complex, and reacted for 2 h according to the base complementary pairing principle to obtain a dsDNA-MSNs complex rich in A and T bases.

[0036] The structure of the primer P2 with a modified 5'-terminal carboxyl group is as follows: 5'-COOH-C6-ATATATAT-3'.

[0037] (3) Electrochemical detection

[0038] The indium tin electrode (ITO) was placed in an aqueous solution containing 5mM HAuCl4 and 0.1M H2SO4, and the deposition voltage was 0.2V and the deposition charge was 0.04C. The three-dimensional gold nanocluster modified indium tin electrode (GNC / ITO) was prepared by electrochemical constant potential deposition method. 20μL of 5μM capture probe CP (5' end modified with thiol) aqueous solution was added to 20μL of 10mM TCEP (tris (2-hydroxyethyl) phosphine hydrochloride) aqueous solution, and the solution was allowed to stand for 30min to break the disulfide bond to obtain an activated CP solution. GNC / ITO was placed in the activated CP solution and incubated at 37°C overnight, so that the capture probe CP modified with thiol at the 5' end was assembled on the GNC / ITO through covalent bonds to obtain a CP / GNC / ITO electrode. Then, 10 μL of 1 mM MCH (6-mercaptohexanol) aqueous solution was dropped onto the surface of the CP / GNC / ITO electrode and allowed to stand for 2 h to block the active sites. Subsequently, the dsDNA-MSNs complex rich in A and T bases obtained in step (2) was mixed with 20 μL of 5 μM auxiliary probe HP aqueous solution, and the CP / GNC / ITO with blocked active sites was immersed therein, and the HP and dsDNA-MSNs complex was assembled onto the CP / GNC / ITO surface by chain hybridization reaction to obtain a dsDNA-MSNs / HP / CP / GNC / ITO electrode. The chain hybridization reaction lasted for 2 h. 30 μL of 50 mM ascorbic acid aqueous solution and 30 μL of 10 mM anhydrous copper sulfate aqueous solution were sequentially added to the dsDNA-MSNs / HP / CP / GNC / ITO electrode and reacted at 37°C for 50 min to promote the formation of Cu NCs (copper nanoclusters); after the reaction at 37°C, the reacted dsDNA-MSNs / HP / CP / GNC / ITO was detected by differential pulse voltammetry (DPV) using a CHI650E workstation. The detection solution was 0.1 M PBS buffer (pH 5.5) containing 0.1 M NaCl, the potential range was -0.2 V to 0.5 V, and the pulse amplitude was 0.05 V.

[0039] The structure of the capture probe CP modified with a thiol group at the 5' end is as follows: 5'-SH-C6-ATATATAT-3'.

[0040] The sequence of the auxiliary probe HP is as follows: 5'-GTGTATATATAT-3'.

[0041] The sequences of the present invention are shown in Table 1.

[0042] Table 1 Sequences and numbers of nucleic acid fragments

[0043]

[0044] Test results such as Figure 2 As shown in (A), the DPV oxidation peak at a peak potential of 0.16 V corresponds to CuNPs, and the peak intensity increases with the increase of miRNA-141 concentration. In the range of 1fM-100nM, the differential pulse voltammetry current shows a good linear relationship with different concentrations of miRNA-141, and the detection limit is 0.35fM. Figure 2 (B) shows that the standard curve equation is ΔI = 18.187 + 1.140lgC (R 2 =0.994), ΔI is the peak current difference between the target with different concentrations and the blank sample (ultrapure water without miRNA-141), in μA, and C is the concentration of miRNA-141, in molar (M). According to the linear equation, the concentration of the sample to be tested can be quantitatively analyzed.

[0045] 2. Test the samples to be tested

[0046] The sample to be tested is detected according to steps (1), (2) and (3) in preparing the standard curve, except that the miRNA-141 solution in step (1) is replaced by the sample to be tested. The peak current difference between the sample to be tested and the blank sample obtained by differential pulse voltammetry (DPV) is substituted into the standard curve ΔI=18.187+1.1401 gC to calculate the concentration of miRNA-141 in the sample to be tested.

[0047] Example 2 Characteristics of the method of the present invention

[0048] (1) Feasibility analysis of the method of the present invention

[0049] Electrodes at different assembly stages were prepared according to step (3) of preparing the standard curve in Example 1: blank working electrode ITO, GNC / ITO, CP / GNC / ITO, HP / CP / GNC / ITO, dsDNA-MSNs / HP / CP / GNC / ITO.

[0050] Among them, HP / CP / GNC / ITO was prepared by the following method: CP / GNC / ITO with blocked active sites was immersed in 20 μL of 5 μM auxiliary probe HP aqueous solution, and HP was assembled on the CP / GNC / ITO surface by chain hybridization reaction to obtain HP / CP / GNC / ITO electrode.

[0051] Electrochemical impedance spectroscopy (EIS) was used to characterize the electrodes at different assembly stages in the presence of 0.1 M KCl and 5.0 mM [Fe(CN)6] 3- / 4-The electrochemical performance in 0.1M PBS buffer (pH 7.4) was measured at a voltage of 5.0mV and a frequency range of 0.1Hz-100kHz. Figure 3 As can be seen in (A), the EIS signal (Ret) of GNC / ITO (curve b) is greatly reduced compared with ITO (indium tin electrode, curve a), because GNC (gold nanoclusters) have good conductivity and can effectively accelerate electron transfer. Then, CP (curve c) and HP (curve d) were introduced into the GNC / ITO surface, respectively, which significantly increased the Ret response, which was due to the negative charge of nucleic acids. In addition, after assembly with dsDNA-MSNs, Ret still increased significantly (curve e), indicating that AT-rich dsDNA-MSNs greatly blocked electron transfer. The above results show that the preparation of the biosensor is relatively successful.

[0052] The DSN-mediated RCA strategy is crucial for the good performance of the prepared detection electrode. Therefore, this cyclic amplification strategy was confirmed by gel electrophoresis analysis ( Figure 3 (B)). The complex of DNA1, 2 and 3 with P1 was prepared by the following method: 5 μL of a 100 μM mixture of single-stranded DNA 1, 2 and 3 (abbreviated as ssDNA mixture) rich in A and T bases was added to the PBS buffer containing the 5'-terminal amino-modified primer P1, and placed at 37°C for 2 hours. DNA1, 2 and 3 were bound to P1 respectively through base complementary pairing. The bands of DNA1 (lane 1), DNA2 (lane 2) and DNA3 (lane 3) were clear. After DNA1 / 2 / 3 and P1 were complementary to each other (the complex of DNA1, 2 and 3 with P1), the migration speed of band 4 was significantly slower than that of bands 1, 2 and 3. Subsequently, after the action of miRNA-141 and DSN, P1 was digested, and ssDNA and miRNA-141 were released at the same time (lane 5). These results successfully verified the feasibility of using DSN-mediated RCA for this detection electrode.

[0053] (2) Recovery rate experiment

[0054] The method in Example 1 was used to detect miRNA-141 in actual complex samples to verify its practical feasibility. The standard addition method was used to test the recovery rate of different concentrations of miRNA-141 (0.01, 10, 10000 pM) in 10-fold diluted serum samples, and the final results are listed in Table 2. The recovery rate of miRNA-141 detected by this method was 95-105%, and the relative standard deviation was less than 4.7%, indicating that this method has high accuracy and great potential for monitoring miRNA in actual serum samples.

[0055] Table 2 Recovery rate test results

[0056] sample Addition amount (pM) Detection amount (pM) Recovery rate (%) Relative standard deviation (%) 1 0.01 0.0105 105 4.7 2 10 9.7 97 3.4 3 10000 9500 95 3.8

[0057] (3) Specificity analysis of the method of the present invention

[0058] In order to evaluate the specificity of the method of the present invention, blank samples (ultrapure water without miRNA-141), 5 μL 100 pM miRNA-21 aqueous solution, 5 μL 100 pM let-7a aqueous solution, 5 μL 100 pM miRNA-155 aqueous solution, 5 μL 100 pM miRNA-141 aqueous solution and equal volume mixtures of the above solutions were used as detection objects, and detection was carried out using the method in Example 1.

[0059] Figure 2 (C) shows the peak current bar graph corresponding to the detection results of different samples. The results show that the current response of the detection method of the present invention to miRNA-21, let-7a, and miRNA-155 is similar to the current response of the blank sample, and there is no obvious current response. However, the current response to miRNA-141 and the mixture containing miRNA-141 is obvious. This shows that the detection method of the present invention has a specific selectivity for miRNA-141, mainly due to the unique recognition function between bases.

Claims

1. The electrochemical detection method of miRNA-141 comprises the following steps: (1) Testing with different concentrations of miRNA-141 solution to prepare a standard curve; (2) Detecting the sample to be tested and calculating the concentration of miRNA-141 in the sample to be tested according to the standard curve.

2. The method according to claim 1, characterized in that: The method for detecting a sample to be tested or a miRNA-141 solution of different concentrations comprises the following steps: (1) Carboxyl-modified Fe3O4 nanoparticles react with primer P1 modified with 5'-terminal amino group to obtain P1-Fe3O4 complex; DNA1, 2 and 3 are bound to the P1-Fe3O4 complex by the base complementary pairing principle to obtain a hybrid complex; the hybrid complex and double-stranded specific nuclease are reacted with a sample to be tested or a miRNA-141 solution of different concentrations at 60-70° C. for 20-40 min to perform a DSN-mediated target cyclic amplification reaction to obtain ssDNA released after the action of DSN; (2) The 5'-end carboxyl-modified primer P2 is combined with the surface amino-modified mesoporous silica nanospheres and reacts with the ssDNA released after the DSN in step (1) to obtain a dsDNA-MSNs complex rich in A and T bases; (3) The capture probe CP modified with a thiol group at the 5' end is covalently assembled onto an indium tin electrode modified with three-dimensional gold nanoclusters to obtain a CP / GNC / ITO electrode; the CP / GNC / ITO is immersed in a solution containing an auxiliary probe HP and a dsDNA-MSNs complex, and the HP and dsDNA-MSNs complex are assembled onto the surface of the CP / GNC / ITO to obtain a dsDNA-MSNs / HP / CP / GNC / ITO electrode; then, ascorbic acid solution and anhydrous copper sulfate solution are sequentially added to the surface of the dsDNA-MSNs / HP / CP / GNC / ITO at 35-40°C for 40-60 minutes to promote the formation of copper nanoclusters; then, the dsDNA-MSNs / HP / CP / GNC / ITO after the reaction is detected by differential pulse voltammetry using a CHI650E workstation.

3. The method according to claim 2, characterized in that The sequence of primer P1 with amino modification at the 5' end is as follows: 5'-NH2-C6-CCATCTTTACCAGACAGTGTTA-3'; the sequence of DNA1 is as follows: 5'-ACACATATATAT-3'; the sequence of DNA2 is as follows: 5'-CTGGATATATAT-3'; the sequence of DNA3 is as follows: 5'-AGATATATATAT-3'; the sequence of primer P2 with carboxyl modification at the 5' end is as follows: 5'-COOH-C6-ATATATAT-3'; the sequence of HP is as follows: 5'-GTGTATATATAT-3'; the sequence of capture probe CP with thiol modification at the 5' end is as follows: 5'-SH-C6-ATATATAT-3'.

4. The method according to claim 3, characterized in that: The horizontal coordinate of the standard curve is lgC, C is the concentration of miRNA-141, and the vertical coordinate is the peak current difference between different concentrations of miRNA-141 and blank samples; the blank sample is an ultrapure aqueous solution without miRNA-141.

Images