Electrochemical sensor for detecting miRNA markers and application thereof

Abstract

The invention belongs to the technical field of electrochemical detection, and relates to an electrochemical sensor for detecting miRNA markers and application of the electrochemical sensor. According to the electrochemical sensor constructed by the invention, a conjugate of MOFs (Metal-Organic Frameworks) and a miRNA aptamer is used as a carrier to extract miRNA; the aptamer is combined with the target miRNA to fulfill the aim of enriching the target miRNA; then, the aptamer which is not combined with the miRNA is released from the MOFs under the strong coordination action between phosphate and metal ions, and then the modified screen-printed electrode is used for determination, so that the extraction efficiency and the detection specificity are improved, the reagent is safe, non-toxic and environment-friendly, and the pretreatment process is also simplified. The miRNA detection kit is low in detection cost, high in sensitivity, strong in specificity and short in detection time, is convenient to construct point-of-care testing (POCT) equipment, realizes diagnosis of early cancers, myocardial infarction and rheumatoid arthritis in hospitals and homes, and has important practical application value.

Description

Technical Field

[0001] The invention belongs to the technical field of electrochemical detection, and relates to an electrochemical sensor for detecting miRNA markers and application thereof. Background Art

[0002] MicroRNA (miRNA) is a class of endogenous non-coding single-stranded RNA with a length of about 20 nucleotides. MiRNA is widely involved in regulating biological processes such as immune system stimulation, fat metabolism, cell proliferation, differentiation and death. Its abnormal regulation is closely related to the onset of diseases such as myocardial infarction, cancer, and rheumatoid arthritis. For example, miR-21 is abnormally expressed in the serum of breast cancer patients, the content of miR-499 in circulating blood increases rapidly after myocardial infarction, and the expression level of miR-146a is related to arthritis. Therefore, the detection of miRNA in body fluids is of great significance for the diagnosis, monitoring and prognosis of diseases. However, the high similarity within the miRNA family and the large concentration differences in different individuals make miRNA detection still a huge challenge, so there is an urgent need to develop highly sensitive, highly specific and low-cost detection methods.

[0003] Quantitative real-time polymerase chain reaction (qPCR) is the gold standard for detecting miRNA content. However, due to the short primer sequence of miRNA, the required dissolution temperature is low, which reduces the accuracy of the qPCR method. At the same time, qPCR instruments are expensive, the cost of a single test is high, and the detection time is long, making it difficult to develop into a point-of-care testing (POCT) device. In recent years, new methods for detecting miRNA based on fluorescence analysis, electrochemical detection, electrochemiluminescence, and Raman spectroscopy have continued to emerge. Among them, biosensors made of nanomaterials such as metal-organic frameworks (MOFs), gold nanoparticles, and lipid nanoparticles have good optical, electrical, and magnetic properties, which can greatly improve the sensitivity and accuracy of detecting miRNA and reduce the detection cost.

[0004] For example, the patent document with the publication number CN109844514B provides a method for preparing an electrochemical sensor for non-coding RNA and its application. The reduced graphene oxide loaded with sulfonated calixarene is prepared by a one-step method, the iron oxide nanomaterial is synthesized by a wet heat method, and gold nanoparticles are added to synthesize Au@SCX8-RGO and Au@Fe3O4 composite materials respectively; after confirming the successful construction of the material by material characterization, the target is electrochemically detected by screen-printed electrodes using substances such as toluidine blue as electrical signal substances. The electrochemical biosensor prepared by this method can realize the detection of the expression amount of 3'UTR transcripts of non-coding regions of different lengths generated by the selective polyadenylation (APA) phenomenon. However, most electrochemical methods still face the problem that the sample pretreatment process is cumbersome and involves toxic reagents, which is not conducive to the application and promotion of the method. Summary of the invention

[0005] The object of the present invention is to provide an electrochemical sensor for detecting miRNA markers and its application, which can realize the rapid detection of miRNA markers, including miRNA markers for diseases such as tumors, myocardial infarction and rheumatoid arthritis, thereby facilitating early, rapid and convenient detection of related diseases.

[0006] To achieve the above object, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides an electrochemical sensor for detecting miRNA markers, the electrochemical sensor comprising an enrichment reagent, a pretreatment reagent and a sensor module;

[0008] The enrichment reagents include: Figure 1AAs shown, a MOF carrier and an aptamer FL-apt, wherein the aptamer FL-apt is adsorbed on the surface of the MOF carrier, wherein the aptamer contains a nucleotide sequence that is completely complementary to the target miRNA and has a fluorescent marker molecule at the 5' end; wherein the fluorescent marker molecule is a fluorescent molecule that is stable in aqueous solution, including but not limited to Texas Red Red (TR), 5 (6) -carboxyfluorescein FAM, cyanine fluorescent dye Cy3, cyanine fluorescent dye Cy5, hexachlorofluorescein HEX, tetrachlorofluorescein TET, 5-carboxytetramethylrhodamine TAMRA; the MOFs are metal organic framework materials that are slightly soluble or difficult to dissolve in aqueous solution, including but not limited to UiO-66-NH2, PCN-224, PCN-222, ZIF-8 / 67 / 90, MIL-53 / 88B / 101, and the MOFs can be synthesized by any one of a hydrothermal method, a volatilization method, a diffusion synthesis method, and an ultrasonic method; further preferably, the preparation step of the enrichment reagent comprises: adding an aptamer solution with a concentration of 100 to 500 nM to a MOFs solution with a concentration of not more than 300 μg / mL, and incubating for 5 to 30 minutes to obtain the enrichment reagent. Preferably, if the MOFs is UiO-66-NH2, the optimal concentration of the MOFs solution is 50 μg / mL.

[0009] The pretreatment reagent is a phosphate solution with a concentration not higher than 6 mM; and preferably, the phosphate is sodium tripolyphosphate (STPP), sodium pyrophosphate (TSPP) or sodium hexametaphosphate (SHMP).

[0010] The manufacturing steps of the sensor module include: Figure 1BAs shown, gold nanoparticles AuNPs are electrochemically deposited on the surface of a screen-printed electrode (SPE); SH-pDNA, a thiol DNA chain that can capture FL-apt, is incubated on the electrode surface; after the electrode is cleaned and dried, 6-mercapto hexanol (MCH) is incubated on the surface; after the electrode is cleaned and dried, it can be used to detect the pretreated test solution. Preferably, the SH-pDNA is a single-stranded DNA that is completely complementary to the FL-apt modified with a thiol group; preferably, the step of electrochemically depositing gold nanoparticles AuNPs on the surface of the screen-printed electrode is: placing the SPE in a 0.1% HAuCl4 solution and depositing AuNPs using the it method; further preferably, the it deposition potential is -0.2V and the time is 100 to 500s; preferably, the step of incubating the thiol DNA chain that can capture FL-apt on the electrode surface is: drop-coating SH-pDNA on the electrode surface, incubating at -20°C for 20 to 60 minutes, and incubating at room temperature for 5 minutes. Preferably, the concentration of the SH-pDNA is 50 to 500 nM. Preferably, the concentration of the MCH is 1 mM, and the incubation time of the MCH is 5 to 60 minutes.

[0011] In a second aspect, the present invention also provides the use of the electrochemical sensor in the preparation of a miRNA detection kit, the application comprising: enrichment treatment, pretreatment, and sensor incubation;

[0012] The enrichment treatment steps are: Figure 1C As shown, the sample to be tested is mixed with the enrichment reagent and incubated for 5 to 30 minutes. During this process, FL-apt binds to the miRNA in the sample to be tested;

[0013] The pre-processing steps are: Figure 1D As shown, the phosphate solution is added to the mixed solution after the enrichment treatment, and the mixture is incubated for 5 to 30 minutes. During this process, the phosphate releases FL-apt from the MOF structure. After incubation, the mixture is centrifuged and the supernatant is taken as the pre-treated test solution. The supernatant contains the double-stranded FL-apt bound to the miRNA and the single-stranded FL-apt not bound to the miRNA.

[0014] The sensing incubation steps are as follows: Figure 1E As shown, the test solution after pretreatment is incubated on the surface of the sensor module, and then washed with ultrapure water and tested by cyclic voltammetry (CV) or differential pulse voltammetry (DPV) to test the SPE in [Fe(CN)6] before and after incubation of the test solution.3- / 4- The electrochemical signal generated in the solution is used to calculate the concentration of miRNA in the sample to be tested based on the linear relationship between the electrochemical signal difference ΔΙ and the miRNA concentration. Preferably, the [Fe(CN)6] 3- / 4- The concentration of the solution is 5mM. Preferably, the test parameters of the DPV are: the scanning range is -0.2 to 0.6V, and the pulse amplitude is 0.1V. Preferably, the concentration of miRNA in the sample to be tested is determined by selecting a series of miRNA standard solutions with a concentration gradient, and performing the enrichment treatment, pretreatment, sensor incubation and DPV test steps one by one to obtain the corresponding ΔΙ. A standard curve is established based on the functional relationship between ΔΙ and the logarithm of the miRNA concentration, so that the ΔΙ used to detect the sample to be tested for unknown miRNA concentration can directly calculate the concentration of its miRNA.

[0015] Preferably, the incubation time of the pretreated test solution is 1 to 30 minutes.

[0016] The advantages of the present invention are:

[0017] In the commercial detection method of miRNA, it is necessary to pre-treat the biological matrix with chemical reagents to extract nucleic acids. However, the problems of low recovery rate, large loss, low sample purity and poor repeatability are prone to occur during the extraction process. In particular, the reagents used for nucleic acid extraction are mostly highly toxic organic solvents, which pose safety hazards. The present invention innovatively uses a combination of MOFs and miRNA aptamers as a carrier to extract miRNA; MOFs are a type of porous material formed by the coordination of organic ligands and metal ions, which has the advantages of large specific surface area, pores, adjustable particle size, simple synthesis and easy surface modification. The present application uses MOFs as a carrier to adsorb the nucleic acid aptamer of miRNA, and the aptamer binds to the target miRNA to achieve the purpose of enriching the target miRNA; then, the aptamer that is not bound to the miRNA is released from the MOFs by the strong coordination between phosphate and metal ions, and then the modified screen-printed electrode, i.e., the sensor module, is measured, which not only improves the extraction efficiency and detection specificity, but also makes the reagents safe and non-toxic, environmentally friendly, and simplifies the pretreatment process.

[0018] The electrochemical sensor constructed by the present invention can be used to directly detect miRNA, rather than traditional protein markers such as carcinoembryonic antigen, alpha-fetoprotein, human epidermal growth factor receptor 2, etc. The advantage of detecting miRNA is that miRNA is involved in the whole process of disease occurrence and development, and its content can sensitively predict tumors, as well as the risk of myocardial infarction or rheumatoid arthritis, increasing the possibility of early cure. Currently, there is a lack of miRNA detection technology that can be applied to on-site rapid detection scenarios. The present invention constructs an electrochemical biosensor on a screen-printed electrode, which can be used in conjunction with a printed circuit board to create a POCT device. The SPE is combined with a printed circuit board to construct a small miRNA detector, which is convenient for users to understand their health information in real time at home and prepare for timely medical treatment.

[0019] The electrochemical sensor constructed by the present invention does not use high-cost reagents or raw materials such as enzymes, and the constructed raw materials and reagents can be stably stored, so it has the technical advantage of low cost. In addition, the electrochemical sensor provided by the present application has a short detection time for detecting miRNA and has high practical application value.

[0020] The miRNA aptamer combined with MOF in the electrochemical sensor constructed by the present invention carries a fluorescent group, which not only increases the enrichment density of the aptamer on the MOF, but also improves the release efficiency of the aptamer by phosphate, which is beneficial to improve the difference of the electrochemical detection signal before and after incubation of the test solution, thereby improving the detection sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1A This is a schematic diagram of the configuration process of the enrichment reagent in the electrochemical sensor of the present invention;

[0022] Figure 1B This is a schematic diagram of the manufacturing process of the sensor module in the electrochemical sensor of the present invention;

[0023] Figure 1C This is a schematic diagram of the enrichment processing steps of the present invention;

[0024] Figure 1D This is a schematic diagram of the pretreatment steps of the present invention;

[0025] Figure 1E This is a schematic diagram of the sensing incubation of the present invention;

[0026] Figure 2 is a transmission electron micrograph of UiO-66-NH2 synthesized in Example 1;

[0027] Figure 3A The fluorescence emission spectrum of the supernatant after TR-apt-21 incubation in Example 1, and the fluorescence emission spectrum of the supernatant after TR-apt-21 and UiO-66-NH2 co-incubation;

[0028] Figure 3B This is a diagram showing the optimal concentration screening results of UiO-66-NH2 in Example 1;

[0029] Figure 3C The fluorescence emission spectrum of the supernatant after incubation with TR-apt-21 in Application Example 1, the fluorescence emission spectrum of the supernatant after co-incubation with TR-apt-21 and UiO-66-NH2, and the fluorescence emission spectrum of the supernatant after co-incubation with TR-apt-21, UiO-66-NH2, and STPP;

[0030] Figure 3D This is the result diagram of the verification of the optimal concentration of STPP in Application Example 1;

[0031] Figure 4A This is the CV characterization result diagram of the electrode step-by-step modification process;

[0032] Figure 4B This is the DPV characterization result diagram of the electrode step-by-step modification process;

[0033] Figure 5 This is a comparison chart of the test results of comparative test 1;

[0034] Fig. 6A This is a graph showing the screening results of the optimal concentration of SH-pDNA-21 in comparative experiment 2;

[0035] Figure 6B This is a graph showing the screening results of the optimal incubation time of MCH in comparative experiment 2;

[0036] Figure 6C This is a graph showing the screening results of the optimal incubation time of TR-apt-21 in comparative experiment 2;

[0037] Fig. 7A This is a graph showing the ΔΙ change of the system after adding different concentrations of miR-21 in Test Example 1;

[0038] Figure 7B The DPV images of the system after adding different concentrations of miR-21 in Test Example 1;

[0039] Figure 7C This is a graph showing the repeatability test results of the sensing method in Test Example 2;

[0040] Fig.7D This is a graph showing the specificity test results of the sensing method in Test Example 3;

[0041] Fig. 7E This is a graph showing the stability test results of the sensing method in Test Example 4;

[0042] Figure 7FThis is a graph showing the results of using the sensing method to detect normal mouse serum and melanoma mouse serum in Test Example 5. DETAILED DESCRIPTION

[0043] In order to make the contents of the present invention easier to understand, the technical solutions of the present invention are further described below in combination with specific implementation methods with reference to the accompanying drawings, but the present invention is not limited thereto.

[0044] The raw materials for synthesizing UiO-66-NH2 are ZrCl4, aminoterephthalic acid, benzoic acid and DMF. Among them, ZrCl4 was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., aminoterephthalic acid was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., benzoic acid was purchased from Shanghai Lingfeng Chemical Reagent Co., Ltd., and DMF was purchased from Shanghai Lingfeng Chemical Reagent Co., Ltd.;

[0045] SPE was purchased from Changsha Sanjun Electronic Technology Co., Ltd.;

[0046] STPP solution was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.;

[0047] HAuCl4 solution was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.;

[0048] [Fe(CN)6] 3- / 4- (5 mM) solution was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.;

[0049] MCH solution was purchased from Shanghai MacLean Biochemical Technology Co., Ltd.;

[0050] Serum samples of melanoma mice (Tumor) and healthy mice (Normal) were from B6 mice purchased from Charles River Laboratories. The melanoma mouse model was established by implanting B16-F10 cells on the back of B6 mice. B16-F10 cells were obtained from China Center for Type Culture Collection.

[0051] The sequence of miR-21 is 5′-UAGCUUAUCAGACUGAUGUUGA-3′;

[0052] The sequence of apt-21 is 5'-TCAACATCAGTCTGATAAGCTA-3', and the sequence of apt-21 is shown as SEQ ID NO.1 in the sequence table;

[0053] TR-apt-21 is a single-stranded DNA that is completely complementary to miR-21 and modified with the fluorescent group Texas Red; the structure of TR-apt-21 is 5'-(Texas Red)-TCAACATCAGTCTGATAAGCTA-3';

[0054] FAM-apt-21 is a single-stranded DNA that is completely complementary to miR-21 and modified with the fluorescent group FAM; the structure of FAM-apt-21 is 5'-(FAM)-TCAACATCAGTCTGATAAGCTA-3';

[0055] Cy3-apt-21 is a single-stranded DNA that is completely complementary to miR-21 and modified with the fluorescent group Cy3; the structure of Cy3-apt-21 is 5'-(Cy3)-TCAACATCAGTCTGATAAGCTA-3'.

[0056] The above sequences were custom-made from Sangon Bioengineering Co., Ltd.

[0057] Example 1 Construction of electrochemical sensor

[0058] Configuration of enrichment reagent: See the open literature ACS Appl. Mater. Interfaces 2019, 11, 40, 36316-36323, the classic hydrothermal method was used to synthesize UiO-66-NH2, such as Figure 2 The figure shows the transmission electron micrograph of the synthesized UiO-66-NH2. 50 μL TR-apt-21 solution (400 nM) was added to 50 μL UiO-66-NH2 solution (200 μg / mL), and the mixture was incubated for 5 min to allow UiO-66-NH2 to adsorb TR-apt-21 and obtain an enrichment reagent.

[0059] In order to verify the adsorption of UiO-66-NH2 in the enrichment reagent on TR-apt-21 and the concentration optimization of the UiO-66-NH2 solution, the enrichment reagent was centrifuged at 10000 rpm for 6 min, and the supernatant diluted 10 times was taken for fluorescence test. ex is 584nm, λ em is 610nm; Figure 3A As shown in the figure, the weakening of the fluorescence value at 610 nm in the fluorescence spectrum indicates that UiO-66-NH2 has a strong adsorption effect on TR-apt-21. The amount of TR-apt-21 not adsorbed by UiO-66-NH2 is determined according to the fluorescence value at 610 nm of the supernatant after 5-fold dilution, and the concentration of UiO-66-NH2 solution is optimized, as shown in the figure. Figure 3B , the optimal concentration of UiO-66-NH2 solution is 50μg / mL (final concentration).

[0060] Modification of screen-printed electrode (SPE): SPE was placed in 0.1% HAuCl4 solution and AuNPs were deposited by it method. The it parameters were: deposition potential of -0.2V and deposition time of 200s. Then 10μL SH-pDNA-21 was dripped onto the electrode surface and incubated at -20°C for 30min and at room temperature for 5min. The sequence of SH-pDNA-21 is 5'-(SH-C6)-TAGCTTATCAGACTGATGTTGA, as shown in SEQ ID NO.2 in the sequence table. After rinsing the modified electrode, 10μL MCH (1mM) was further dripped onto the electrode.

[0061] Example 2

[0062] The electrochemical sensor constructed in Example 2 is different from that in Example 1 only in that the aptamer in the enrichment reagent is FAM-apt-21, which is a completely complementary paired DNA single strand of miR-21 modified with the fluorescent group FAM, and accordingly, the enrichment reagent is prepared by adding 50 μL of FAM-apt-21 solution (400 nM) to 50 μL of LuiO-66-NH2 solution (200 μg / mL) and incubating for 5 min;

[0063] Example 3

[0064] The electrochemical sensor constructed in Example 3 is different from that in Example 1 only in that the aptamer in the enrichment reagent is Cy3-apt-21, which is a fully complementary paired DNA single strand of miR-21 modified with the fluorescent group Cy3, and accordingly, the enrichment reagent is prepared by adding 50 μL of Cy3-apt-21 solution (400 nM) to 50 μL of LuO-66-NH2 solution (200 μg / mL) and incubating for 5 min;

[0065] Comparative Example 1

[0066] The electrochemical sensor constructed in Comparative Example 1 is different from that in Example 1 only in that the aptamer in the enrichment reagent is apt-21, which is a completely complementary paired DNA single strand of miR-21 without modified fluorescent group, and accordingly, the enrichment reagent is prepared by adding 50 μL of apt-21 solution (400 nM) to 50 μL of LuO-66-NH2 solution (200 μg / mL) and incubating for 5 min;

[0067] Application Example 1

[0068] Enrichment treatment: 50 μL of miR-21 solution (400 fM) was mixed with the enrichment solution in Example 1 and incubated for 5 min;

[0069] The enriched samples were pretreated with sodium tripolyphosphate (STPP): 50 μL STPP solution (4 mM) was added to the mixed solution after the enrichment treatment, incubated for 10 min, and then centrifuged at 10,000 rpm for 6 min. The supernatant was taken to obtain a mixed solution of double-stranded TR-apt-21 and miR-21 binding and single-stranded TR-apt-21 not binding to miR-21, i.e., the test solution; the fluorescence of the test solution diluted 10 times was tested, λ ex is 584nm, λ em is 610nm, such as Figure 3C As shown in the figure, the recovery of the fluorescence value at 610 nm in the fluorescence spectrum indicates that STPP can competitively bind to the binding sites of TR-apt-21 and UiO-66-NH2, releasing TR-apt-21 into the solution. The optimal concentration of the STPP solution can be determined based on the degree of recovery of the fluorescence value of the test solution after 5-fold dilution, as shown in Figure 3D , the optimal concentration is 1.25mM (final concentration).

[0070] Sensing incubation and detection: Take 10 μL of the test solution pretreated with the optimal conditions, drop it on the modified screen-printed electrode (SPE), wash it with ultrapure water, and then place it on [Fe(CN)6] 3- / 4- CV and DPV tests were performed in a 5mM solution. The CV parameters were: initial potential -0.6V, upper limit potential 0.8V, lower limit potential -0.6V, and scanning speed 0.1V / s; the DPV parameters were: scanning range -0.2~0.6V, and pulse amplitude 0.1V. Figure 4A and Figure 4B As shown in the figure, after AuNPs were deposited on SPE, the excellent conductivity of AuNPs increased the peak current of SPE, while the modification of SH-pDNA-21 and MCH increased the surface resistance of the electrode and decreased the peak current; after adding the test solution, the double-stranded structure formed by the combination of TR-apt-21 and SH-pDNA-21 in the test solution further hindered the [Fe(CN)6] 3- / 4- The peak current continues to decrease due to the redox transformation.

[0071] Comparative experiment 1: Fluorophore optimization

[0072] The electrochemical sensors constructed in the above comparative example 1 and embodiments 1 to 3 were respectively used to detect the tumor marker miR-21, and the specific operation steps are as follows:

[0073] Enrichment treatment: 50 μL of miR-21 solution (400 fM) was mixed with the enrichment solutions in Comparative Example 1 and Examples 1 to 3, respectively, and incubated for 5 min, which were recorded as None group, Texas Red group, FAM group and Cy3 group, respectively;

[0074] Pretreatment: Sodium tripolyphosphate (STPP) was used to pretreat the enriched samples: 50 μL STPP solution (1.25 mM) was added to the mixed solution after the enrichment treatment, incubated for 10 min, and then centrifuged at 10,000 rpm for 6 min. The supernatant was taken, namely the None group, Texas Red group, FAM group and Cy3 group.

[0075] Sensing incubation: 10 μL of the pre-treated test solution of None group, Texas Red group, FAM group and Cy3 group was dropped on the electrode. After incubation for 20 min, the electrode was cleaned and placed in [Fe(CN)6] 3- / 4- DPV test was performed in a 5mM solution. The DPV parameters were: scanning range -0.2 to 0.6V, pulse amplitude 0.1V. Figure 5 As shown in the figure, compared with the None group, the ΔΙ of the other three groups increased significantly, indicating that good electrochemical effects were achieved after the fluorescent group was modified at the tail of apt-21. Among the Texas Red group, FAM group and Cy3 group, the ΔΙ of the Texas Red group was the largest, indicating that the best modification group was Texas Red.

[0076] Comparative test 2: Optimization of electrochemical conditions

[0077] The concentration of miR-21 in the test solution was evaluated based on the current difference ΔΙ before and after the addition of the test solution. In order to improve the sensitivity of the analysis method and increase ΔΙ, based on Application Example 1, the concentration of SH-pDNA-21, the incubation time of MCH, and the incubation time of the test solution were optimized by controlling a single variable. Fig. 6A As shown, the optimal concentration of SH-pDNA-21 was 100 nM. Figure 6B As shown in Figure 2, the optimal incubation time for MCH is 20 min. Figure 6C As shown, the optimal incubation time for the test solution is 20 min.

[0078] Test Example 1: Linearity, Range, Detection Limit

[0079] Based on Application Example 1, the constructed electrochemical sensing method was used to detect miR-21 standard solutions with final concentrations of 0.0001, 0.001, 0.01, 0.1, 1, and 10 nM to obtain the corresponding ΔΙ. A standard curve was established based on the functional relationship between ΔΙ and the logarithm of the miR-21 concentration. Fig. 7A , Figure 7B As shown, the standard curve showed a good linear relationship when the miR-21 concentration was 0.0001-10 nM, and the equation was ΔΙ = -1.118log 10 [miR-21]+9.531, R2 The calculated detection limit is 34.13 fM (S / N=3).

[0080] Test Example 2: Repeatability

[0081] Based on Application Example 1, the constructed electrochemical sensing method was used to detect a miR-21 standard solution with a final concentration of 10 nM, that is, 50 μL of a miR-21 solution with a final concentration of 100 fM in Application Example 1 was replaced with 50 μL of a miR-21 standard solution with a final concentration of 10 nM, and the rest of the operations were the same. The experiment was repeated 6 times in parallel to obtain the corresponding ΔΙ. Figure 7C As shown, the RSD of ΔΙ for 6 experiments was 4.13%, indicating that the analytical method had good repeatability.

[0082] Test case 3: Exclusivity

[0083] Based on Application Example 1, the miR-21 standard solution in Application Example 1 was replaced with miR-21, miR-155, NC, miR-192 solutions with a final concentration of 10 nM, a blank solution (Control) without miRNA, and a mixed solution (Mixture), respectively. The mixed solution here refers to a mixed solution containing miR-21, miR-155, NC, and miR-192 with a concentration of 10 nM each.

[0084] Among them, the sequence of miR-155 is 5′-UUAAUGCUAAUCGUGAUAGGGGU-3′;

[0085] The sequence of NC is 5′-UUGUACUACACAAAAGUACUG-3′;

[0086] The sequence of miR-192 is 5′-CUGCCAAUUCCAUAGGUCACAG-3′.

[0087] like Fig.7D As shown, compared with the Control group, only the ΔΙ of miR-21 and Mixture groups was significantly reduced, indicating that the analysis method has good specificity.

[0088] Test Example 4: Stability

[0089] Based on Application Example 1, the modified electrode was placed in a 4°C refrigerator for 0, 2, 4, 6, and 8 days, respectively, and then used to detect a test solution containing 10 nM miR-21. Fig. 7E As shown, there is no obvious change in ΔΙ within 4 days, indicating that the electrochemical sensor has good stability within 4 days.

[0090] Test Example 5: Actual Biological Sample Testing

[0091] Based on Application Example 1, the constructed electrochemical sensing method was used to detect serum samples of melanoma mice (Tumor) and healthy mice (Normal), that is, the miR-21 standard solution in Application Example 1 was replaced with serum samples of melanoma mice (Tumor) and healthy mice (Normal), respectively; Figure 7F As shown, the ΔΙ of the normal mouse group was significantly higher than that of the melanoma mouse group, indicating that the miR-21 content in the serum of melanoma mice was higher than that of normal mice, which was in line with expectations, indicating that this analysis method has the potential to be promoted and applied to clinical testing.

Claims

1. An electrochemical sensor for detecting miRNA markers, the electrochemical sensor comprising an enrichment reagent, a pretreatment reagent and a sensor module; The enrichment reagent includes: a MOF carrier and an aptamer FL-apt, wherein the aptamer FL-apt is adsorbed on the surface of the MOF carrier, the aptamer contains a nucleotide sequence that is completely complementary to the target miRNA and has a fluorescent marker molecule at the 5' end; the fluorescent marker molecule is a fluorescent molecule that is stable in aqueous solution; the MOFs are metal organic framework materials that are slightly soluble or difficult to dissolve in aqueous solution; The pretreatment reagent is a phosphate solution with a concentration not higher than 6 mM; The manufacturing steps of the sensor module include: electrochemically depositing gold nanoparticles AuNPs on the surface of a screen-printed electrode SPE; incubating SH-pDNA on the electrode surface, wherein the SH-pDNA is used to capture FL-apt; after the electrode is cleaned and dried, incubating 6-mercapto hexanol (MCH) on the surface; after the electrode is cleaned and dried, it can be used to detect the pretreated test solution.

2. An electrochemical sensor for detecting miRNA markers according to claim 1, characterized in that: The fluorescent labeling molecule is any one of Texas Red (TR), 5(6)-carboxyfluorescein FAM, cyanine fluorescent dye Cy3, cyanine fluorescent dye Cy5, hexachlorofluorescein HEX, tetrachlorofluorescein TET, and 5-carboxytetramethylrhodamine TAMRA.

3. An electrochemical sensor for detecting miRNA markers according to claim 1, characterized in that: The MOFs is any one of UiO-66-NH2, PCN-224, PCN-222, ZIF-8 / 67 / 90, and MIL-53 / 88B / 101.

4. An electrochemical sensor for detecting miRNA markers according to claim 1, characterized in that: The preparation step of the enrichment reagent comprises: adding an aptamer solution with a concentration of 100 to 500 nM to a MOFs solution with a concentration not higher than 300 μg / mL, and incubating for 5 to 30 minutes to obtain the enrichment reagent.

5. An electrochemical sensor for detecting miRNA markers according to claim 1, characterized in that: The phosphate is any one of sodium tripolyphosphate (STPP), sodium pyrophosphate (TSPP) or sodium hexametaphosphate (SHMP).

6. An electrochemical sensor for detecting miRNA markers according to claim 1, characterized in that: The SH-pDNA is a single-stranded DNA that is completely complementary to the FL-apt modified with thiol groups.

7. An electrochemical sensor for detecting miRNA markers according to claim 1, characterized in that: The step of electrochemically depositing gold nanoparticles AuNPs on the surface of the screen-printed electrode is as follows: placing the SPE in a 0.1% HAuCl4 solution and depositing the AuNPs using an it method.

8. An electrochemical sensor for detecting miRNA markers according to claim 1, characterized in that: The step of incubating SH-pDNA on the electrode surface is: drop-coating SH-pDNA on the electrode surface, incubating at -20°C for 20 to 60 minutes, or incubating at room temperature for 5 minutes.

9. An electrochemical sensor for detecting miRNA markers according to claim 1, characterized in that: The concentration of the SH-pDNA is 50-500 nM; the concentration of the MCH is 1 mM, and the incubation time of MCH is 5-60 min.

10. Use of the electrochemical sensor according to claim 1 in preparing a miRNA detection kit, the use comprising: Enrichment treatment, pretreatment, sensing incubation; The enrichment treatment step is: mixing the sample to be tested with the enrichment reagent, and incubating for 5 to 30 minutes, during which FL-apt binds to the miRNA in the sample to be tested; The pretreatment step is: adding the phosphate solution to the mixed solution after the enrichment treatment, incubating for 5 to 30 minutes, during which the phosphate releases FL-apt from the MOF structure, centrifuging after incubation, and taking the supernatant as the pretreated test solution, the supernatant contains the double-stranded FL-apt bound to the miRNA, and also contains the single-stranded FL-apt not bound to the miRNA; The sensing incubation step comprises: incubating the pretreated test solution on the surface of the sensor module, washing with ultrapure water, and then performing cyclic voltammetry (CV) or differential pulse voltammetry (DPV) testing to test the SPE before and after incubation of the test solution in [Fe(CN)6] 3- / 4- The electrochemical signal generated in the solution is used to calculate the concentration of miRNA in the sample to be tested based on the linear relationship between the electrochemical signal difference ΔΙ and the miRNA concentration.

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