Preparation method and application of an electrochemical biosensor based on MOF / nuclear pore membrane sandwich structure

Abstract

The application relates to a preparation method and application of an electrochemical biosensor based on a MOF / nuclear pore membrane sandwich structure and belongs to the technical field of biosensing. The electrochemical biosensor based on the MOF / nuclear pore membrane sandwich structure comprises a nuclear pore membrane substrate, a MOF functional layer, a conductive enhancement layer and a biological recognition layer. The nuclear pore membrane substrate provides stable physical support for the MOF functional layer by taking the nuclear pore membrane as the substrate, simultaneously guides the directional transmission of target molecules along the pore channel and reduces diffusion resistance; the MOF functional layer is modified by in-situ growth of MOF materials on the inner wall of the nuclear pore membrane channel, the density of active sites is increased, then a continuous MOF layer is formed on the surface of the nuclear pore membrane, selective separation and sensing are realized and the current signal is amplified; the conductive enhancement layer is modified by a conductive material in the MOF channel to form a continuous conductive network, the electrode interface resistance is reduced, the transmission of electrons from the reaction site to the external circuit is accelerated, a conductive coating is prepared on the surface of the nuclear pore membrane as the electrode interface signal, the signal-to-noise ratio is reduced and the stability is improved; and the biological recognition layer is used for specific recognition of target molecules by fixing DNA probes on the surface of the MOF. The electrochemical biosensor based on the MOF / nuclear pore membrane sandwich structure can utilize the mechanical support and ordered pore channel of the nuclear pore membrane and improve the sensing performance through the functional surface of the MOF, and is suitable for high-sensitivity and high-selectivity biological molecule detection.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biosensing, and particularly relates to an electrochemical biosensor based on a metal-organic framework (MOF) and nuclear pore membrane sandwich structure. BACKGROUND

[0002] An electrochemical biosensor is a high-tech that is grown from the mutual penetration of biology, chemistry, physics, medicine, materials, and electronic technology, and is a new type of detection technology developed by combining the specific recognition of biological molecules and high-sensitivity sensing technology, and has been widely applied to various aspects of clinical testing. However, the traditional electrochemical biosensor has the following deficiencies: 1. Limitation of signal amplification strategy: Although early modification of electrodes by nanomaterials (such as gold nanoparticles and carbon nanotubes) can partially improve the sensitivity, the materials have poor dispersibility and insufficient active sites; 2. Insufficient sensitivity and selectivity: Traditional electrochemical sensors rely on single signal output (such as current or potential), which is easily disturbed by the background, resulting in limited sensitivity. In addition, non-specific adsorption in complex samples also reduces selectivity; 3. Low efficiency of biological molecule immobilization: The electron transfer efficiency of enzymes is low, and the enzymes are prone to fall off from the electrode surface, resulting in poor stability of the sensor. Metal-organic frameworks (MOFs) are a kind of porous nanomaterial composed of metal-containing inorganic secondary structural units and organic linkers, and the functional properties thereof can be increased by changing the surface structure. MOF materials have attracted more and more attention in the field of biosensing due to their high specific surface area, large number of binding sites, and high porosity. The high porosity of MOF provides sufficient space for the loading of biological molecules (such as aptamers and enzymes), and the ordered pore channels can promote electron transfer. MOF can achieve specific recognition through ligand design, and the rigid framework of MOF can protect the loaded biological molecules from inactivation. However, the electrochemical biosensor based on MOF has the deficiency of poor stability, and therefore the sandwich structure of MOF and nuclear pore membrane greatly improves the stability of the sensor through multi-level pores and functional partitioning. The electrochemical biosensor based on the sandwich structure of MOF and nuclear pore membrane provides a new idea for high-sensitivity and high-stability biological detection through the synergistic innovation of materials and structure, and is expected to be more widely applied in the fields of disease diagnosis, environmental monitoring, and food safety in the future. SUMMARY

[0003] The electrochemical biosensor based on the sandwich structure of MOF and nuclear pore membrane is an innovative strategy that combines high specific surface area, adjustable pore function, and uniform pore size structure. This composite membrane can not only utilize the mechanical support and ordered pores of the nuclear pore membrane, but also improve the sensing performance through the functionalized surface of MOF, and is suitable for high-sensitivity and high-selectivity biological molecule detection.

[0004] To achieve the above object, the application adopts the following technical solutions: taking a nuclear pore membrane as a substrate, modifying MOF material in the inner wall of the nuclear pore membrane channel by in-situ growth, modifying conductive material in the MOF channel to form a continuous conductive network, preparing a conductive coating on the surface of the nuclear pore membrane as an electrode interface signal, electrodeposition of a MOF layer with carboxyl groups on the nuclear pore membrane, then activating the composite membrane, fixing DNA probes on the surface of the MOF for specific recognition of target molecules, and then sealing the active sites.

[0005] The nuclear pore membrane of the technical solution is a polyethylene terephthalate (PET) membrane, the shape of the nanopore channel is columnar, conical or irregular, and the etching solution is a sodium hydroxide solution.

[0006] The MOF functional layer of the technical solution is divided into an inner wall of the channel and a surface of the channel, wherein the inner wall of the channel is modified with MIL-68(In) nano MOF material, and the surface of the channel is modified with UIO-66 nano MOF material.

[0007] The conductive enhancement layer of the technical solution is filled with carbon nanotubes in the channel to form a three-dimensional conductive network, and a silver nano layer is sprayed on the surface of the nuclear pore membrane as an electrode interface.

[0008] The biological recognition layer of the technical solution is DNA probes fixed on the surface of the MOF for specific recognition of target molecules.

[0009] The activator of the technical solution is EDC and NHS, and the active site sealing agent is bovine serum albumin.

[0010] The application discloses a preparation method of an electrochemical biosensor based on a MOF / nuclear pore membrane sandwich structure, which is prepared by a metal organic framework (MOF) and nuclear pore membrane composite structure, can utilize mechanical support and ordered pores of the nuclear pore membrane, and can improve sensing performance through a functionalized surface of the MOF, and is suitable for target detection with high sensitivity and high selectivity.

[0011] Further, in the step (1), 100-200 mg of In(NO3)3.5H2O and the nuclear pore membrane are dissolved in 20-60 mL of N, N-dimethylformamide (DMF) and completely dissolved in ultrasonic waves; then 100-200 mg of terephthalic acid is added and dissolved in ultrasonic waves; the reaction is carried out in an 80-120 DEG C oil bath for 20-30 min, and then the system is cooled to room temperature; after centrifugation, the precipitate is washed with ethanol for three times, so that the MIL-68(In) in-situ grown in the nuclear pore membrane pore wall is obtained.

[0012] Further, in the step (2), the carbon nanotube is prepared by an electrodeposition method, and the silver nano layer is only sprayed on one side.

[0013] Further, in the step (3), the MOF is UIO-66 nano MOF, the UIO-66 nano MOF is synthesized by a hydrothermal method, ZrCl4 is used as a metal precursor, terephthalic acid is used as a ligand, and DMF is used as a solvent; the UIO-66 crystal is prepared by reacting at 100-120 DEG C for 20-24 h; and the MOF layer with the carboxyl functional group is electrodeposited on the reverse side of the silver nano layer.

[0014] Further, in the step (4), the concentration of EDC is 10-20 mg / L, the concentration of NHS is 3-5 mg / L, the concentration of all DNA aqueous solution is (2-5 µM / L, pH 7.0-8.0), the concentration of bovine serum albumin aqueous solution is (8 mg / mL-10 mg / mL, pH 7.0-8.0), the soaking time is 1-2 h, and the soaking is carried out at room temperature, and the amino-functionalized probe is covalently connected with the MOF layer with carboxyl functional groups.

[0015] In addition, the application further provides an application of an electrochemical biosensor based on a metal organic framework (MOF) and nuclear pore membrane composite structure, and the main steps are as follows: step (1) placing the composite membrane on ITO conductive glass as a working electrode, adding a platinum wire as a counter electrode and Ag / AgCl as a reference electrode, adding an electrolyte solution with the same concentration in an electrolytic cell, connecting an external load to form a loop, testing I-V current, and obtaining a current value I0 before incubation; step (2) then pumping out the electrolyte solution in the electrolytic cell, washing the composite membrane with deionized water, mixing the target standard sample and hybridization buffer uniformly, and then adding them into the electrolytic cell, incubating at room temperature, after incubation, pumping out the incubation solution, washing the residual target on the surface of the composite membrane with deionized water, adding an electrolyte solution, testing the I-V current of the target standard sample, and obtaining a current value I of the target standard sample after incubation; and step (3) comparing the electrochemical changes before and after incubation to analyze whether the target and the probe are combined.

[0016] Further, in the application of the electrochemical biosensor based on the MOF / nuclear pore membrane sandwich structure, the electrolyte solution is 1-2 mol / L of potassium chloride, the hybridization buffer is tris buffer with a concentration of 0.1-0.5 µmol / L, the concentration of the target is 0.5-2 µmol / L, and the target is a nucleic acid, and all tests are carried out at room temperature.

[0017] The detection principle of the application is: taking a nuclear pore membrane substrate, the MOF material is modified in the inner wall of the nuclear pore membrane channel by in-situ growth, the density of active sites is increased, then a continuous MOF layer is formed on the surface of the nuclear pore membrane, selective separation and sensing are realized, then the carbon nanotubes are filled in the nuclear pore membrane channel to form a three-dimensional conductive network to enhance the electrical signal output, a conductive coating is prepared on the surface of the nuclear pore membrane as an electrode interface, finally the DNA probe is fixed on the surface of the MOF for specific recognition of target molecules, and the active sites are blocked with bovine serum albumin; when the target enters the whole system, it will first specifically hybridize with the probe DNA on the surface of the MOF nanomaterial UIO-66, due to the contribution of the negatively charged DNA nucleotide phosphate backbone, the surface charge and charge density change greatly, which further affects the transmembrane movement of ions, converts the concentration of the target into an electrical current signal output, the current signal is amplified by the UIO-66 nanometer MOF material and transmitted to the nuclear pore membrane channel, then the signal is further amplified by the MOF nanomaterial MIL-68(In) in the channel, then the conductive layer reduces the resistance to accelerate the transfer of electrons from the reaction site to the external circuit output, thereby realizing the ultra-sensitive and quantitative detection of biomolecules; the hybridization between the probe and the target is monitored by recording the current-voltage (I-V) curve.

[0018] In the MOF / nanopore membrane composite structure-based electrochemical biosensor, the substrate, functional layer, conductive enhancement layer and biological recognition layer each play its own role, and through synergistic effect, the detection target is realized with high sensitivity and high selectivity. The following is the principle of action and synergistic mechanism of each layer: 1. Substrate (nanopore membrane) principle of action: mechanical support and pore guidance: the nanopore membrane (such as polycarbonate PC, PET) forms uniform vertical pores through nanopore etching technology, providing stable physical support for the MOF functional layer, while guiding the directional transmission of target molecules along the pores, reducing diffusion resistance; 2. Functional layer (MOF) principle of action: (1) molecular recognition and enrichment: the super-high specific surface area and adjustable pore size of MOF enrich target molecules through size sieving and surface adsorption dual mechanisms; (2) signal amplification: MOFs have high conductivity, and their high surface area and porous structure can enhance the electron transmission efficiency at the electrode interface, thereby amplifying the current signal; (3) functional modification platform: the ligand functional groups of MOF provide sites for covalently fixing biological molecules; 3. Conductive enhancement layer principle of action: (1) electron transmission channel construction: conductive materials form a continuous conductive network on the surface of the nanopore membrane or in the pores of the MOF, reducing the electrode interface resistance and accelerating the transmission of electrons from the reaction site to the external circuit; (2) signal stability improvement: the conductive layer can buffer the semiconductor properties of MOF, reduce electrochemical noise, and improve the signal-to-noise ratio. (3) Mechanical protection: the conductive layer covering the MOF surface can prevent it from dissolving or collapsing in an aqueous environment; 4. Biological recognition layer principle of action: specific molecular recognition: biological probes fixed on the MOF surface selectively capture target molecules through the lock-and-key mechanism, causing changes in interface charge distribution or mass.

[0019] Synergistic mechanism: (1) mass transfer-reaction-signal transmission integration: the vertical pores of the nanopore membrane accelerate the diffusion of target molecules to the MOF functional layer, MOFs have high conductivity and high-efficiency electron transmission efficiency to amplify the current signal, and the biological recognition layer ensures specificity, forming a closed loop of "directional mass transfer-molecular recognition-signal amplification-signal output"; (2) balance between stability and sensitivity; (3) the mechanical strength of the nanopore membrane protects the fragile MOF layer, the high-activity surface of the MOF enhances the sensitivity, and the conductive layer compensates for the conductivity defects of the MOF, and the three work together to achieve long-term stable detection.

[0020] The principle of action of each layer is essentially a precise match of "structure-function": substrate: physical support and mass transfer optimization; functional layer: molecular recognition and signal amplification; conductive layer: electron transmission and signal stability; biological layer: specific capture and signal triggering. The synergy of the four breaks through the bottleneck of traditional sensors in sensitivity, selectivity and stability, providing an innovative solution for the detection of complex real samples.

[0021] This invention utilizes a metal-organic framework (MOF) and nuclear pore membrane composite structure as the detection membrane material through an electrochemical method. Based on the unique current response of DNA hybridization, it provides a new method for electrochemical detection. Compared with the prior art, the beneficial effects of this invention are at least as follows: (1) High sensitivity: The high specific surface area of ​​MOF (>1000 m² / g) provides a large number of active sites, and the detection limit is as low as 0.01 fM; (2) Strong selectivity: The pore size of MOF is adjustable (0.01–3 µm), effectively eliminating interfering substances; (3) High stability: The mechanical support of the nuclear pore membrane prevents the MOF layer from breaking and is resistant to acid and alkali environments; (4) Fast response: The vertically arranged channels (nuclear pore membrane) and MOF nanosheets (thickness <100 nm) synergistically accelerate mass transfer, and the response time is <5 seconds. Attached Figure Description

[0022] Figure 1 This is an electron microscope image of the nuclear pore membrane.

[0023] Figure 2 This is a current diagram illustrating the fabrication process of an electrochemical biosensor based on a MOF / nuclear pore membrane sandwich structure.

[0024] Figure 3 The A-plot current graph and the B-plot standard curve are shown for the response of target substances at different concentrations.

[0025] Figure 4 This is a specific identification map.

[0026] Figure 5 Figure for stability investigation. Detailed Implementation

[0027] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, the scope of protection of the present invention is not limited thereto. Any changes made to the technical solutions of the present invention by those skilled in the art should fall within the scope of protection of the present invention.

[0028] The nucleic acid sequence listings involved in the following embodiments are shown in Table 1.

[0029]

[0030] Embodiment 1: A preparation method of an electrochemical biosensor based on a MOF / nuclear pore membrane sandwich structure, which can utilize the mechanical support and ordered pores of the nuclear pore membrane and improve the sensing performance through the functionalized surface of the MOF by a metal organic framework (MOF) and nuclear pore membrane composite structure, and is suitable for high sensitivity and high selectivity target detection, the specific preparation steps of the electrochemical biosensor are as follows: Step (1) dissolve the nuclear pore membrane and In(NO3)3.5H2O in N,N-dimethylformamide (DMF), completely dissolve in the ultrasonic wave, then add terephthalic acid, dissolve with ultrasonic wave, and perform hydrothermal reaction in a reaction kettle in an oven, cool to room temperature, wash the precipitate with ethanol three times after centrifugation, and in-situ growth of MIL-68(In) on the inner wall of the nuclear pore membrane pore is obtained; Step (2) immerse the nuclear pore membrane in a solution containing carbon nanotubes, after the reaction is completed, wash the nuclear pore membrane with deionized water to remove the surface residues, and then spray a silver nanolayer on the surface of the nuclear pore membrane as an electrode interface; Step (3) place the nuclear pore membrane sprayed with the silver nanolayer on the ITO conductive glass for electrochemical deposition, and deposit a MOF layer with carboxyl functional group modification on one side of the conductive nuclear pore membrane, and wash the other side with deionized water to remove residues; Step (4) place the composite membrane in the activation solution for activation, then wash and immerse in the probe solution with amino group to fix the DNA probe in the MOF pore, and then wash and block the active sites with bovine serum albumin.

[0031] Further, in the step (1), 100 mg of In(NO3)3.5H2O and the nuclear pore membrane are dissolved in 20 mL of N,N-dimethylformamide (DMF) and completely dissolved in the ultrasonic wave; then 100 mg of terephthalic acid is added and dissolved with ultrasonic wave; react in a 100℃ oil bath for 20 min, cool to room temperature, and wash the precipitate with ethanol three times after centrifugation, and in-situ growth of MIL-68(In) in the nuclear pore membrane pore is obtained;

[0032] Further, in the step (2), the preparation method of the carbon nanotube is an electrodeposition method, and the silver nanolayer is only sprayed on one side;

[0033] Further, in the step (3), the MOF is UIO-66 nanometer MOF, and the synthesis of the UIO-66 nanometer MOF is hydrothermal synthesis, ZrCl4 is used as the metal precursor, terephthalic acid is used as the ligand, and DMF is used as the solvent, and the UIO-66 crystal is prepared by reacting at 100℃ for 20 h, and the MOF layer with carboxyl functional group modification is modified to the reverse side of the silver nanolayer by electrodeposition;

[0034] Further, in the step (4), the activating agent is EDC and NHS, the concentration of EDC is 10 mg / L, the concentration of NHS is 3 mg / L, the concentration of all DNA aqueous solution is (2 µM / L, pH 7.0), the concentration of bovine serum albumin aqueous solution is (8 mg / mL, pH 7.0), the soaking time is 1 h, and the operation is carried out at room temperature, and the amino-modified probe is covalently connected with the MOF layer with carboxyl functional groups.

[0035] In addition, the application further provides an application of an electrochemical biosensor based on a metal organic framework (MOF) and nuclear pore membrane composite structure, and the main steps are as follows: (1) placing the composite membrane on ITO conductive glass as a working electrode, adding a platinum wire as a counter electrode and Ag / AgCl as a reference electrode, adding electrolyte solutions with the same concentration in an electrolytic cell, connecting an external load to form a loop, testing I-V current, and obtaining the current value I0 before incubation; (2) then, the electrolyte solution in the electrolytic cell is sucked out, the composite membrane is washed with deionized water, the target standard sample and hybridization buffer are uniformly mixed and then added to the electrolytic cell, and after incubation at room temperature, the incubation liquid is sucked out, the residual target on the surface of the composite membrane is washed away with deionized water, the electrolyte solution is added, the I-V current of the target standard sample is tested, and the current value I0 of the target standard sample after incubation is obtained; and (3) comparing the electrochemical changes before and after incubation to analyze whether the target and the probe are combined.

[0036] Further, in the application of the electrochemical biosensor based on the MOF / nuclear pore membrane sandwich structure, the electrolyte solution is 1-2 mol / L potassium chloride, the hybridization buffer is tris buffer, the concentration is 0.1-0.5 µmol / L, the concentration of the target is 0.5-2 µmol / L, the target is a nucleic acid, and all the tests are carried out at room temperature.

[0037] In this embodiment, an electron microscope (SEM) is used as a powerful technology to help observe the morphology and structure of the prepared material. Figure 1 The results show that the membrane has a through columnar nanopore, the porosity is 30%, under the same conditions, the pore diameters of 100 groups of nanopores are counted, the nanopore diameters constructed are 0.1-5 µm, the shape of the nanopore is cylindrical, the pore density is 1×10 3 ~1×10 15 / cm 2 .

[0038] Example 4: Experimental verification of the principle

[0039] In order to prove the feasibility of the technical scheme, different modified electrodes are used in this embodiment to prepare an electrochemical biosensor. The results are as follows:Figure 2 As shown, after coating the nuclear pore membrane on the ITO conductive glass, current detection was performed, and a small current value was observed, indicating that the nuclear pore membrane was almost insulating; after modifying the MOF layer, current detection was performed, and a small increase in current was observed; after modifying the conductive layer, current detection was performed, and a large increase in current was observed; after modifying the recognition layer, current detection was performed, and a small increase in current was observed; when the target object was subjected to current detection, a sharp increase in current was observed, indicating that when the target object was present, specific recognition with the probe occurred, thereby causing a change in current.

[0040] Example 5 Sensitivity experiment

[0041] In order to evaluate the sensitivity of the target object detected by the technical solution, the current of the target object with different concentrations was measured under the best experimental conditions. As shown in Figure 3 As shown in FIG. A, the current increased with the increase of the concentration of the target object, and as shown in Figure 3 FIG. B, there is a certain functional relationship between the change of the current and the size of the target object concentration, so the target object can be analyzed by the electrochemical biosensor, and the electrochemical biosensor combined with different biological recognition elements will construct an electrochemical biosensor with different detection functions.

[0042] Example 6 Specificity experiment

[0043] In order to study the specificity of the electrochemical biosensor, as shown in Figure 5 , five mismatches and one specific target object were used as controls for the specificity experiment; the results are shown in Figure 4 As shown, the current difference of the target object under the same reaction conditions is much larger than that of the non-target object.

[0044] Example 7 Stability experiment

[0045] In order to study the stability of the electrochemical biosensor, as shown in Figure 5 , five parallel current responses were performed in a PBS buffer solution with a concentration of 0.1 mol / L and a pH of 7, and it was found that the electrochemical biosensor had excellent stability.

[0046] It should be noted that the above examples are only used to illustrate the technical solutions of the present application and not to limit them. Although the present application has been described in detail with reference to the examples given, those skilled in the art can modify or equivalently replace the technical solutions of the present application according to their needs, without departing from the spirit and scope of the present application.

Claims

1. A MOF / nuclear pore membrane sandwich structure-based electrochemical biosensor, characterized in that: The nuclear pore membrane base, MOF functional layer, conductive enhancement layer and biological recognition layer are included; the MOF material is modified in the nuclear pore membrane channel inner wall by in-situ growth, the conductive material is modified in the MOF channel to form a continuous conductive network, the conductive coating is prepared on the surface of the nuclear pore membrane as an electrode interface to form a conductive enhancement layer; the MOF layer with carboxyl is modified on the surface of the nuclear pore membrane by electrochemical deposition to form a MOF functional layer; then the composite membrane is activated by an activator, the DNA probe is fixed on the MOF surface for specific recognition of target molecules, and then the active site is closed by a blocking agent to form a biological recognition layer; The nuclear pore membrane is a polyethylene terephthalate (PET) membrane, the shape of the nano-channel is columnar, conical or irregular, and the etching solution is sodium hydroxide solution. The MOF functional layer is divided into the channel inner wall and the channel surface, wherein the MIL-68(In) nano MOF material is modified on the channel inner wall, and the UIO-66 nano MOF material is modified on the channel surface. The conductive enhancement layer is filled with carbon nanotubes in the channel to form a three-dimensional conductive network, and the silver nano layer is sprayed on the surface of the nuclear pore membrane as an electrode interface. The biological recognition layer is the DNA probe fixed on the MOF surface for specific recognition of target molecules. The activator is EDC and NHS, and the active site blocking agent is bovine serum albumin.

2. A method for preparing an electrochemical biosensor based on a MOF / nanopore membrane sandwich structure, characterized in that, The specific preparation steps of the electrochemical biosensor are as follows: step (1) dissolving the nuclear pore membrane and In(NO3)3·5H2O in N, N-dimethylformamide (DMF) and completely dissolving in the ultrasonic wave, then adding terephthalic acid and dissolving in the ultrasonic wave, and performing hydrothermal reaction in a reaction kettle in an oven, cooling to room temperature, washing the precipitate with ethanol three times after centrifugation, to obtain the MIL-68(In) in-situ grown in the channel inner wall of the nuclear pore membrane; step (2) immersing the nuclear pore membrane in a solution containing carbon nanotubes, washing the nuclear pore membrane with deionized water to remove the surface residues after the reaction is completed, and then spraying a silver nano layer on the surface of the nuclear pore membrane as an electrode interface; step (3) placing the nuclear pore membrane with the sprayed silver nano layer on the ITO conductive glass for electrochemical deposition, and depositing the MOF layer with a carboxyl functional group on one side of the conductive nuclear pore membrane, and washing the other side to remove residues; step (4) placing the composite membrane in the activator solution for activation, then washing and immersing in the probe solution with amino to fix the DNA probe in the MOF channel, and then washing and blocking the active site with bovine serum albumin.

3. The method for fabricating an electrochemical biosensor based on a MOF / nuclear pore membrane sandwich structure according to claim 2, characterized in that, In step (2), the carbon nanotubes are prepared by electrochemical deposition, and the silver nano layer is sprayed on only one side.

4. The method for fabricating an electrochemical biosensor based on a MOF / nuclear pore membrane sandwich structure according to claim 2, characterized in that, In step (3), the MOF is UIO-66 nano MOF, and the MOF layer with a carboxyl functional group is deposited on the opposite side of the silver nano layer.

5. The method for fabricating an electrochemical biosensor based on a MOF / nuclear pore membrane sandwich structure according to claim 2, characterized in that, In the step (4), the amino-functionalized probe is covalently linked to the MOF layer with carboxyl functional groups.

6. Use of a MOF / nanopore membrane sandwich structure-based electrochemical biosensor according to claim 1, characterized in that, The specific detection steps are as follows: step (1) placing the composite film on ITO conductive glass as a working electrode, adding a platinum wire as a counter electrode, Ag / AgCl as a reference electrode, adding an electrolyte solution of the same concentration in an electrolytic cell, connecting an external load to form a circuit, testing I-V current, and obtaining the current value I0 before incubation; step (2) then aspirating the electrolyte solution in the electrolytic cell, washing the composite film with deionized water, mixing the target standard sample and hybridization buffer uniformly, and then adding them into the electrolytic cell, incubating at room temperature, after incubation, aspirating the incubation solution, washing the residual target on the surface of the composite film with deionized water, adding the electrolyte solution, testing the I-V current of the target standard sample, and obtaining the current value I of the target standard sample after incubation; step (3) comparing the electrochemical changes before and after incubation to analyze whether the target and the probe are combined.

7. Use of a MOF / nanopore membrane sandwich structure based electrochemical biosensor according to claim 6, characterized in that, The electrolyte solution is 1-2 mol / L potassium chloride, the hybridization buffer is tris buffer, the concentration is 0.1-0.5 μmol / L, the concentration of the target is 0.5-2 μmol / L, the target is a nucleic acid, and all tests are carried out at room temperature.

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