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

Abstract

The invention discloses a preparation method and application of an electrochemical biosensor based on an MOF / nuclear track membrane sandwich structure, and belongs to the technical field of biosensing. The invention discloses an electrochemical biosensor based on an MOF / nuclear track membrane sandwich structure. The electrochemical biosensor comprises a nuclear track membrane substrate, an MOF functional layer, a conductive enhancement layer and a biological recognition layer, the nuclear track membrane substrate is used for providing stable physical support for the MOF functional layer by taking a nuclear track membrane as a substrate, and guiding target molecules to be directionally transmitted along a pore channel at the same time, so that diffusion resistance is reduced; an MOF functional layer: modifying an MOF material on the inner wall of a nuclear track membrane pore channel through in-situ growth to increase the active site density, and then forming a continuous MOF layer on the surface of the nuclear track membrane to realize selective separation and sensing and amplify a current signal; according to the conductive enhancement layer, a conductive material is modified in an MOF pore channel to form a continuous conductive network, electrode interface resistance is reduced, transmission of electrons from a reaction site to an external circuit is accelerated, a conductive coating is prepared on the surface of the nuclear pore membrane to serve as an electrode interface signal, the signal-to-noise ratio is reduced, and stability is improved; and the biological recognition layer is used for fixing a DNA probe on the surface of the MOF and is used for specifically recognizing target molecules. The electrochemical biosensor based on the MOF / nuclear track membrane interlayer not only can utilize the mechanical support and ordered pore channels of the nuclear track membrane, but also can improve the sensing performance through the functionalized surface of the MOF, and is suitable for high-sensitivity and high-selectivity biomolecule detection.

Description

Technical Field

[0001] The present invention belongs to the technical field of biosensors, and in particular relates to an electrochemical biosensor based on a metal organic framework (MOF) and a nuclear pore membrane sandwich structure. Background Art

[0002] Electrochemical biosensors are a high-tech technology that has grown from the mutual penetration of multiple disciplines such as biology, chemistry, physics, medicine, materials, and electronic technology. It is a new type of detection technology that combines the specific recognition of biomolecules with highly sensitive sensing technology and has been widely used in various aspects of clinical testing. However, traditional electrochemical biosensors have the following shortcomings: 1. Limitations of signal amplification strategies: Although the early modification of electrodes by nanomaterials (such as gold nanoparticles and carbon nanotubes) can partially improve the sensitivity, the material has poor dispersion and insufficient active sites; 2. Insufficient sensitivity and selectivity: Traditional electrochemical sensors rely on a single signal output (such as current or potential) and are easily affected by background interference, resulting in limited sensitivity. In addition, nonspecific adsorption in complex samples will also reduce selectivity; 3. Low efficiency of biomolecule immobilization: The enzyme has low electron transfer efficiency and is easily detached from the electrode surface, resulting in poor sensor stability. Metal-organic frameworks (MOFs) are porous nanomaterials composed of metal-containing inorganic secondary structural units and organic connectors. Their functional properties 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, a large number of binding sites, and high porosity. The high porosity of MOF provides sufficient space for the loading of biomolecules (such as aptamers and enzymes). At the same time, its ordered pores can promote electron transfer. MOF can achieve specific recognition through ligand design, and the rigid framework of MOF can protect the loaded biomolecules from inactivation. However, electrochemical biosensors based on MOF have the disadvantage of poor stability. Therefore, the sandwich structure of nuclear pore membrane and MOF is used to greatly improve the stability of the sensor through multi-level pores and functional partitioning. The electrochemical biosensor with MOF nuclear pore membrane sandwich structure provides a new idea for highly sensitive and stable biological detection through the collaborative innovation of materials and structures. In the future, it is expected to be more widely used in disease diagnosis, environmental monitoring, food safety and other fields. Summary of the invention

[0003] The purpose of the present invention is to provide an electrochemical biosensor based on a sandwich structure of a metal organic framework (MOF) and a nuclear pore membrane, which is an innovative strategy combining high specific surface area, adjustable pore function and uniform pore 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-mentioned purpose, the present invention adopts the following technical scheme: taking the nuclear pore membrane as the substrate, the MOF material is modified on the inner wall of the nuclear pore membrane pore channel by in-situ growth, the conductive material is modified in the MOF pore channel to form a continuous conductive network, a conductive coating is prepared on the surface of the nuclear pore membrane as an electrode interface signal, a MOF layer with a carboxyl group is modified by electro-deposition on the nuclear pore membrane, and then the composite membrane is activated, and a DNA probe is fixed on the MOF surface for specific identification of the target molecule, and then the active site is blocked.

[0005] The nuclear pore membrane of the technical solution is a polyethylene terephthalate (PET) membrane, the shape of the nanopore 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 the inner wall of the channel and the 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 a pore filled with carbon nanotubes to form a three-dimensional conductive network, and a silver nanolayer is sprayed on the surface of the nuclear pore membrane as an electrode interface.

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

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

[0010] A method for preparing 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 through a metal organic framework (MOF) and a nuclear pore membrane composite structure, and can also improve the sensing performance through the functionalized surface of MOF, and is suitable for high-sensitivity and high-selectivity target detection. The specific preparation steps of the electrochemical biosensor are as follows: Step (1) The nuclear pore membrane and In(NO 3 ) 3 ·5H 2O is dissolved in N, N-dimethylformamide (DMF) and completely dissolved in ultrasound; terephthalic acid is then added and dissolved in ultrasound; a hydrothermal reaction is carried out in a reaction kettle in an oven, cooled to room temperature, and the precipitate is washed three times with ethanol after centrifugation to obtain MIL-68 (In) grown in situ on the inner wall of the pore channel of the nuclear pore membrane; step (2) immersing the nuclear pore membrane in a solution containing carbon nanotubes, waiting for the reaction to be completed, washing the nuclear pore membrane with deionized water to remove the residue on the surface, and then spraying a silver nanolayer on the surface of the nuclear pore membrane as an electrode interface; step (3) placing the nuclear pore membrane sprayed with the silver nanolayer on an ITO conductive glass for electrochemical deposition, electrochemically depositing a MOF layer modified with a carboxyl functional group on one side of the conductive nuclear pore membrane, and washing the other side with deionized water to remove the residue; step (4) placing the composite membrane in (3) in an activator solution for activation, and then immersing it in an amino probe solution after washing to fix the DNA probe in the MOF pore channel, and then blocking the active site with bovine serum albumin after washing.

[0011] Furthermore, in step (1), 100-200 mg of In(NO 3 ) 3 ·5H 2 O and the nuclear pore membrane are dissolved in 20-60 mL of N, N-dimethylformamide (DMF) and completely dissolved in ultrasound; then 100-200 mg of terephthalic acid is added and dissolved in ultrasound; react in an oil bath at 80-120°C for 20-30 min, cool to room temperature, centrifuge and wash the precipitate three times with ethanol to obtain MIL-68(In) grown in situ in the pores of the nuclear pore membrane;

[0012] Furthermore, in step (2), the carbon nanotubes are prepared by electrodeposition, and the silver nanolayer is sprayed on only one side.

[0013] Furthermore, in step (3), the MOF is UIO-66 nano-MOF, and the synthesis of UIO-66 nano-MOF is a hydrothermal method, with ZrCl 4 As the metal precursor, terephthalic acid as the ligand, and DMF as the solvent, UIO-66 crystals were prepared by reacting at 100-120°C for 20-24 h, and a MOF layer modified with carboxyl functional groups was electrodeposited to modify the reverse side of the silver nanolayer.

[0014] Furthermore, 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 solutions 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 immersion time is 1-2 h, and the process is carried out at room temperature. The amino probe is only covalently linked to the MOF layer with carboxyl functional groups.

[0015] In addition, the present invention also provides an application of an electrochemical biosensor based on a metal organic framework (MOF) and a nuclear pore membrane composite structure, the main steps of which are as follows: Step (1) placing the composite membrane on an 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 of the same concentration to an electrolytic cell, connecting an external load to form a loop, and testing the IV current to obtain the current value I before incubation. 0 ; Step (2) then suck out the electrolyte solution in the electrolytic cell, wash the composite membrane with deionized water, mix the target standard sample and hybridization buffer evenly and add them to the electrolytic cell, incubate at room temperature, suck out the incubation solution after incubation, wash the residual target on the surface of the composite membrane with deionized water, add the electrolyte solution, test the IV current of the target standard sample, and obtain the current value I of the target standard after incubation; Step (3) compare the electrochemical changes before and after incubation to analyze whether the target and the probe are bound.

[0016] Furthermore, 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 with a concentration of 0.1~0.5 µmol / L, the concentration of the target is 0.5~2 µmol / L, the target is nucleic acid, and all tests are performed at room temperature.

[0017] The detection principle of the present invention is as follows: using a nuclear pore membrane substrate, MOF material is modified on the inner wall of the nuclear pore membrane pore channel by in-situ growth to increase the density of active sites, and then a continuous MOF layer is formed on the surface of the nuclear pore membrane to achieve selective separation and sensing. Then, carbon nanotubes are filled in the pores of the nuclear pore membrane to form a three-dimensional conductive network to enhance the output of electrical signals. A conductive coating is prepared on the surface of the nuclear pore membrane as an electrode interface. Finally, a DNA probe is fixed on the MOF surface to specifically identify the target molecule, and then the active site is blocked with bovine serum albumin. When the target enters the entire system, it will first undergo specific hybridization and binding with the probe DNA on the surface MOF nanomaterial UIO-66. Due to the contribution of the negatively charged DNA nucleotide phosphate backbone, the surface charge and charge density undergo tremendous changes, which in turn affects the transmembrane movement of ions and converts the concentration of the target into a current signal output. The current signal is amplified by the UIO-66 nano-MOF material and transmitted to the nuclear pore membrane channel, and then further amplified by the MOF nanomaterial MIL-68 (In) in the channel. The signal is then reduced through the conductive layer to accelerate the transfer of electrons from the reaction site to the external circuit output, thereby achieving ultra-sensitive and quantitative detection of biological molecules; the hybridization between the probe and the target is monitored by recording the current-voltage (IV) curve.

[0018] In the electrochemical biosensor based on the MOF / nuclear pore membrane composite structure, the substrate, functional layer, conductive enhancement layer and biorecognition layer each perform their respective functions and achieve the detection target of high sensitivity and high selectivity through synergistic action. The following are the working principles of each layer and their synergistic mechanism: 1. Working principle of substrate (nuclear pore membrane): Mechanical support and pore guidance: The nuclear pore membrane (such as polycarbonate PC, PET) forms uniform vertical pores through nuclear track etching technology, providing stable physical support for the MOF functional layer, while guiding the target molecules to be transported along the pores in a directional manner, reducing diffusion resistance; 2. Working principle of functional layer (MOF): (1) Molecular recognition and enrichment: The ultra-high specific surface area and adjustable pore size of MOF enrich the target molecules through the dual mechanism of size screening and surface adsorption; (2) Signal amplification: MOFs It has high conductivity. Its high surface area and porous structure can enhance the electron transfer efficiency at the electrode interface, thereby amplifying the current signal; (3) Functional modification platform: The ligand functional groups of MOF provide sites for covalent fixation of biological molecules; 3. Principle of the conductive enhancement layer: (1) Construction of electron transfer channel: The conductive material forms a continuous conductive network on the surface of the nuclear pore membrane or in the MOF pore, reducing the electrode interface resistance and accelerating the transfer of electrons from the reaction site to the external circuit; (2) Improved signal stability: 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 covers the surface of MOF to prevent it from dissolving or structural collapse in the aqueous environment; 4. Principle of the biological recognition layer: Specific molecular recognition: The biological probe fixed on the surface of MOF selectively captures the target molecule through a lock-and-key mechanism, triggering changes in the interface charge distribution or mass.

[0019] Synergistic mechanism: (1) Integration of mass transfer, reaction and signal conduction: the vertical pores of the nuclear pore membrane accelerate the diffusion of target molecules to the MOF functional layer. MOFs have high conductivity and efficient electron transfer efficiency and can amplify current signals. The biorecognition 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 nuclear pore membrane protects the brittle MOF layer, the highly active surface of MOF improves sensitivity, and the conductive layer compensates for the conductive defects of MOF. The three work together to achieve long-term and stable detection.

[0020] The working principle 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: electronic transmission and signal stabilization; biological layer: specific capture and signal triggering. The four layers work together to break through the bottleneck of traditional sensors in sensitivity, selectivity and stability, and provide innovative solutions for the detection of complex actual samples.

[0021] The present invention uses an electrochemical method and a metal organic framework (MOF) and a nuclear pore membrane composite structure as a detection membrane material. Based on the unique current response of DNA hybridization, a new method for electrochemical detection is provided. Compared with the prior art, the present invention has at least the following beneficial effects: (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 MOF pore size is adjustable (0.01–3 µm), which effectively excludes interferences; (3) high stability: the mechanical support of the nuclear pore membrane prevents the MOF layer from rupturing and is resistant to acid and alkali environments; (4) fast response: the vertically arranged pores (nuclear pore membrane) and MOF nanosheets (thickness <100 nm) synergistically accelerate mass transfer, and the response time is <5 seconds. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0024] Figure 3 Figure A shows the current graph of the response to different concentrations of the target, and Figure B shows the standard curve.

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

[0026] Figure 5 This is a stability exploration diagram. DETAILED DESCRIPTION

[0027] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments, but the protection scope of the present invention is not limited thereto, and all changes made to the technical solution of the present invention by professionals in this field should fall within the protection scope of the present invention.

[0028] In the following examples, the nucleic acid sequence table involved is shown in Table 1

[0029] Example 1: A method for preparing 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 MOF through the metal organic framework (MOF) and the 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) The nuclear pore membrane and In(NO 3 ) 3 ·5H 2O is dissolved in N, N-dimethylbenzene amide (DMF) and completely dissolved in ultrasound, and then terephthalic acid is added and dissolved in ultrasound, and a hydrothermal reaction is carried out in a reactor in an oven, cooled to room temperature, and the precipitate is washed three times with ethanol after centrifugation to obtain MIL-68 (In) grown in situ in the inner wall of the pore channel of the nuclear pore membrane; step (2) immersing the nuclear pore membrane in a solution containing carbon nanotubes, waiting for the reaction to be completed, washing the nuclear pore membrane with deionized water to remove the residue on the surface, and then spraying a silver nanolayer on the surface of the nuclear pore membrane as an electrode interface; step (3) placing the nuclear pore membrane sprayed with the silver nanolayer on an ITO conductive glass for electrochemical deposition, electrochemically depositing a MOF layer modified with a carboxyl functional group on one side of the conductive nuclear pore membrane, and washing the other side with deionized water to remove the residue; step (4) placing the composite membrane in (3) in an activator solution for activation, and then immersing it in an amino probe solution after washing to fix the DNA probe in the MOF pore channel, and then blocking the active site with bovine serum albumin after washing.

[0030] Furthermore, in step (1), 100 mg of In(NO 3 ) 3 ·5H 2 O and the nuclear pore membrane were dissolved in 20 mL of N, N-dimethylformamide (DMF) and completely dissolved in ultrasound; 100 mg of terephthalic acid was added and dissolved in ultrasound; the mixture was reacted in a 100°C oil bath for 20 min, cooled to room temperature, centrifuged and the precipitate was washed three times with ethanol to obtain the in situ grown MIL-68(In) in the pores of the nuclear pore membrane;

[0031] Furthermore, in step (2), the carbon nanotubes are prepared by electrodeposition, and the silver nanolayer is sprayed on only one side;

[0032] Furthermore, in step (3), the MOF is UIO-66 nano-MOF, and the synthesis of UIO-66 nano-MOF is a hydrothermal method, with ZrCl 4 UIO-66 crystals were prepared by using terephthalic acid as metal precursor, terephthalic acid as ligand and DMF as solvent at 100℃ for 20 h, and then MOF layer modified with carboxyl functional group was electrodeposited to modify the reverse side of silver nanolayer.

[0033] Furthermore, in step (4), the activators are 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 solutions is (2 µM / L, pH 7.0), the concentration of bovine serum albumin aqueous solution is (8 mg / mL, pH 7.0), the immersion time is 1 h, and the process is carried out at room temperature. The amino probe is only covalently linked to the MOF layer with carboxyl functional groups.

[0034] Example 2: In addition, the present invention also provides an application of an electrochemical biosensor based on a metal organic framework (MOF) and a nuclear pore membrane composite structure, the main steps of which are as follows: (1) placing the composite membrane on an 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 of the same concentration to an electrolytic cell, connecting an external load to form a loop, and testing the IV current to obtain the current value I before incubation. 0 (2) The electrolyte solution in the electrolytic cell is then sucked out, the composite membrane is washed with deionized water, the target standard sample and hybridization buffer are mixed evenly and added to the electrolytic cell, and incubated at room temperature. After incubation, the incubation solution is sucked out, and the residual target on the surface of the composite membrane is washed with deionized water. The electrolyte solution is added, and the IV current of the target standard sample is tested to obtain the current value I of the target standard after incubation. 0 ; (3) Compare the electrochemical changes before and after incubation to analyze whether the target and the probe are bound.

[0035] Furthermore, 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 with a concentration of 0.1~0.5 µmol / L, the concentration of the target is 0.5~2 µmol / L, the target is nucleic acid, and all tests are performed at room temperature.

[0036] Example 3: This example uses scanning electron microscopy (SEM) as a powerful technique to help observe the morphology and structure of the prepared materials. The nuclear pore membrane was examined by scanning electron microscopy. Figure 1 The results showed that the membrane had penetrating columnar nanopores with a porosity of 30%. Under the same conditions, the pore sizes of 100 groups of pores were statistically analyzed, and the pore sizes of the constructed nanopores were 0.1~5 µm. The shape of the nanopores was cylindrical, and the pore density was 1×10 3 ~1×10 15 / cm 2 .

[0037] Example 4 Experimental verification of the principle

[0038] In order to prove the feasibility of this technical solution, this implementation case used different modified electrodes to prepare electrochemical biosensors. Figure 2As 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; when the MOF layer was modified and current detection was performed, a small increase in current was observed; when the conductive layer was modified and current detection was performed, a large increase in current was observed; when the recognition layer was modified and current detection was performed, a small increase in current was observed; when current detection was performed on the target, a dramatic current increase was observed, indicating that when the target is present, specific recognition occurs with the probe, resulting in a current change.

[0039] Example 5 Sensitivity Experiment

[0040] In order to evaluate the sensitivity of the present technical solution in detecting the target, the current of the target with different concentrations was measured under the optimal experimental conditions. Figure 3 As shown in Figure A, the current increases with the increase of the concentration of the target. Figure 3 As shown in Figure B, there is a certain functional relationship between the change in current and the concentration of the target. Therefore, the target can be analyzed by the electrochemical biosensor. The electrochemical biosensor is combined with different biorecognition elements to construct an electrochemical biosensor with different detection functions.

[0041] Example 6 Specificity Experiment

[0042] In order to study the specificity of the electrochemical biosensor, Figure 5 As shown, this embodiment uses five mismatches and one specific target as a control to perform a specific experiment; the results are shown in Figure 4 As shown, under the same reaction conditions, the current difference of the target object is much greater than that of the non-target object.

[0043] Example 7 Stability Test

[0044] In order to study the stability of the electrochemical biosensor, Figure 5 As shown, five parallel current responses were carried out in a PBS buffer solution with a concentration of 0.1 mol / L and a pH of 7, and the results showed that the electrochemical biosensor had excellent stability.

[0045] It should be noted that the above examples are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention is described in detail with reference to the given examples, those skilled in the art may modify or replace the technical solution of the present invention as needed without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. An electrochemical biosensor based on a MOF / nuclear pore membrane sandwich structure, characterized in that: It includes a nuclear pore membrane substrate, a MOF functional layer, a conductive enhancement layer and a biorecognition layer; with the nuclear pore membrane as the substrate, the MOF material is modified on the inner wall of the nuclear pore membrane pore channel by in-situ growth, the conductive material is modified in the MOF pore channel to form a continuous conductive network, and a conductive coating is prepared on the surface of the nuclear pore membrane as an electrode interface signal to form a conductive enhancement layer; the MOF layer with carboxyl groups is modified by electro-deposition on the surface of the nuclear pore membrane to form a MOF functional layer; then the composite membrane is activated with an activator, a DNA probe is fixed on the MOF surface for specific recognition of the target molecule, and then the active site is blocked with a blocking agent to form a biorecognition layer.

2. A method for preparing an electrochemical biosensor based on a MOF / nuclear pore membrane sandwich structure, characterized in that: The electrochemical biosensor is prepared by using MIL-68 (In) as the MOF material grown in situ in the pores, UIO-66 as the continuous MOF layer on the pore surface, the conductive material as carbon nanotubes, the conductive coating as silver nanocoating, and the DNA probe as the biorecognition molecule. 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-dimethylbenzene amide (DMF), completely dissolving them in ultrasound, then adding terephthalic acid, dissolving them in ultrasound, performing hydrothermal reaction in an oven with a reactor, cooling to room temperature, centrifuging, and washing the precipitate three times with ethanol to obtain the MIL-68 (I2O) grown in situ in the inner wall of the nuclear pore membrane pores. n); step (2) immersing the nuclear pore membrane in a solution containing carbon nanotubes, waiting for the reaction to be completed, washing the nuclear pore membrane with deionized water to remove the residue on the surface, and then spraying a silver nanolayer on the surface of the nuclear pore membrane as an electrode interface; step (3) placing the nuclear pore membrane sprayed with the silver nanolayer on an ITO conductive glass for electrochemical deposition, electro-depositing a MOF layer modified with carboxyl functional groups on one side of the conductive nuclear pore membrane, and washing the other side with deionized water to remove the residue; step (4) placing the composite membrane in (3) in an activator solution for activation, and then immersing it in an amino probe solution after washing to fix the DNA probe in the MOF pore channel, and then blocking the active site with bovine serum albumin after washing.

3. The method for preparing an electrochemical biosensor based on a MOF / nuclear pore membrane sandwich structure according to claim 2, characterized in that: In the step (1), the nuclear pore membrane is a polyethylene terephthalate (PET) membrane, and the shape of the nanopores is columnar, conical, or irregular.

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

5. The method for preparing an electrochemical biosensor based on a MOF / nuclear pore membrane sandwich structure according to claim 2, characterized in that: In the step (3), the MOF is UIO-66 nano-MOF, and a MOF layer modified with carboxyl functional groups is electro-deposited to modify the reverse side of the silver nano-layer.

6. The method for preparing 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 probe is only covalently linked to the MOF layer with carboxyl functional groups.

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

8. The use of an electrochemical biosensor based on a MOF / nuclear pore membrane sandwich structure according to claim 7, characterized in that: The electrolyte solution is 1~2 mol / L potassium chloride, the hybridization buffer is tris buffer with a concentration of 0.1~0.5µmol / L, the concentration of the target is 0.5~2 µmol / L, the target is nucleic acid, and all tests are performed at room temperature.

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