Electrochemical biosensor based on nanoparticle modified multilayer porous membrane and application of electrochemical biosensor

Abstract

The invention provides an electrochemical biosensor based on a nanoparticle modified multilayer porous membrane and application of the electrochemical biosensor. The nano particle modified multilayer porous membrane is composed of a plurality of modified nano particles among membranes with nano pore channels. The electrochemical biosensor constructed based on the nanoparticle modified multilayer porous membrane has the following advantages: (1) good conductivity and stability; and (2) multiple capture probes are modified on the surfaces of the nano pore channels of the multi-layer porous membrane, so that multiple different targets can be respectively detected, and multiple different targets can also be detected at the same time. And (3) enzyme-free double-target sensitive normal-temperature amplification-free detection can be carried out. (4) preparation steps are simple, consumed time is short, and operability is high; and (5) the sensitivity and the specificity are high.

Description

Technical Field

[0001] The invention belongs to the field of electrochemical biosensors, and in particular relates to an electrochemical biosensor based on nanoparticle-modified multilayer porous membranes and applications thereof. Background Art

[0002] Electrochemical biosensor is 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. It has the characteristics of rapidity, sensitivity, and ease of operation, and has been widely used in various aspects of clinical testing. Compared with traditional detection technologies, electrochemical biosensor technology has the following advantages: (1) high sensitivity and high precision, which can realize quantitative analysis of biological target molecules; (2) high specificity, the biomolecule recognition element can selectively bind to the biological target molecule; (3) short detection time, which takes about 15 to 60 minutes to complete a single detection; (4) real-time detection, which can dynamically monitor the entire process of biomolecular reaction in real time. Nanoparticles have strong antioxidant properties, are basically non-toxic, have good biocompatibility, and have strong conductivity. They are widely used in electrochemical biosensors. Signal amplification through nanoparticles with good performance is crucial to improving the sensitivity of detection methods. The use of nanoparticles and multi-layer porous membranes to construct electrochemical biosensors can greatly improve the sensitivity of analytical methods and the performance of biosensors, thereby achieving high-performance detection of target substances. However, traditional nanoparticle-modified electrochemical biosensors have two major defects: first, traditional nanoparticle-modified electrochemical biosensors require nanoparticles to be modified on the surface of electrodes, and the modification of electrodes usually needs to be performed one by one, which greatly increases the preparation cost and limits their application in large-scale production of electrochemical biosensors. Second, traditional nanoparticle-modified electrochemical biosensors have complex preparation steps, are not capable of detecting multiple targets simultaneously or detecting different targets separately, and have low specificity.

[0003] Therefore, it is of great significance to develop an electrochemical sensor that is simple to prepare, low in cost, highly sensitive, highly specific, and capable of multi-target detection. Summary of the invention

[0004] The purpose of the present invention is to provide an electrochemical biosensor based on nanoparticle-modified multilayer porous membrane and its application, which has the advantages of high sensitivity, high specificity, rapid detection and the ability to detect multiple targets simultaneously.

[0005] The technical scheme of the present invention is specifically as follows: an electrochemical biosensor based on nanoparticle-modified multilayer porous membrane, which uses a polymer membrane bombarded by heavy ions as a substrate, first sensitizes it with ultraviolet light, and then chemically etches it to prepare nanopores of different shapes, and prepares a film containing multiple nanopores; nanoparticles are modified in the nanopores of the porous membrane, and then polyethyleneimine is modified on the surface of the pores through self-assembly, and then capture probes are modified on the surface of the nanopores to prepare a nanopore composite membrane functionalized with nanoparticle-modified capture probe DNA, and the active sites of the nanopores are blocked with bovine serum albumin; finally, multiple nanopore composite membranes functionalized with different capture probe DNAs are assembled together, thereby obtaining an electrochemical biosensor based on nanoparticle-modified multilayer porous membrane.

[0006] The multilayer porous membrane of the technical solution is a polyethylene terephthalate (PET) membrane with a thickness of 10-50 µm, a pore size of 0.1-5 µm on its surface, and a shape of a nanopore of columnar, conical, or irregular shape with a pore density of 1×10 3 ~1×10 15 / cm 2 .

[0007] The ultraviolet light wavelength of the technical solution is 300-400 nm, the etching solution is a sodium hydroxide solution with a concentration of 6 mol / L-9 mol / L, and the nanoparticles are gold nanoparticles with a diameter of 10-20 nm.

[0008] The etched nanopore surface of the technical solution has a negative charge due to the presence of carboxyl groups, and combines with the positively charged PEI through electrostatic interaction. At this time, the nanopore is positively charged, and then combines with the negatively charged capture probe DNA again through electrostatic interaction, thereby obtaining a capture probe DNA functionalized nanopore.

[0009] According to the above scheme, the target can be nucleic acid or protein.

[0010] The capture probe of the technical solution is a biological sequence that can specifically bind to the target, and can identify and capture the target; the target binds to the capture probe DNA to generate an electric current response, thereby achieving ultra-sensitive detection of the target.

[0011] An electrochemical biosensor based on nanoparticle-modified multilayer porous membrane, which detects a target object based on a DNA hybridization reaction by using a nanoparticle-modified multilayer capture probe DNA nanopore membrane as a detection material through an electrochemical method, wherein the nanoparticle-modified multilayer capture probe DNA nanopore membrane is prepared according to the following steps: (1) bombarding a polymer membrane with heavy ions accelerated by an accelerator, and then irradiating it with an ultraviolet lamp for 2 hours to sensitize it; (2) etching with an appropriate chemical etching solution to etch the tracks on the film into nanopores of different shapes, thereby preparing a porous membrane; (3) soaking the porous membrane in an aqueous solution containing nanoparticles for 10 minutes, then taking out the porous membrane modified with nanoparticles, rinsing it with water, and drying it in air to prepare a porous membrane modified with nanoparticles; (4) The particle-modified porous membrane was immersed in a polyethyleneimine (PEI) aqueous solution for 12 hours to self-assemble into a multilayer porous membrane modified with nanoparticles. The membrane was washed with deionized water and dried in air. (5) The nanoparticle-modified porous membrane was immersed in a capture probe DNA solution and incubated at room temperature for 2 hours. After incubation, it was washed with deionized water to remove unbound capture probe DNA to obtain a nanoparticle-modified capture probe DNA functionalized nanoporous composite membrane. (6) The nanoparticle-modified capture probe DNA functionalized nanoporous composite membrane was immersed in a bovine serum albumin (BSA) aqueous solution and incubated for 2 hours. It was washed with deionized water and dried in air. Finally, multiple nanoporous composite membranes functionalized with different capture probe DNA were assembled together to prepare an electrochemical biosensor of a nanoparticle-modified multilayer porous membrane.

[0012] Wherein, the polymer film is a polyethylene terephthalate (PET) film, a polycarbonate (PC) film or a polyimide (PI) film, and the wavelength of the ultraviolet light is 300-400 nm.

[0013] The chemical etching solution is a sodium hydroxide solution with a concentration of 6 mol / L to 9 mol / L, and the shape of the nanopores is columnar, conical, or irregular.

[0014] Wherein, the nanoparticles are gold nanoparticles with a diameter of 10 to 20 nm.

[0015] The concentration of the PEI aqueous solution is 1 mg / mL~3 mg / mL, pH 7.0~8.0; and the incubation conditions are incubation at room temperature.

[0016] The concentration of the capture probe DNA aqueous solution is 2 µmol / L~5 µmol / L, pH 7.0~8.0; the concentration of the BSA aqueous solution is 8 mg / mL~10 mg / mL, pH 7.0~8.0.

[0017] Among them, the concentration of BSA aqueous solution is 8 mg / mL~10 mg / mL, pH 7.0~8.0.

[0018] In addition, the present invention also provides an application of an electrochemical biosensor based on a nanoparticle-modified multilayer porous membrane, the main steps of which are as follows: placing the nanoparticle-modified multilayer porous membrane in the middle of an H-type electrolytic cell, adding an electrolyte solution of the same concentration to the H-type electrolytic cell, placing a pair of Ag / AgCl electrodes in two half electrolytic cells, connecting an external load to form a loop, connecting an external load to form a loop, testing IV current, and obtaining a current value I0 before incubation; then sucking out the electrolyte solution in the electrolytic cell, washing the composite membrane with deionized water, mixing a target standard sample and a hybridization buffer evenly, and adding them to the electrolytic cell, incubating at room temperature for 1 hour, and after incubation, sucking out the target standard sample and a hybridization buffer solution. The incubation solution is taken out, and the residual target on the surface of the composite membrane is washed away with deionized water. An 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, and then the process is repeated for multiple times to obtain the current values ​​I0 and I of the target standard samples with different concentrations before and after incubation. Finally, the current change rate (I-I0) / I0 of the target standard samples with different concentrations before and after incubation under the same conditions is calculated, and a standard curve is drawn with the logarithm of the target concentration as the horizontal axis and the current change rate as the vertical axis; then the above steps are tested to obtain the current values ​​I0 and I of the sample to be tested before and after incubation, and the concentration of the target in the sample to be tested is calculated according to the standard curve.

[0019] The electrolyte solution is a 0.1 mol / L~0.5 mol / L potassium chloride solution, the hybridization buffer is a tris buffer with a concentration of 0.1 µmol / L~0.5 µmol / L, and the concentration of the target is 0.5 µmol / L~2 µmol / L.

[0020] The above-mentioned application of the electrochemical biosensor based on nanoparticle modified multilayer porous membrane.

[0021] The detection principle of the present invention is as follows: a PET film with nanopores is prepared by chemical etching. After chemical etching, ionized carboxyl groups are exposed on the surface of the unmodified PET nanopores, resulting in a negative charge on the surface; positively charged PEI self-assembles on the surface of the nanopores through electrostatic interaction, triggering the polarity of the nanopores to change from negative to positive; then, the negatively charged capture probe DNA is further assembled on the positively charged channel surface, and the negatively charged BSA is blocked to reduce the positive charge on the surface; when the target DNA hybridizes with the capture probe DNA, due to the contribution of the negatively charged DNA nucleotide phosphate backbone, the surface charge and charge density undergo a huge change, which in turn affects the transmembrane movement of ions in the nanopores, converting the concentration of the target into a current signal output, thereby achieving ultra-sensitive and quantitative detection of biomolecules; the hybridization between the probe and the target is monitored by recording the current-voltage (IV) curve, such as Figure 2 shown.

[0022] The present invention uses an electrochemical method to utilize capture probe DNA nanopores of a multilayer porous membrane modified with nanoparticles as a detection membrane material, and provides a new method for electrochemical detection based on the unique current response of DNA hybridization. Compared with the prior art, the present invention has at least the following beneficial effects: (1) multiple different targets can be detected separately or simultaneously; (2) the present invention increases the conductivity by introducing nanoparticles, thereby effectively amplifying the current signal to a certain extent and improving the detection sensitivity; (3) the detection sensitivity is high, and its detection limit can reach 10 fM, with high specificity and no response to miRNAs of other sequences; (4) the preparation method of capture probe DNA nanopores of a multilayer porous membrane modified with nanoparticles has a mild reaction, an easy-to-control process, and simple operation; and (5) the reagents are all commercially available conventional reagents, which are low in cost and easy to industrialize. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Schematic diagram of the electrochemical biosensor detection device.

[0024] Figure 2 Schematic diagram of the electrochemical sensing principle of the electrochemical biosensor based on nanoparticle-modified multilayer porous membrane to detect target objects.

[0025] Figure 3 Schematic diagram of nanoparticle-modified multilayer porous membrane.

[0026] Figure 4 These are SEM characterization images of the nanopore (Figure a is the overall picture of the nanopore; Figure b is the large-mouth end face of the nanopore).

[0027] Figure 5 This is the IV curve diagram of the preparation process of the electrochemical biosensor based on nanoparticle modified multilayer porous membrane.

[0028] Figure 6 Optimize the experimental conditions (time, pH, temperature).

[0029] Figure 7 This is a stability curve of the electrochemical biosensor based on nanoparticle-modified multilayer porous membrane.

[0030] Figure 8 It is a specificity experiment diagram (IV curve of two samples responding to different targets).

[0031] Fig. 9 It is a selective experiment diagram (a bar graph of the current change rate in response to different targets of two samples).

[0032] Fig.10 The IV curves are responses to different concentrations of the target.

[0033] Fig.11 The current change rate curves in response to different concentrations of the target and the corresponding standard curves. DETAILED DESCRIPTION

[0034] In order to make the contents of the present invention easier to understand, the technical solution of the present invention is further described below in conjunction with specific implementation methods, but the present invention is not limited thereto.

[0035] In the above embodiment, the formula of the DNA fixation buffer is: 10 mM Tris, 1 mM EDTA·2Na, 50 mM NaCl, 1 mM KCl, pH 8.00.

[0036] In the following examples, the nucleic acid sequences (5'-3') involved are shown in the following table; Example

[0037] 1. A method for preparing an electrochemical biosensor based on nanoparticle-modified multilayer porous membrane, the specific steps of which are as follows: (1) bombarding a membrane material (PET membrane with a thickness of about 10 μm to 50 μm) with heavy ions on a linear accelerator to obtain a membrane containing heavy ion tracks, which is retained for later use; (2) irradiating the membrane containing heavy ion tracks with an ultraviolet lamp having a wavelength of 360 nm for 1 hour to obtain a sensitized heavy ion track membrane; (3) immersing the sensitized heavy ion track membrane in a pool containing an etching solution (6 mol / L sodium hydroxide solution) for 2 hours to obtain a polymer membrane containing nanopores;

[0038] The above polymer film was examined by electron microscopy. Figure 4The 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 .

[0039] Next, the porous membrane was immersed in an aqueous solution containing nanoparticles for 10 minutes, and then the porous membrane modified with nanoparticles was taken out, rinsed with water, and dried in the air to prepare a porous membrane modified with nanoparticles; the porous membrane modified with nanoparticles was immersed in a polyethyleneimine (PEI) aqueous solution for 12 hours, self-assembled into a multilayer porous membrane modified with nanoparticles, and the membrane was washed with deionized water and dried in the air; the porous membrane modified with nanoparticles was immersed in a capture probe DNA solution, incubated at room temperature for 2 hours, and after incubation, washed with deionized water to remove unbound capture probe DNA to obtain a nanoparticle-modified capture probe DNA functionalized nanoporous composite membrane; the nanoparticle-modified capture probe DNA functionalized nanoporous composite membrane was immersed in a bovine serum albumin (BSA) aqueous solution, incubated for 2 hours, washed with deionized water, and dried in the air, and finally, a plurality of nanoporous composite membranes functionalized with different capture probe DNAs were assembled together to prepare an electrochemical biosensor of a nanoparticle-modified multilayer porous membrane, the schematic diagram of which is as follows Figure 3 , using IV test to characterize it, IV test during the preparation process such as Figure 5 As shown;

[0040] The electrolyte solution used in the IV test in the above steps is 0.1 mol / L potassium chloride solution; after each modification step, the nanoporous PET membrane needs to be rinsed with deionized water, wiped dry with filter paper, and then the IV test is performed.

[0041] 2. Optimize the experiment; place the electrochemical biosensor in a deionized water environment, adjust the pH value of the electrolyte solution to 4, 5, 6, 7, 8, 9, and 10, perform IV testing at the same time every day for seven consecutive days, and make a bar graph of the current-PH relationship with the current value I at 1 V voltage ( Figure 6-2 ), indicating that the electrochemical biosensor has the best performance when the pH is 7. The electrochemical biosensor was placed in a deionized water environment, and the incubation time was adjusted to 10, 20, 30, 40, 50, and 60 minutes, respectively. The IV test was performed at the same time every day for six consecutive days, and a bar graph of the current-time relationship was made with the current value I at a voltage of 1 V ( Figure 6-1), indicating that the electrochemical biosensor tends to be stable after 30 minutes of hybridization, so 30 minutes of hybridization is taken as the optimal experimental condition. The electrochemical biosensor was placed in a deionized water environment, and the incubation temperature was adjusted to 20, 30, 35, 37, 40, 50, and 70°C, and IV tests were performed at the same time every day for seven consecutive days. The current value I at 1 V voltage was used to make a bar graph of the current-temperature relationship ( Figure 6-3 ), indicating that the electrochemical biosensor has the best performance when the hybridization temperature is 37°C.

[0042] 3. Stability test: The electrochemical biosensor was placed in a deionized water environment for IV testing to examine its rectification and ion current fluctuation. The test was continued for 270 seconds, and the current value I in the nanopore at 1 V was plotted as a bar graph of the current-time relationship ( Figure 7 ), indicating that the electrochemical biosensor has good stability.

[0043] 4. Specificity experiment: The interfering substances miR-21, miR-26a, miR-27a, miR-122 and the target substances miR-192 and miR-223 were used to verify the selectivity of the electrochemical biosensor, and the concentration was 2 µmol / L. The target substance miR-192 and the hybridization buffer were tris buffer at a concentration of 0.1 µmol / L, and added to the electrochemical biosensor device, and reacted for 30 minutes at 37°C. After the reaction was completed, it was washed with deionized water and wiped dry, and then the electrolyte solution was added with a concentration of 0.1 mol / L and a potassium chloride solution with a pH of 7.0. The IV test was performed using an electrochemical workstation, and the current and voltage changes were recorded. Then, the target substance miR-223 and the interfering substances miR-21, miR-26a, miR-27a, and miR-122 were reacted and tested in the same conditions as the control group.

[0044] like Figure 8 As shown, when the target is present, the current of the electrochemical biosensor increases significantly, and when the interferent is present, the current of the electrochemical biosensor does not change significantly. As mentioned above, I0 and I are the currents in the nanopore before and after the response to the target to be measured (i.e., the current values ​​before and after incubation), and the current change rate at a voltage of -1 V (I-I0) / I0 (i.e., the change rate of the current before and after incubation) is calculated to evaluate the selective response performance of the electrochemical biosensor. Fig. 9As shown in the figure, the current change rate of the electrochemical biosensor at -1 V after reacting with miR-192 and miR-223 reached 1.11 and 1.10, which were significantly higher than those of other miRNAs. This result shows that due to the high sequence dependence of nucleic acid hybridization, the electrochemical sensor has good selectivity for the detection of targets.

[0045] 5. The present invention is based on an electrochemical biosensor, and uses the nanopore signal functionalized with its capture probe DNA as a template. By regulating the nanopore DNA hybridization reaction, it can achieve highly sensitive, rapid, and quantitative detection of biological molecules. Taking miR-192 as an example, the present invention has the following specific detection steps: 300 µL of a hybridization buffer, which is a tris buffer, and different concentrations of miR-192 standards are placed in deionized water to obtain 2 mL of incubation solution, wherein the hybridization solution is composed of 10 mM Tris, 1 mM EDTA·2Na, 50 mM NaCl, 1 mM KCl, pH 8.0.

[0046] The electrochemical biosensor was immersed in the incubation solution and reacted at 37°C for 30 minutes. After the reaction, the incubation solution was aspirated, rinsed with deionized water, dried with filter paper, and an electrolyte solution of 0.1 mol / L potassium chloride solution with a pH of 7.0 was added to perform IV testing. The results were as follows: Fig.10 As shown in the figure, as the concentration increases, the ion current also increases. The current change rate values ​​before and after incubation are calculated respectively, and a standard curve is drawn based on the concentration of the target and the current change rate values ​​( Fig.11 ), so that the concentration of the target substance in the sample to be tested can be calculated based on the standard curve.

[0047] The linear regression equation of the standard curve is (I-I0) / I0=2.64722+0.22835×log C (I0 and I are the current values ​​measured at -1 V before and after incubation, respectively), and the linear correlation coefficient R 2 =0.9989.

[0048] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. An electrochemical biosensor based on nanoparticle-modified multilayer porous membrane, characterized in that: A polymer membrane bombarded by heavy ions is used as a substrate, which is sensitized by ultraviolet light irradiation and then chemically etched to prepare nanopores of different shapes, thereby preparing a thin film containing multiple nanopores; nanoparticles are modified in the nanopores of the porous membrane, and then polyethyleneimine is modified on the surface of the pores by self-assembly, and then capture probes are modified on the surface of the nanopores to prepare a nanopore composite membrane functionalized with nanoparticle-modified capture probe DNA, and the active sites of the nanopores are blocked with bovine serum albumin; finally, multiple nanopore composite membranes functionalized with different capture probe DNAs are assembled together, thereby obtaining an electrochemical biosensor based on nanoparticle-modified multilayer porous membranes.

2. An electrochemical biosensor based on nanoparticle-modified multilayer porous membrane according to claim 1, characterized in that: After chemical etching to form a nanopore with negatively charged carboxyl groups on its surface, positively charged polyethyleneimine is self-assembled on the nanopore surface through electrostatic action; then the negatively charged capture probe DNA is further assembled in the positively charged pore, and the active sites of the nanopore containing nanoparticle-modified capture probe DNA functionalized nanopore are blocked with negatively charged bovine serum albumin; finally, multiple nanopore composite membranes functionalized with different capture probe DNAs are assembled together to obtain an electrochemical biosensor based on nanoparticle-modified multilayer porous membrane.

3. The electrochemical biosensor based on nanoparticle modified multilayer porous membrane according to claim 1, characterized in that: The polymer film is a polyethylene terephthalate (PET) film, a polycarbonate (PC) film or a polyimide (PI) film, and the shape of the nanopores is columnar, conical or irregular, with a thickness of 10-50 μm, a pore size of 0.1-5 μm on the surface, and a pore density of 1×10 3 ~1×10 15 Pieces / cm 2 .

4. The method for preparing an electrochemical biosensor based on nanoparticle-modified multilayer porous membrane according to claim 1, characterized in that: The specific steps of the electrochemical biosensor are as follows: (1) bombarding the polymer film with heavy ions accelerated by an accelerator, and then irradiating it with an ultraviolet lamp for 2 hours to sensitize it; (2) etching with an appropriate chemical etching solution to etch the tracks on the film into nanopores of different shapes, that is, to prepare a porous membrane; (3) soaking the porous membrane in an aqueous solution containing nanoparticles for 10 minutes, then taking out the porous membrane modified with nanoparticles, rinsing it with water, and drying it in the air to prepare a porous membrane modified with nanoparticles; (4) soaking the porous membrane modified with nanoparticles in an aqueous solution of polyethyleneimine (PEI) for 12 hours to self-assemble it into a multilayer porous membrane modified with nanoparticles, The membrane was washed with deionized water and dried in air; (5) the porous membrane modified with nanoparticles was immersed in the capture probe DNA solution and incubated at room temperature for 2 hours. After incubation, it was washed with deionized water to remove the unbound capture probe DNA to obtain the nanoparticle-modified capture probe DNA functionalized nanoporous composite membrane; (6) the nanoparticle-modified capture probe DNA functionalized nanoporous composite membrane was immersed in a bovine serum albumin (BSA) aqueous solution, incubated for 2 hours, washed with deionized water, and dried in air. Finally, multiple nanoporous composite membranes functionalized with different capture probe DNA were assembled together to prepare an electrochemical biosensor with nanoparticle-modified multilayer porous membrane.

5. The method for preparing an electrochemical biosensor based on nanoparticle-modified multilayer porous membrane according to claim 4, characterized in that: In the step (1), the polymer film is a polyethylene terephthalate (PET) film, a polycarbonate (PC) film or a polyimide (PI) film, and the wavelength of the ultraviolet light is 300-400 nm.

6. The method for preparing an electrochemical biosensor based on nanoparticle-modified multilayer porous membrane according to claim 4, characterized in that: In the step (2), the chemical etching solution is a sodium hydroxide solution with a concentration of 6 mol / L to 9 mol / L, and the shape of the nanopores is columnar, conical, or irregular.

7. The method for preparing an electrochemical biosensor based on nanoparticle-modified multilayer porous membrane according to claim 4, characterized in that: The nanoparticles in step (3) are gold nanoparticles with a diameter of 10 to 20 nm.

8. The method for preparing an electrochemical biosensor based on nanoparticle-modified multilayer porous membrane according to claim 4, characterized in that: In step (4), the concentration of the PEI aqueous solution is 1 mg / mL to 3 mg / mL, and the pH is 7.0 to 8.0; and the incubation condition is at room temperature.

9. The method for preparing an electrochemical biosensor based on nanoparticle-modified multilayer porous membrane according to claim 4, characterized in that: In step (5), the concentration of the capture probe DNA aqueous solution is 2 µmol / L~5 µmol / L, pH 7.0~8.0; the concentration of the BSA aqueous solution is 8 mg / mL~10 mg / mL, pH 7.0~8.

0.

10. The method for preparing an electrochemical biosensor based on nanoparticle-modified multilayer porous membrane according to claim 4, characterized in that: In step (6), the concentration of the BSA aqueous solution is 8 mg / mL to 10 mg / mL, and the pH is 7.0 to 8.

0.

11. An application of an electrochemical biosensor based on nanoparticle-modified multilayer porous membrane, characterized in that: The specific detection process is as follows: the electrochemical biosensor shown in claim 1 is placed in the middle of an H-type electrolytic cell, an electrolyte solution of the same concentration is added to the H-type electrolytic cell, a pair of Ag / AgCl electrodes are placed in two half electrolytic cells, an external load is connected to form a loop, and the IV current is tested to obtain the current value I0 before incubation; then the electrolyte solution in the electrolytic cell is sucked out, the composite membrane is washed with deionized water, the target standard sample and the hybridization buffer are mixed evenly and added to the electrolytic cell, incubated at room temperature for 1 hour, and 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 target substance is added to the standard sample, and the electrolyte solution is added to test the IV current of the target standard sample to obtain the current value I of the target standard sample after incubation, and then the process is repeated for multiple times to obtain the current values ​​I0 and I of the target standard samples with different concentrations before and after incubation. Finally, the current change rate (I-I0) / I0 of the target standard samples with different concentrations before and after incubation under the same conditions is calculated, and a standard curve is drawn with the logarithm of the target concentration as the horizontal axis and the current change rate as the vertical axis; then the above steps are followed to test to obtain the current values ​​I0 and I of the sample to be tested before and after incubation, and the concentration of the target in the sample to be tested is calculated according to the standard curve.

12. The use of an electrochemical biosensor based on nanoparticle-modified multilayer porous membrane according to claim 11, characterized in that: The electrolyte solution is a 0.1 mol / L~0.5 mol / L potassium chloride solution, the hybridization buffer is a tris buffer with a concentration of 0.1 µmol / L~0.5 µmol / L, the concentration of the target is 0.5 µmol / L~2 µmol / L, and the target is a nucleic acid or a protein.

Images