An electrochemical biosensor based on nanoparticle-modified porous membrane loaded with metal film and its application

Abstract

The present invention provides an electrochemical biosensor based on a nanoparticle-modified porous membrane loaded with a metal film and its application, belonging to the field of electrochemical biosensors. The electrochemical biosensor of the nanoparticle-modified porous membrane loaded with a metal film is composed of a porous membrane modified with nanoparticles as a substrate, a metal film deposited on the porous membrane, and then the nanopores are successfully functionalized by using the covalent bond between the carboxyl functional group in the nanopore and the capture probe with an amino group, and then the active site is blocked with bovine serum albumin to obtain an electrochemical biosensor of the nanoparticle-modified porous membrane loaded with a metal film. The sensor has the advantages of low impedance, high measurement accuracy, high conductivity, simple preparation steps, and short detection time.

Description

Technical Field

[0001] The invention belongs to the field of electrochemical biosensors, and in particular relates to an electrochemical biosensor based on a nanoparticle-modified porous membrane loaded with a metal film and an application thereof. Background Art

[0002] Electrochemical biosensors belong to the emerging discipline of electrochemical bioelectronics, which is an interdisciplinary field involving biology, chemistry, materials, medicine, nanotechnology, microelectronics, information technology and other disciplines. Electrochemical biosensors use electrochemically active sensor elements - electrodes to analyze electroactive substances produced by biological components or biological receptors, and can directly convert the generated electroactive substances into electronic signals for detection. It has the characteristics of rapidity, sensitivity, and easy operation, and has been widely used in various aspects of inspection. However, conventional electrochemical biosensors have poor conductivity, large charge transfer resistance, and low detection accuracy. Therefore, it is necessary to design an electrochemical biosensor with strong conductivity, small charge transfer resistance, and high detection accuracy. Summary of the invention

[0003] The purpose of the present invention is to provide an electrochemical biosensor based on nanoparticle-modified porous membrane loaded with metal film and its application, which is a new technology for detection application by placing this composite film as a working electrode on an ITO glass electrode to realize a classic three-electrode system.

[0004] The technical scheme of the present invention is as follows: a method for preparing an electrochemical biosensor based on a nanoparticle-modified porous membrane loaded with a metal film comprises the following steps: (1) pretreatment of the porous membrane: first, on a linear accelerator, a polymer is irradiated with a single heavy ion to obtain a thin film with an ion track, and then it is sensitized by ultraviolet light irradiation; (2) preparation of the porous membrane: the polymer membrane is embedded between two chambers of a conductivity cell, one chamber is filled with an etching solution, and the other chamber is filled with a stop solution, and then a voltage is applied to the membrane, and the etching process stops at a desired current value corresponding to a certain tip diameter, thereby obtaining a thin film containing multiple nanopores; (3) preparation of a nanoparticle-modified porous membrane loaded with a metal film: first, the thin film containing multiple nanopores is immersed in a nanoparticle aqueous solution, and then rinsed and dried in air to obtain a nanoparticle-modified porous membrane; then the nanoparticle-modified porous membrane is placed on a working electrode, and a counting electrode and a reference electrode are placed together with the working electrode. In the electrolytic cell, the metal oxide is electrodeposited using an electrochemical workstation, and the porous membrane loaded with the metal film is then rinsed with deionized water and dried at room temperature; (4) Preparation of capture probe functionalized nanoporous composite membrane: The nanoparticle-modified porous membrane loaded with the metal film is immersed in an aqueous solution of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) for 2 hours, and then thoroughly rinsed with pure water. After rinsing, it is dried, and then the treated membrane is immersed in an amino capture probe solution overnight. Finally, the modified membrane is washed three times with distilled water and dried at room temperature; (5) Preparation of electrochemical biosensor of nanoparticle-modified porous membrane loaded with metal film: Finally, the capture probe functionalized composite membrane is immersed in an aqueous solution of bovine serum albumin (BSA), then rinsed with deionized water, and dried in air, thus preparing an electrochemical biosensor of nanoparticle-modified porous membrane loaded with metal film.

[0005] Furthermore, in step (1), the polymer film is a polyethylene terephthalate (PET) film, a polycarbonate (PC) film or a polyimide (PI) film, the shape of the nanopores is conical, the thickness is 10-50 µm, the pore size of the surface pores is 0.1-5 µm, and the pore density is 1×10 3 ~1×10 15 Pieces / cm 2 The wavelength of ultraviolet light is 300~400 nm, and the irradiation time is 2 hours.

[0006] Furthermore, in step (2), the chemical etching solution is a sodium hydroxide solution with a concentration of 6 mol / L to 9 mol / L, the blocking solution is 1 mol / L to 3 mol / L potassium chloride and 1 mol / L to 3 mol / L formic acid, the applied voltage is 1V, and the process is carried out at 30°C. The required current value corresponds to the tip diameter according to dtip =4LI / Πk(c)UD, where L is the hole length; I is the current; U is the applied voltage; d tip and D are the tip diameter and base diameter, respectively; k(c) is the specific conductivity of the electrolyte. For a 1 mol / L~3 mol / L potassium chloride solution at 25°C, k(c) is 0.11173 Ω -1 cm -1 .

[0007] Furthermore, in step (3), the nanoparticles are gold nanoparticles with a diameter of 10-20 nm, and the immersion time is 10 minutes. The working electrode is an ITO conductive glass substrate, the platinum wire is a counter electrode, Ag / AgCl is a reference electrode, the electrochemical workstation is CHI660E, the electrolyte solution is a 4 mol / L-6 mol / L metal oxide solution, and the deposition bath temperature is maintained at 30°C by a thermostat.

[0008] Furthermore, in step (4), the concentration of EDC is 10 mg / L-20 mg / L, the concentration of NHS is 3 mg / L-5 mg / L, the concentration of the probe DNA aqueous solution is 2 µM / L-5 µM / L, the pH is 7.0-8.0, the probe reaction time is 12 hours, and the reaction is carried out at room temperature.

[0009] Furthermore, in the step (5), the concentration of the BSA aqueous solution is 8 mg / mL to 10 mg / mL, the pH is 7.0 to 8.0, the immersion time is 1 hour, and the process is carried out at room temperature.

[0010] The present invention also relates to an electrochemical biosensor based on a nanoparticle-modified porous membrane loaded with a metal film and its application, characterized in that the specific detection process is as follows: (1) All electrochemical tests are carried out in a three-electrode cell, the electrochemical biosensor with a nanoparticle-modified porous membrane loaded with a metal film is placed on an ITO conductive glass to form a working electrode, the counter electrode and the reference electrode are placed together with the working electrode in an electrolytic cell, an electrolyte solution is added to the electrolytic cell, and the frequency is set to 10 -2 ~10 5Hz, AC amplitude of 3 mV~5 mV, and electrochemical impedance spectroscopy was performed using an electrochemical workstation; (2) the composite film modified with the probe was placed on ITO conductive glass to make a working electrode for electrochemical impedance spectroscopy to obtain the impedance response before incubation; (3) the target standard sample was added as an incubation solution into the cDNA-functionalized nanopore of the electrochemical biosensor, and after incubation at 35℃~40℃ for 100~120 minutes, the incubation solution was aspirated, washed with deionized water, and an electrolyte solution was added. The frequency and AC amplitude were the same as before, and an electrochemical impedance spectroscopy was performed; (4) the changes in electrochemical impedance before and after incubation were compared to analyze whether the target and the probe were bound.

[0011] Furthermore, in step 1, the platinum wire is the counter electrode, Ag / AgCl is the reference electrode, the electrochemical workstation is CHI660E, and the electrolyte solution is 1 mol / L~2 mol / L sodium sulfate.

[0012] Furthermore, in step 2, the composite film and the ITO conductive glass are fixed together using a working electrode clamp.

[0013] Furthermore, in step 3, the hybridization buffer is tris buffer with a concentration of 0.1 µmol / L to 0.5 µmol / L, the concentration of the target is 0.5 µmol / L to 2 µmol / L, the target is nucleic acid, protein, biological small molecule, etc., and all tests are performed at room temperature.

[0014] Furthermore, in step 4, the target object combines with the probe to generate impedance, thereby causing a change.

[0015] In addition, the detection principle of the present invention is as follows: a polymer membrane with nanopores is prepared by using ion track-etching technology, and ionized carboxyl functional groups are exposed on the surface of the unmodified polymer membrane nanopores, which can be activated by 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to form amine-reactive ester intermediates; then these active esters are further condensed with capture probes with amino groups by forming covalent bonds, thereby obtaining nanopores with modified capture probes, and finally negatively charged bovine serum albumin is used to block the active sites of the nanopores containing the modified capture probe functionalization, thereby obtaining an electrochemical biosensor based on nanoparticle-modified porous membrane loaded with metal film, and then the target object combines with the capture probe to block the nanopores, increase the electrochemical impedance, and further affect the transmembrane movement of ions in the nanopores, and the hybridization between the probe and the target is detected by performing electrochemical impedance testing on an electrochemical workstation.

[0016] Compared with the prior art, the beneficial effects of the present invention are at least as follows: the electrochemical biosensor of the nanoparticle-modified porous membrane loaded with a metal film provided by the present invention is suitable for detecting nucleic acids, proteins, biological molecules, etc. The present invention utilizes the nanoparticle-modified porous membrane loaded with a metal film as a working electrode; the porous membrane has a plurality of nanopores, which can provide a large number of active sites, promote the electron conversion on the electrode surface, and the nanoparticles further improve the conductivity of the pores. On this basis, loading the metal film can reduce the electrochemical impedance of the nanoparticle-modified porous membrane, thereby maximizing the conductivity; in addition, there are a large number of carboxyl functional groups on the inner wall of the nanopore, which are activated by an activator, and then the inner wall of the nanopore is modified with an amino capture probe, so as to achieve targeted detection of the target object; the present invention also utilizes a blocking agent to block the site where the probe specifically binds to the electrode surface, thereby further improving the specificity and sensitivity of the sensor. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Schematic diagram of the electrochemical biosensor detection device.

[0018] Figure 2 This is an electron microscope image of the porous membrane.

[0019] Figure 3 Schematic diagram of nanoparticle-modified porous membrane loaded with metal film.

[0020] Figure 4 Schematic diagram of the electrochemical sensing principle of the electrochemical biosensor based on nanoparticle-modified porous membrane loaded with metal film to detect the target.

[0021] Figure 5 This is the EIS diagram of the preparation process of the electrochemical biosensor based on nanoparticle-modified porous membrane loaded with metal film.

[0022] Figure 6 The EIS graphs are responses to different concentrations of the target.

[0023] Figure 7 It is a specific identification map.

[0024] Figure 8 This is a stability exploration diagram.

[0025] Fig. 9 Optimize the graph for the experimental conditions (time, pH, temperature). DETAILED DESCRIPTION

[0026] 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.

[0027] In the following examples, the formula of the DNA fixation buffer is: 10 mM Tris, 1 mM EDTA·2Na, 50 mM NaCl, 1 mM KCl, pH 8.00.

[0028] In the following examples, the nucleic acid sequences involved are shown in Table 1;

[0029] Example

[0030] 1. A preparation method of an electrochemical biosensor based on a nanoparticle-modified porous membrane loaded with a metal film, the specific steps of which are as follows: (1) bombarding a membrane material (a PET film with a thickness of about 15 μm) with heavy ions on a linear accelerator to obtain a membrane containing a heavy ion track, which is retained for later use; (2) irradiating the membrane containing a heavy ion track with an ultraviolet lamp having a wavelength of 360 nm for 2 hours to obtain a sensitized heavy ion track membrane; (3) embedding the sensitized heavy ion track membrane between two chambers of a conductivity cell, one chamber filled with an etching solution of 6 mol / L and the other chamber filled with a stop solution (1 mol / L potassium chloride and 1 mol / L formic acid), and then applying a voltage (1 V) to the membrane at 30°C, and the etching process stops at a desired current value corresponding to a certain tip diameter.

[0031] The above polymer film was examined by electron microscopy. Figure 2 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 µm to 5 µm. The shape of the nanopores was conical, and the pore density was 1×10 3 ~1×10 15 / cm 2 .

[0032] 2. (1) Immerse the porous membrane in a solution containing nanoparticles for 5 minutes, then rinse and air dry, place the porous membrane modified with nanoparticles on the working electrode, place the counter electrode and the reference electrode together with the working electrode in an electrolytic cell, and use an electrochemical workstation to perform electrodeposition on the metal oxide. After that, rinse the film in deionized water and dry it at room temperature to obtain a nanoparticle-modified porous membrane loaded with a metal film, as shown in the schematic diagram. Figure 3; (2) The nanoparticle-modified porous membrane loaded with metal film is immersed in an aqueous solution of 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) for 2 hours, and then rinsed thoroughly with pure water. After rinsing, it is dried, and then the treated membrane is immersed in a capture probe solution with amino groups overnight. Finally, the modified membrane is washed three times with distilled water and dried at room temperature; (3) The capture probe-functionalized composite membrane is immersed in an aqueous solution of bovine serum albumin (BSA), then rinsed with deionized water, and dried in air to prepare an electrochemical biosensor of a nanoparticle-modified porous membrane loaded with metal film, and finally a DNA probe-functionalized nanopore of a nanoparticle-modified porous membrane loaded with metal film containing closed active sites is obtained, which is characterized by EIS test. During the preparation process, the EIS test is as follows: Figure 5 As shown;

[0033] In the above steps, the working electrode is a metal film loaded with a porous membrane modified with nanoparticles fixed on an ITO conductive glass, the platinum wire is the counter electrode, the Ag / AgCl is the reference electrode, the electrochemical workstation is CHI660E, the electrolyte solution is a 4 mol / L metal oxide solution, and the deposition bath temperature is maintained at 30°C by a thermostat. The concentration of EDC is 15 mg / L, the concentration of NHSS is 3 mg / L, the concentration of the probe DNA aqueous solution is (2 µM / L, pH 7.0), the probe reaction time is 12 hours, and it is carried out at room temperature. The concentration of the BSA aqueous solution is (10 mg / mL, pH 7.0), the immersion time is 1 hour, and the electrolyte solution used for the EIS test is 4 mol / L potassium chloride solution at room temperature; after each modification step, the nanoporous membrane needs to be rinsed with deionized water, the filter paper is wiped dry, and then the EIS test is carried out.

[0034] The present invention is based on an electrochemical biosensor, and uses its cDNA functionalized nanopore signal as a template. By regulating the nanopore DNA hybridization reaction, it can achieve highly sensitive, rapid, and quantitative detection of biological molecules. Taking miRNA-21 as an example, the present invention has the following specific detection steps: 300 µL of a hybridization buffer, tris buffer, and miRNA-21 standards of different concentrations 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 8.00.

[0035] 3. Soak the electrochemical biosensor in the incubation solution and react at 37°C for 1 hour. After the reaction, aspirate the incubation solution, rinse with deionized water, dry with filter paper, add 1 mol / L sodium sulfate solution with a pH of 7.0 as the electrolyte solution, and perform EIS test. The results are as follows: Figure 6 As shown in the figure, as the concentration increases, the resistance also increases. The resistance change rate before and after incubation is calculated, and the standard curve is drawn based on the concentration of the target and the current change rate. Figure 6 ), so that the concentration of the target substance in the sample to be tested can be calculated based on the standard curve.

[0036] Among them, the linear regression equation of the standard curve (R 1 -R 0 ) / R 0 =37.035+2.276×log C(R 0 and R 1 are the resistance values ​​measured at 1 V before and after incubation, respectively, and the linear correlation coefficient R 2 =0.99764.

[0037] 4. Specificity experiment: Completely non-complementary DNA, single-base mismatch, three-base mismatch, fifteen-base mismatch and complementary DNA were used to verify the selectivity of the electrochemical biosensor; the composite membrane with modified probes was immersed in a tris hybridization buffer containing a concentration of 2 µmol / L of the target to be tested and a concentration of 0.1 µmol / L, and reacted at 37°C for 1 hour. After the reaction was completed, it was washed with deionized water and wiped dry. The incubated composite membrane was then fixed on the working electrode, and then an electrolyte solution with a concentration of 1 mol / L and a sodium sulfate solution with a pH of 7.0 was added. The EIS test was performed using an electrochemical workstation, and the resistance change value was recorded; then the target 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.

[0038] like Figure 7 As shown in Figure 2, when the target is present, the resistivity 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, R 0 and R 1 The resistance values ​​before and after the nanopore and the target object are respectively (i.e., the resistance values ​​before and after incubation), and the current change rate (R 1 -R 0 ) / R 0 (i.e., the rate of change of resistance before and after incubation) was calculated to evaluate the selective response performance of the electrochemical biosensor; Figure 7 As shown in the figure, the current change rate of the electrochemical biosensor at -1 V after reacting with miR-192 reached 3.09, which was significantly higher than that 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 the target.

[0039] 5. Stability experiment: After the electrochemical biosensor is hybridized with complementary DNA, the electrochemical biosensor is placed in a deionized water environment for EIS test to examine its resistance change rate. Then the electrochemical biosensor under the same conditions is tested five times in sequence to obtain the relationship between the resistance change rate and the number of detection cycles. Figure 8 As shown; the results show that the sensor has excellent stability.

[0040] 6. Optimize the experiment; place the electrochemical biosensor in a deionized water environment, adjust the pH value of the electrolyte solution to 5, 6, 7, 8, and 9, perform EIS test at the same time every day for five consecutive days, and make a bar graph of the relationship between the resistance change rate and pH at a voltage of 1 V ( Figure 9 ), 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 20, 40, 60, 80, and 100 minutes, respectively. EIS tests were performed at the same time every day for six consecutive days, and a bar graph of the relationship between the resistance change rate and time was made with the resistance change rate at 1 V voltage ( Figure 9 ), indicating that the electrochemical biosensor tends to be stable after 60 minutes of hybridization time, so 60 minutes of hybridization time is taken as the optimal experimental condition; the above electrochemical biosensor is placed in a deionized water environment, and the incubation temperature is adjusted to 25, 30, 35, 40, 50, and 45°C in sequence. EIS tests are performed at the same time every day for six consecutive days, and a bar graph of the relationship between the resistance change rate and temperature is made with the resistance change rate at 1 V voltage ( Figure 9 ), indicating that the electrochemical biosensor has the best performance when the hybridization temperature is 37°C.

[0041] 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 porous membrane loaded with metal film, characterized in that: A polymer membrane containing multiple nanopores is used as a substrate, nanoparticles are modified on the inner wall of the nanopores of the polymer membrane, and metal oxides are then deposited on the tip surface of the nanopores of the polymer membrane to prepare a nanoparticle-modified porous membrane loaded with a metal membrane, and then capture probe DNA is modified on the surface of the nanopores of the membrane, and finally the active sites are blocked to obtain an electrochemical biosensor based on a nanoparticle-modified porous membrane loaded with a metal membrane; The porous membrane has multiple nanopores, which can provide a large number of active sites and promote electronic conversion on the electrode surface. The nanoparticles further improve the conductivity of the pores. On this basis, loading the metal film can reduce the electrochemical impedance of the nanoparticle-modified porous membrane, thereby maximizing the conductivity. At the same time, the active sites are blocked by the blocking agent to further improve the specificity and sensitivity of the sensor.

2. The method for preparing an electrochemical biosensor based on a nanoparticle-modified porous membrane loaded with a metal film according to claim 1, characterized in that: The main steps are as follows: (1) on a linear accelerator, the polymer film is irradiated with heavy ions, and each side of the polymer film is exposed to ultraviolet light to sensitize it; (2) the polymer film is embedded between two chambers of a conductivity cell, one chamber is filled with an etching solution, and the other chamber is filled with a blocking solution, and then a voltage is applied to the film, and the etching process stops at a desired current value corresponding to a certain tip diameter; (3) the porous membrane is immersed in an aqueous solution containing nanoparticles, and then the porous membrane modified with nanoparticles is taken out, rinsed with water, and dried in air; (4) the porous membrane modified with nanoparticles is placed on the working electrode, and then the counting electrode and the reference electrode are placed in an electrolytic cell together with the working electrode, and the metal oxide is electrodeposited using an electrochemical workstation, and then the working electrode is used to perform electrochemical deposition. The porous membrane loaded with the metal film is rinsed in deionized water and dried at room temperature; (5) the metal film loaded with the nanoparticle-modified porous membrane is immersed in an aqueous solution of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) for 2 hours, and then rinsed thoroughly with pure water. After rinsing, it is dried, and then the treated membrane is immersed in a capture probe solution with an amino group overnight. Finally, the modified membrane is washed three times with distilled water and dried at room temperature; (6) Finally, the capture probe-functionalized composite membrane is immersed in an aqueous solution of bovine serum albumin (BSA), then rinsed with deionized water, and dried in air, thereby preparing an electrochemical biosensor of a nanoparticle-modified porous membrane loaded with a metal film.

3. The method for preparing an electrochemical biosensor based on nanoparticle-modified porous membrane loaded with metal film according to claim 2, 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, the shape of the nanopores is conical, the thickness is 10 to 50 μm, the pore size of the surface pores is 0.1 to 5 μm, and the pore density is 1×10 3 ~1×10 15 Pieces / cm 2 The wavelength of ultraviolet light is 300-400nm, and the irradiation time is 2 hours.

4. The method for preparing an electrochemical biosensor based on a nanoparticle-modified porous membrane loaded with a metal film according to claim 2, 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, the blocking solution is 1 mol / L to 3 mol / L potassium chloride and 1 mol / L to 3 mol / L formic acid, the applied voltage is 1 V, and the etching is carried out at 30° C. The required current value corresponds to the tip diameter according to d tip =4LI / Πk(c)UD, where L is the hole length; I is the current; U is the applied voltage; d tip and D are the tip diameter and base diameter respectively; k(c) is the specific conductivity of the electrolyte, and for a 1 mol / L potassium chloride solution at 25°C, k(c) is 0.11173Ω -1 cm -1 .

5. The method for preparing an electrochemical biosensor based on a nanoparticle-modified porous membrane loaded with a metal film according to claim 2, characterized in that: The nanoparticles in step (3) are gold nanoparticles with a diameter of 10 to 20 nm, and the immersion time is 10 minutes.

6. The method for preparing an electrochemical biosensor based on a nanoparticle-modified porous membrane loaded with a metal film according to claim 2, characterized in that: In the step (4), the working electrode is an ITO conductive glass substrate, the platinum wire is a counter electrode, Ag / AgCl is a reference electrode, the electrochemical workstation is CHI660E, the electrolyte solution is a 4 mol / L to 6 mol / L metal oxide solution, and the deposition bath temperature is maintained at 30° C. by a thermostat.

7. The method for preparing an electrochemical biosensor based on nanoparticle-modified porous membrane loaded with metal film according to claim 2, characterized in that: In the step (5), the concentration of EDC is 10 mg / L to 15 mg / L, the concentration of NHS is 3 mg / L to 5 mg / L, the concentration of the probe DNA aqueous solution is 2 μmol / L to 5 μmol / L, the pH is 7.0 to 8.0, and the probe reaction time is 12 hours at room temperature.

8. The method for preparing an electrochemical biosensor based on a nanoparticle-modified porous membrane loaded with a metal film according to claim 2, characterized in that: In the step (6), the concentration of the BSA aqueous solution is 8 mg / mL to 10 mg / mL, the pH is 7.0 to 8.0, the immersion time is 1 hour, and the process is carried out at room temperature.

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