A method for preparing an electrochemical immunosensor electrode for silk fibroin detection

Abstract

This invention relates to the field of protein detection sensors, and discloses a method for preparing an electrochemical immunosensor electrode for silk fibroin detection. The invention involves obtaining a nitrogen-containing carbon material with a graphite layer structure after heat treatment of silk fibroin; directly synthesizing nano-cobalt tetroxide at the carbon material sites using cobalt nitrate hexahydrate; and combining the composite active material with nickel foam as an electrode in the immunosensor. The porous structure and internal permeability of the nickel foam not only facilitate the loading of more active materials and antibodies, but also promote sufficient contact between the active substances and the analytes, allowing for more effective transmission of the electrical signal to the substrate. The electrode material prepared by this method has high current density, high sensitivity, low cost, no pollution or danger, good stability, and a simple modification process, making it particularly suitable for detecting silk fibroin and silk artifacts.

Description

Technical Field

[0001] This invention relates to the field of protein detection sensors, and more particularly to a method for preparing an electrochemical immunosensor electrode for silk fibroin detection. Background Technology

[0002] The main component of silk artifacts is fibroin after the sericin has been removed. However, due to degradation and aging caused by time and external environment, the content of fibroin is low. As cultural relics are non-renewable cultural heritage, the amount of samples taken for testing is limited. Traditional Fourier transform infrared spectroscopy and Raman spectroscopy are easily affected by impurities, while X-ray diffraction technology requires multiple measurements to ensure experimental reliability and requires a large amount of samples per measurement. Electrochemical immunosensors can play a significant role in the detection of cultural relics.

[0003] Common materials for electrochemical immunosensors primarily utilize noble metal nanoparticles, such as gold nanoparticles (AuNps), to form gold-sulfur bonds and load antibodies. Electrodes include glassy carbon electrodes and gold electrodes. While these immunosensor systems are relatively mature, they suffer from limited material selection, high cost, and low current response in electrochemical testing. Research indicates that materials possessing nanomaterial properties, high biocompatibility, and antibody loading capabilities have the potential to become immunosensor materials. Our research aims to broaden our horizons by combining these findings with other areas of electrochemistry to select materials with these characteristics to fabricate a novel immunosensor system that surpasses traditional materials in certain aspects. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a method for preparing an electrode for an electrochemical immunosensor used in the detection of silk fibroin. This invention uses silk fibroin as a carbon source, and after a special heat treatment process, obtains a nitrogen-containing carbon material with high porosity, large specific surface area, and a complete pseudo-graphite layer structure. This invention directly synthesizes nano-cobalt tetroxide with a large specific surface area and small dimensionality at the sites of the carbon material using cobalt nitrate hexahydrate. This nano-cobalt tetroxide, when combined with the nitrogen-containing porous pseudo-graphite layer structure carbon material, serves as a composite active material, compensating for the shortcomings of cobalt tetroxide such as expansion decay, easy detachment, and low capacitance of the carbon material. This invention combines the composite active material with nickel foam as an electrode in an immunosensor. The porous structure and internal permeability of the nickel foam not only facilitate the loading of more active materials and antibodies but also promote sufficient contact between the active substances and the analytes, allowing for more effective transmission of the electrical signal to the substrate. The electrode material prepared by this method has high current density, high sensitivity, low cost, no pollution or danger, good stability, and a simple modification process, making it particularly suitable for detecting silk fibroin and silk artifacts.

[0005] The specific technical solution of this invention is: a method for preparing an electrochemical immunosensor electrode for silk fibroin detection, comprising the following steps:

[0006] Step 1: Clean and dry the natural silk, soak it in a saturated KCl solution for activation, take it out, wash and dry it, and then perform heat treatment graphitization. Specifically, starting from room temperature, heat the temperature in air at a rate of 5-8℃ / min to 280-320℃, hold it for 1.5-2.5h, introduce nitrogen gas and heat the temperature at a rate of 2-5℃ / min to 850-950℃, hold it for 1.5-2h, cool it, and crush the resulting product to obtain a nitrogen-containing porous pseudo-graphite layer structure carbon material.

[0007] The principle of step 1 of this invention is as follows: Selecting nitrogen-rich silk fibroin as the carbon source for preparing nitrogen-containing porous pseudo-graphite layer structure carbon materials. Further research in this invention has revealed that graphitization of silk fibroin under different high-temperature heat treatment processes yields carbon materials with different compositions and microstructures. Therefore, this invention has specifically designed a heat treatment graphitization process to obtain the desired nitrogen-containing porous pseudo-graphite layer structure carbon material.

[0008] Specifically: Under normal circumstances, silk fibroin requires 2800℃ to generate the highly developed graphite stacked structure desired in this invention, but this structure results in decreased crystallinity and increased defects. This invention discovers that pretreatment of silk fibroin with KCl solution, upon heating, etches carbon into a honeycomb structure, creating mesopores, increasing active sites and improving conductivity. Simultaneously, the pretreated silk fibroin exhibits a graphite-like structure earlier (around 900℃) and better crystallinity. Next, this invention first heats the material in air to around 300℃, causing mild dehydration, graphitization, and ashing of the biomass material. This removes some impurities while obtaining a semi-graphitized material with a rich porous structure. Then, nitrogen gas is introduced, and the temperature is further increased to around 900℃, during which the β-sheet structure gradually transforms into a conjugated sp structure around 350℃. 2 The hybrid carbon hexagonal structure ultimately yields a pseudo-graphite crystalline layer structure. Furthermore, this invention reveals that a slower heating rate at this stage can increase the material's crystallinity and reduce defects.

[0009] In summary, this invention can obtain a carbon material with high porosity, large specific surface area and complete pseudo-graphite layer structure by pretreating natural silk and controlling parameters such as heating rate, temperature and holding time in stages during heat treatment. The material also contains a certain amount of nitrogen elements that form CN bonds with carbon atoms, which can enhance the conductivity of the material.

[0010] Step 2: Mix hexadecyltrimethylammonium bromide (CTAB) with cobalt nitrate hexahydrate [Co(NO3)2·6H2O], add anhydrous ethanol and deionized water, and stir until dissolved.

[0011] Step 3: Mix the solution obtained in Step 2 with the nitrogen-containing porous pseudo-graphite layer structure carbon material obtained in Step 1 and carry out a hydrothermal reaction. Filter the resulting product with anhydrous ethanol and then vacuum dry it to obtain the composite active material.

[0012] CTAB, acting as a surfactant, enables the direct synthesis of cobalt tetroxide (CTO) from cobalt nitrate hexahydrate at sites on carbon materials in deionized water and anhydrous ethanol solutions. The hydrothermal reaction significantly improves the uniformity and crystallinity of the material, and different water bath temperatures affect the morphology of CTO. This invention prepares nano-cobalt tetroxide with a large specific surface area and small dimensions by controlling the temperature. Furthermore, its composite with nitrogen-containing porous pseudo-graphite layer structure carbon materials, used as a composite active material, can to some extent compensate for the shortcomings of CTO as an electrode material, such as expansion decay, easy detachment, and low capacitance of carbon materials.

[0013] Step 4: Mix the composite active material obtained in Step 3 with the conductive agent and binder, add organic solvent and grind, uniformly coat the resulting ground product onto the surface of the shredded nickel foam, and vacuum dry to obtain the composite electrode.

[0014] The binder is used to fix the composite active material onto the nickel foam to prevent or reduce detachment, and the conductive agent can further increase conductivity; biomass carbon material has double-layer capacitance characteristics, and cobalt tetroxide has pseudocapacitive characteristics, so the composite active material of the present invention can combine the advantages of both.

[0015] This invention combines composite active materials with nickel foam as electrodes for an immunosensor. The porous structure and internal permeability of nickel foam not only facilitate the loading of more composite active materials and antibodies, but also enable the active substances and the analytes to fully contact each other, allowing the electrical signals to be transmitted to the substrate more effectively.

[0016] Step 5: Take bovine serum albumin (BSA) and add it to PBS buffer to obtain a diluent, then mix it with the antibody to obtain a silk fibroin monoclonal antibody diluent.

[0017] Step 6: Drop the silk fibroin monoclonal antibody dilution solution onto the surface of the composite electrode, and after incubation, obtain the electrochemical immunosensor electrode for silk fibroin detection.

[0018] The carbon material obtained by high-temperature treatment of silk fibroin has carboxyl groups on its surface, which can form hydrogen bonds with the amino groups in the antibody to form a strong adsorption effect, thereby loading the antibody. The non-chemical bonds do not affect the conductivity of the material. At the same time, the surface carboxyl groups can be activated to combine with the amino groups to form chemical bonds to load the antibody more firmly.

[0019] As a preferred option, in step 1: anhydrous ethanol is used to clean the silk for 20-40 minutes, and the activation temperature is 50-70℃ for 4-8 hours.

[0020] Preferably, in steps 2 and 3, the ratio of hexadecyltrimethylammonium bromide, cobalt nitrate hexahydrate, anhydrous ethanol, deionized water, and nitrogen-containing porous pseudo-graphite layer structure carbon material is 0.02-0.03g:0.05-0.07g:25-35mL:4-6mL:0.1g; and the stirring time is 5-15min.

[0021] Preferably, in step 3, the hydrothermal reaction temperature is 170-190℃, the holding time is 80-100min, and the reaction is allowed to cool naturally after completion.

[0022] Preferably, in step 3, the vacuum drying temperature is 20-30℃.

[0023] Preferably, in step 3: the conductive agent is conductive carbon black, the binder is polyvinylidene fluoride, and the organic solvent is N-methylpyrrolidone.

[0024] Preferably, in step 4, the mass ratio of the composite active material to the conductive agent and polyvinylidene fluoride is 8:0.8-1.2:0.8-1.2.

[0025] Preferably, in step 4: the grinding time is not less than 30 minutes; the vacuum drying temperature is 70-90℃.

[0026] Preferably, in step 5: the weight ratio of bovine serum albumin to PBS buffer is 1:80-120; the concentration of silk fibroin monoclonal antibody diluent is 1.3-1.4 mg / mL; and the concentration of silk fibroin monoclonal antibody diluent is 1.3-1.4 mg / mL.

[0027] Preferably, in step 6, the incubation temperature is 35-40℃ and the time is 0.5-1.5h.

[0028] Compared with the prior art, the present invention has the following technical effects:

[0029] (1) The present invention uses silk fibroin as a carbon source. After a special heat treatment process, a carbon material with high porosity, large specific surface area and complete pseudo-graphite layer structure can be obtained. The material also contains nitrogen elements that form CN bonds with carbon atoms to enhance the conductivity of the material.

[0030] (2) This invention controls the temperature to directly synthesize cobalt nitrate hexahydrate into nano-cobalt tetroxide with large specific surface area and small dimension at the sites of carbon materials. Furthermore, when combined with nitrogen-containing porous pseudo-graphite layer structure carbon materials, it can be used as a composite active material to a certain extent to compensate for the shortcomings of cobalt tetroxide when used as an electrode material, such as expansion decay, easy shedding, and small capacity of carbon materials.

[0031] (3) In this invention, composite active materials are combined with nickel foam as electrodes in an immunosensor. The porous structure and internal permeability of nickel foam not only facilitate the loading of more active materials and antibodies, but also facilitate the full contact between the active substances and the analytes, so that the electrical signals can be transmitted to the substrate more effectively.

[0032] (4) The electrode prepared by this invention has a higher current than other biosensors at the same potential in cyclic voltammetry and differential volt pulse tests.

[0033] (5) The materials used in this invention have high biocompatibility, are green and environmentally friendly, and have a simple preparation process with no danger. Attached Figure Description

[0034] Figure 1 This is a SEM image of C / Co3O4 obtained in Example 1;

[0035] Figure 2 The Raman spectra of C and C / Co3O4 obtained in Example 1;

[0036] Figure 3 The cyclic voltammetry curves of Ni and the composite electrode in Example 2 at a scan rate of 50 mV / s are shown.

[0037] Figure 4 The image shows the differential voltage pulse test results in Example 2 for bare nickel foam, after loading materials, and after loading antibodies, within a voltage window of 0.1V-0.4V.

[0038] Figure 5 SEM image of the product prepared in Comparative Example 1;

[0039] Figure 6 The XRD pattern is shown for the material prepared by heat treatment at 300℃ in Comparative Example 2. Detailed Implementation

[0040] The present invention will be further described below with reference to embodiments.

[0041] Example 1

[0042] Step 1: Ultrasonically clean the natural silk and nickel foam with anhydrous ethanol for 30 minutes, then dry them in a vacuum drying oven. Set aside the nickel foam. Activate the precursor by soaking the silk in a saturated KCl solution in a 60°C constant temperature oven for 6 hours. Rinse the activated silk multiple times with deionized water and dry it. Place it in a porcelain boat and transfer it to a muffle furnace. Starting from room temperature (20°C), increase the temperature by 6°C per minute to 300°C and hold for 2 hours. Then, introduce nitrogen gas and raise the temperature to 900°C at a rate of 5°C / min, hold for 2 hours, and allow it to cool down with the furnace. Place the graphitized material from the porcelain boat into a mortar for grinding, weighing, and storage.

[0043] Step 2: Mix 6 mM (0.06 g) of cobalt nitrate hexahydrate [Co(NO3)2·6H2O] with 2 mM (0.0258 g) of hexadecyltrimethylammonium bromide (CTAB), add 30 ml of anhydrous ethanol, then add 5 ml of deionized water using a pipette, and stir in a water bath until the solid is completely dissolved.

[0044] Step 3: Transfer the solution from Step 2 into a hydrothermal reactor with a polytetrafluoroethylene liner and add 0.1g of the carbon material obtained in Step 1. Set the oven temperature to 180℃ and maintain the temperature for 90 minutes. After removing the reactor, allow it to cool naturally before opening the oven. Filter the resulting product using anhydrous ethanol and then transfer it to a vacuum drying oven. Close the vent, turn on the pump, and open the vacuum valve. Set the temperature to 25℃ and the drying time to 12 hours. The final composite active material is obtained.

[0045] The morphology of the composite active material prepared in Example 1 was characterized using scanning electron microscopy, such as... Figure 1 This study demonstrated that activation and graphitization increased the active binding sites and mesopores in carbon materials, resulting in the successful preparation and composite formation of nanoscale, sheet-like cobalt tetroxide with a large specific surface area on the surface of carbon materials. Raman spectroscopy was used to characterize the material's composition, such as... Figure 2 The comparison of characteristic absorption peaks proved the successful preparation of the composite active material.

[0046] Example 2

[0047] Step 1: Take 0.07g of bare nickel foam, 0.008g of the composite active material from Example 1, 0.001g of polyvinylidene fluoride (PVDF), and 0.001g of conductive carbon black. Add them to an agate mortar and mix them in small batches with N-methylpyrrolidone (NMP) to dissolve them. Continue for 30 minutes until the mixture becomes oily. Then, use a glass rod to coat the nickel foam multiple times until it is finished.

[0048] Step 2: Transfer the coated nickel foam from Step 1 into a vacuum drying oven, adjust the temperature to 80℃, and dry for 12 hours. Weigh 0.072g of the finished product, which is equivalent to 1.6mg of loaded active material.

[0049] Step 3: Take 0.2g potassium chloride, 0.27g potassium dihydrogen phosphate, 8g sodium chloride and 1.42g disodium hydrogen phosphate, mix them and add deionized water to prepare phosphate buffer (PBS) and adjust the pH of the solution to 7.4.

[0050] Step 4: Mix bovine serum albumin and PBS buffer at a weight ratio of 1:100 in a vortex mixer; then add silk fibroin monoclonal antibody until the concentration of silk fibroin monoclonal antibody is 1.36 mg / mL. Step 5: Take 10 μL of the monoclonal antibody dilution droplet on the surface of the composite electrode obtained in Step 2 and incubate it in a 37°C oven for 1 hour.

[0051] Electrochemical tests were performed on the electrode prepared in Example 2 to verify the self-assembly process. Figure 3 Cyclic voltammetry curves of bare nickel foam with a potential window of -0.2V to 0.6V and a scan rate of 50mV / s after loading the material demonstrate the successful loading and function of the material. Figure 4 The differential voltage pulse test was performed on bare nickel foam, after loading the material, and after loading the antibody in a voltage window of 0.1V-0.4V. The loading material enhanced the conductivity of the electrode. After introducing the antibody and BSA, the conductivity was reduced due to the influence on the pores and surface of the material. This proves that the antibody was successfully loaded onto the electrode and the assembly was successful.

[0052] Comparative Example 1 (The difference from Example 1 is the amount of cobalt nitrate hexahydrate used in step two).

[0053] Step 1: Ultrasonically clean the natural silk and nickel foam with anhydrous ethanol for 30 minutes, then dry them in a vacuum drying oven. Set aside the nickel foam. Activate the precursor by soaking the silk in a saturated KCl solution in a 60°C constant temperature oven for 6 hours. Rinse the activated silk multiple times with deionized water and dry it. Place it in a porcelain boat and transfer it to a muffle furnace. Starting from room temperature (20°C), increase the temperature by 6°C per minute to 300°C and hold for 2 hours. Then, introduce nitrogen gas and raise the temperature to 900°C at a rate of 5°C / min, hold for 2 hours, and allow it to cool down with the furnace. Place the graphitized material from the porcelain boat into a mortar for grinding, weighing, and storage.

[0054] Step 2: Mix 9 mM (0.09 g) of cobalt nitrate hexahydrate [Co(NO3)2·6H2O] with 2 mM (0.0258 g) of hexadecyltrimethylammonium bromide (CTAB), add 30 ml of anhydrous ethanol, then add 5 ml of deionized water using a pipette, and stir in a water bath until the solid is completely dissolved.

[0055] Step 3: Transfer the solution from Step 2 into a hydrothermal reactor with a polytetrafluoroethylene liner and add 0.1g of the carbon material obtained in Step 1. Set the oven temperature to 180℃ and keep it at that temperature for 90 minutes. After removing the reactor, allow it to cool naturally before opening it. Filter the obtained product using anhydrous ethanol and then transfer it into a vacuum drying oven. Close the vent, turn on the pump, and then open the vacuum valve. Set the temperature to 25℃ and the time to 12 hours.

[0056] The product was characterized by SEM to observe its morphology, such as... Figure 5 As shown, when only the concentration of cobalt nitrate hexahydrate [Co(NO3)2·6H2O] is changed, cobalt tetroxide still has a layered structure, but the comparative area of ​​the material and the increase in material diffusion are inferior to those in Example 1.

[0057] Comparative Example 2 (The difference from Example 1 lies in the graphitization process)

[0058] Step 1: Ultrasonically clean the natural silk and nickel foam with anhydrous ethanol for 30 minutes, then dry them in a vacuum drying oven. Set aside the nickel foam. Activate the silk by soaking it in a saturated KCl solution in a 60°C constant temperature oven for 6 hours. Rinse the activated silk multiple times with deionized water and dry it. Place it in a porcelain boat and transfer it to a muffle furnace. Starting from room temperature (20°C), increase the temperature by 6°C per minute to 300°C and hold for 2 hours. Let it cool down with the furnace. Place the heat-treated items in the porcelain boat into a mortar for grinding, weighing, and storage.

[0059] Step 2: Mix 6 mM (0.06 g) of cobalt nitrate hexahydrate [Co(NO3)2·6H2O] with 2 mM (0.0258 g) of hexadecyltrimethylammonium bromide (CTAB), add 30 ml of anhydrous ethanol, then add 5 ml of deionized water using a pipette, and stir in a water bath until the solid is completely dissolved.

[0060] Step 3: Transfer the solution from Step 2 into a hydrothermal reactor with a polytetrafluoroethylene liner and add 0.1g of the carbon material obtained in Step 1. Set the oven temperature to 180℃ and keep it at that temperature for 90 minutes. After removing the reactor, allow it to cool naturally before opening it. Filter the obtained product using anhydrous ethanol and then transfer it into a vacuum drying oven. Close the vent, turn on the pump, and then open the vacuum valve. Set the temperature to 25℃ and the time to 12 hours.

[0061] The crystallinity of the product was observed by XRD characterization, such as... Figure 6 As shown, when only the graphitization temperature and the holding temperature are changed to 300℃, the crystallinity of the material is not very good, the background is very high and there are impurity peaks, indicating that a graphitized structure with high crystallinity cannot be obtained at 300℃.

[0062] Comparative Example 3 (The difference from Example 2 lies in the different proportions of composite active material, binder, and conductive agent)

[0063] Step 1: Take 0.07g of bare nickel foam, 0.008g of the active material from Example 1, 0.002g of polyvinylidene fluoride (PVDF), and 0.001g of conductive carbon black. Add them to an agate mortar and mix them in small batches with N-methylpyrrolidone (NMP) to dissolve them. Continue for 30 minutes until the mixture becomes oily. Then, use a glass rod to coat the nickel foam multiple times until it is finished.

[0064] Step 2: Transfer the nickel foam coated in Step 1 into a vacuum drying oven, adjust the temperature to 80℃, and dry for 12 hours. Weigh 0.0714g of the finished product, which is approximately 1.1mg of loaded active material.

[0065] Step 3: Take 0.1g potassium chloride, 0.135g potassium dihydrogen phosphate, 4g sodium chloride and 0.72g disodium hydrogen phosphate, mix them and add deionized water to prepare phosphate buffer (PBS) and adjust the pH of the solution to 7.4.

[0066] Step 4: Mix bovine serum albumin and PBS buffer at a weight ratio of 1:100 in a vortex mixer; then add silk fibroin monoclonal antibody until the concentration of silk fibroin monoclonal antibody reaches 1.36 mg / mL.

[0067] Step 5: Take 10 μL of monoclonal antibody dilution droplet onto the electrode surface obtained in Step 2 and incubate it in a 37°C oven for 1 hour.

[0068] The results showed that a higher amount of binder would affect the conductivity and diffusion rate of the material.

[0069] Unless otherwise specified, the raw materials and equipment used in this invention are all commonly used in the field; unless otherwise specified, the methods used in this invention are all conventional methods in the field.

[0070] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, alterations, and equivalent transformations made to the above embodiments based on the technical essence of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A method for preparing an electrochemical immunosensor electrode for silk fibroin detection, characterized in that: Includes the following steps: Step 1: Clean and dry the natural silk, soak it in a saturated KCl solution for activation, take it out, wash and dry it, and then perform heat treatment graphitization. Specifically, starting from room temperature, heat the temperature in air at a rate of 5-8℃ / min to 280-320℃, hold it for 1.5-2.5h, introduce nitrogen gas and heat the temperature at a rate of 2-5℃ / min to 850-950℃, hold it for 1.5-2h, cool it, and crush the resulting product to obtain a nitrogen-containing porous pseudo-graphite layer structure carbon material. Step 2: Mix hexadecyltrimethylammonium bromide with cobalt nitrate hexahydrate, add anhydrous ethanol and deionized water, and stir until dissolved; Step 3: Mix the solution obtained in Step 2 with the nitrogen-containing porous pseudo-graphite layer structure carbon material obtained in Step 1 and carry out a hydrothermal reaction. Filter the resulting product with anhydrous ethanol and then vacuum dry it to obtain the composite active material. Step 4: Mix the composite active material obtained in Step 3 with the conductive agent and binder, add organic solvent and grind, uniformly coat the resulting ground product onto the surface of the shredded nickel foam, and vacuum dry to obtain the composite electrode; Step 5: Take bovine serum albumin and add it to PBS buffer at a ratio of 1:100 to obtain a dilution solution, then mix it with the antibody to obtain a silk fibroin monoclonal antibody dilution solution; Step 6: Drop the silk fibroin monoclonal antibody dilution solution onto the surface of the composite electrode, and after incubation, obtain the electrochemical immunosensor electrode for silk fibroin detection.

2. The preparation method according to claim 1, characterized in that: In step 1: Use anhydrous ethanol to clean the silk for 20-40 minutes, and activate it at a temperature of 50-70℃ for 4-8 hours.

3. The preparation method according to claim 1, characterized in that: In steps 2 and 3, the ratio of hexadecyltrimethylammonium bromide, cobalt nitrate hexahydrate, anhydrous ethanol, deionized water, and nitrogen-containing porous pseudo-graphite layer structure carbon material is 0.02-0.03g:0.05-0.07g:25-35mL:4-6mL:0.1g; the stirring time is 5-15min.

4. The preparation method according to claim 1, characterized in that: In step 3: the hydrothermal reaction temperature is 170-190℃, the holding time is 80-100min, and the reaction is allowed to cool naturally after completion.

5. The preparation method according to claim 1 or 4, characterized in that: In step 3: the vacuum drying temperature is 20-30℃.

6. The preparation method according to claim 1, characterized in that: In step 3: the conductive agent is conductive carbon black, the binder is polyvinylidene fluoride, and the organic solvent is N-methylpyrrolidone.

7. The preparation method according to claim 6, characterized in that: In step 4, the mass ratio of the composite active material to the conductive agent and polyvinylidene fluoride is 8:0.8-1.2:0.8-1.

2.

8. The preparation method according to claim 1, 6, or 7, characterized in that: In step 4: the grinding time shall not be less than 30 minutes; the vacuum drying temperature shall be 70-90℃.

9. The preparation method according to claim 1, 6, or 7, characterized in that: In step 5: the weight ratio of bovine serum albumin to PBS buffer is 1:80-120; the concentration of silk fibroin monoclonal antibody in the dilution solution is 1.3-1.4 mg / mL.

10. The preparation method according to claim 1, characterized in that: In step 6: the incubation temperature is 35-40℃, and the time is 0.5-1.5h.

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