Electrospun protein transfer film, method of making and use thereof

Abstract

The application discloses an electrospun protein transfer film and a preparation method and application thereof, and belongs to the technical field of membrane materials. The electrospun protein transfer film is randomly stacked with two fibers with different diameters. By adjusting the number ratio of the two fibers with different diameters to an appropriate range, the wettability of the protein transfer film can be improved while the adsorption and mechanical properties of the protein transfer film are taken into account, so that the contact angle θ1 of the protein transfer film and methanol when being in contact for 0.1 s and the contact angle θ2 of the protein transfer film and methanol when being in contact for 1 s are adjusted, the ratio of θ1 to θ2 is in an appropriate range, and the protein transfer film can meet the requirements of the Western blotting test on the adsorption, wettability and mechanical properties of the transfer film.

Description

Technical Field

[0001] This application relates to the field of membrane materials technology, specifically to an electrospun protein transfer membrane, its preparation method, and its application. Background Technology

[0002] Western blotting is a commonly used molecular biotechnology in cell biology for identifying proteins. It mainly utilizes the specific binding of antibodies to proteins to detect the presence of substances. It can detect small amounts of antigens, determine the molecular weight of antigens, and study the molecular structure and functional properties of antigens. Specifically, Western blotting includes the following main steps: (1) Extracting the target protein from cells or tissues using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE); (2) Transferring the separated protein onto a special membrane, a process also known as protein transfer; (3) Exposing the membrane to a primary antibody specific to the target protein, and then detecting it by adding a labeled secondary antibody. Among these steps, protein transfer is the core step of Western blotting, mainly using electroblotting technology to transfer the proteins separated by SDS-PAGE from the gel onto a protein transfer membrane. Wet transfer is a commonly used protein transfer method. The specific procedure is as follows: The transfer membrane, filter paper, and sponge pad are pre-soaked in transfer buffer; if the transfer membrane is PVDF, it also needs to be pre-activated with methanol. The transfer clamp is opened, and the sponge pad and filter paper are placed in sequence. The electrophoretically deposited gel is spread evenly on the filter paper, with the negative electrode of the gel facing the cathode plate of the transfer clamp. The transfer membrane, filter paper, and sponge pad are then stacked in sequence, and the transfer clamp is fastened, forming a structure consisting of a cathode plate, sponge pad, filter paper, gel, transfer membrane, filter paper, sponge pad, and anode plate. Then, pre-cooled transfer buffer at 4°C is added to the transfer tank, and the assembled transfer clamp is inserted into the electrotransfer tank. Electrotransfer is performed under constant current or constant voltage conditions, allowing the protein to transfer from the gel (negative electrode) to the transfer membrane (positive electrode). Because this step utilizes an electric field to drive protein migration to the membrane surface for fixation, the protein transfer efficiency is affected by the performance of the transfer membrane.

[0003] In protein detection techniques such as Western blotting, the wettability of protein transfer membranes (e.g., PVDF transfer membranes) is crucial, while also requiring a certain level of strength. However, most existing protein transfer membranes exhibit poor wettability, often resulting in uneven wetting within the membrane. This leads to insufficient protein adsorption in unwetted areas, reducing the membrane's capacity or causing non-uniform adsorption on the surface. To improve membrane wettability, some existing techniques modify the protein transfer membrane by introducing hydrophilic groups (such as -OH, -COOH, -NH2, etc.). However, the introduction of these hydrophilic groups reduces the membrane's adsorption capacity and may disrupt the crystalline structure of polymer chains, ultimately degrading the membrane's mechanical properties.

[0004] In view of this, it is indeed necessary to provide a technical solution to solve the above problems, so as to improve the wettability of the protein transfer membrane while taking into account the adsorption and mechanical properties of the protein transfer membrane. Summary of the Invention

[0005] Based on the deficiencies of existing technologies, the purpose of this application is to provide an electrospun protein transfer membrane, its preparation method, and its application. In this electrospun protein transfer membrane, two types of fibers with different diameters are randomly stacked. By adjusting the ratio of the number of these two types of fibers with different diameters, the wettability of the protein transfer membrane can be improved while taking into account both its adsorption and mechanical properties. This allows for the control of the contact angle θ1 between the protein transfer membrane and methanol at 0.1 s and the contact angle θ2 between the protein transfer membrane and methanol at 1 s, ensuring that the ratio of θ1 to θ2 is within a suitable range.

[0006] To achieve the above objectives, the technical solution adopted in this application is as follows:

[0007] In a first aspect of this application, an electrospun protein transfer membrane is provided, comprising a first fiber and a second fiber, wherein the first fiber and the second fiber are randomly stacked, the diameter of the first fiber is D1, the diameter of the second fiber is D2, and the average pore size (PMI) of the protein transfer membrane is R, wherein R, D1, and D2 satisfy: 0.1 μm ≤ R ≤ 1 μm, 1.5 ≤ D1 / R ≤ 5, and D2 / R < 1.5;

[0008] The ratio of the second fiber to the first fiber is A, where A is 2 to 6;

[0009] The contact angle between the protein transfer membrane and methanol is θ1 when they are in contact for 0.1 seconds, and the contact angle between the protein transfer membrane and methanol is θ2 when they are in contact for 1 second. θ1 and θ2 satisfy the condition: 0.2≤θ2 / θ1≤0.6.

[0010] In existing protein transfer membranes manufactured by electrospinning, a fiber structure with uniform diameter is typically used to improve the stability of the protein transfer membrane and ensure that the finished product has more uniform properties upon leaving the factory. However, it is difficult to balance the mechanical and wettability properties of protein transfer membranes with uniform fiber diameters. This is because, with a fixed pore size range, if the fiber diameter of the protein transfer membrane is uniform and too small, it often leads to a certain loss in tensile strength and flexibility. If the fiber diameter is uniform and large, although it can improve the strength to some extent, it will reduce its adsorption and wettability. In particular, as the fiber diameter increases, the specific surface area of ​​the membrane decreases, which in turn leads to a decrease in the wettability of the protein transfer membrane in liquids.

[0011] Compared with the prior art, the protein transfer membrane provided in this application uses two types of fibers with different diameters randomly stacked, allowing for a wider diameter distribution range. The coarser first fiber has a diameter 1.5 to 5 times the average pore size (PMI) of the protein transfer membrane, providing skeletal support and improving the mechanical properties of the membrane. The finer second fiber has a diameter-to-PMI ratio of less than 1.5, resulting in a larger specific surface area and a moderately increased surface roughness. This application utilizes the random stacking of these two types of fibers to form a membrane system that balances wettability, adsorption, and mechanical properties.

[0012] This application improves the wettability of a protein transfer membrane by adjusting the ratio of the number of fibers with different diameters, while taking into account both the adsorption and mechanical properties of the membrane. It controls the contact angle θ1 between the protein transfer membrane and methanol at 0.1 s and the contact angle θ2 between the protein transfer membrane and methanol at 1 s, ensuring that the ratio of θ1 to θ2 is within a suitable range. The initial contact angle θ1 of the protein transfer membrane is the contact angle of the droplet at 0.1 s, reflecting the instantaneous wetting resistance of the protein transfer membrane. The ratio of θ1 to θ2 reflects the ability of the droplet to spread rapidly or resist on the protein transfer membrane, and can serve as a key indicator of the wettability of the protein transfer membrane.

[0013] Preferably, the second fiber comprises a second medium fiber and a second fine fiber, wherein the diameter of the second medium fiber is ≥0.9R and <1.5R, and the diameter of the second fine fiber is <0.9R.

[0014] The inventors discovered that overlapping second fibers with a diameter ≥0.9R and <1.5R can form a better pore structure. Second fibers of this size have better structural stiffness and compressive strength, can share most of the mechanical load, and resist deformation caused by fluid pressure or operating stress, thereby protecting the more fragile second fine fibers and maintaining the stability of the pore shape. Second fine fibers with a diameter less than 0.9R can generate stronger capillary forces, giving the transfer membrane excellent wettability. The second fine fibers can fill the spaces between the thicker first and second fibers, increasing the specific surface area and porosity of the transfer membrane, thereby improving the protein adsorption performance of the transfer membrane.

[0015] More preferably, the ratio of the second medium fiber to the second fine fiber is B, where B is 1 to 2.

[0016] The inventors discovered that a relatively high number of second-type fibers (B > 2) leads to higher mechanical properties of the protein transfer membrane but lower overall wettability; a relatively high number of second-type fine fibers (B < 1) leads to lower porosity of the protein transfer membrane, increased fluid resistance, and decreased adsorption performance. By controlling the ratio of the number of second-type fibers to the number of second-type fine fibers within an appropriate range, both wettability and adsorption performance of the protein transfer membrane can be balanced.

[0017] Further preferred, A and R satisfy:

[0018] If 0.1μm≤R≤0.3μm, then 3.3≤A≤6.0;

[0019] If 0.3μm<R≤1μm, then 2.3≤A≤3.6.

[0020] The inventors discovered through research that for protein transfer membranes with small pore sizes (0.1μm≤R≤0.3μm), the proportion of coarse fibers in the transfer membrane can be appropriately increased, so that the ratio A of the number of first fibers to second fibers is in the range of 3.3 to 6.0. Membrane materials with small pore sizes require better strength and stability, and the overall average fiber diameter will be lower and have a denser structure. Therefore, introducing some coarse fibers can achieve better flow attraction and improve strength.

[0021] For protein transfer membranes with large pore sizes (0.3μm < R ≤ 1μm), the membrane material itself has stronger wettability and needs to maintain the stability of the membrane pores. At the same time, it has higher requirements for flexibility and requires a softer overall structure. The proportion of coarse fibers in the transfer membrane can be appropriately reduced, so that the ratio A of the number of first fibers to second fibers is in the range of 2.3 to 3.6. This can improve the stability of the membrane pores and make the membrane material more flexible, which can reduce the degree of deformation of the membrane material during the winding and packaging process.

[0022] Further preferred, B and R satisfy:

[0023] If 0.1μm≤R≤0.3μm, then 1.3≤B≤1.65;

[0024] If 0.3μm<R≤1μm, then 1.0≤B≤1.4.

[0025] The inventors discovered through research that for protein transfer membranes with small pore sizes (0.1μm≤R≤0.3μm), adjusting the ratio B of the number of second medium fibers to the number of second fine fibers to the range of 1.3 to 1.65 can effectively improve the overall porosity of the transfer membrane, thereby enhancing its wettability and its protein adsorption performance.

[0026] For protein transfer membranes with large pore sizes (0.3 μm < R ≤ 1 μm), adjusting the ratio B of the number of second medium fibers to the number of second fine fibers to the range of 1.0 to 1.4 can effectively ensure the overall mechanical properties of the transfer membrane by making the protein transfer membrane have a higher content of second medium fibers and a lower content of second fine fibers.

[0027] Preferably, the air permeability of the protein transfer membrane meets the following requirements:

[0028] If 0.1 μm ≤ R ≤ 0.3 μm, the air permeability of the protein transfer membrane is 50–70 s·in. 2 / 100mL;

[0029] If 0.3 μm < R ≤ 1 μm, the air permeability of the protein transfer membrane is 10–20 s·in. 2 / 100mL.

[0030] In this application, the air permeability of the protein transfer membrane refers to the time required for 100 mL of air to pass through a 1 square inch protein transfer membrane.

[0031] The inventors discovered through research that controlling the air permeability of the transfer membrane within a suitable range can reflect the porosity of the protein transfer membrane.

[0032] For protein transfer membranes with small pore sizes (0.1 μm ≤ R ≤ 0.3 μm), the permeability of the protein transfer membrane is adjusted to 50–70 s·in. 2 / 100mL, small pore size combined with large air permeability can effectively improve the adsorption capacity of transfer membrane for proteins.

[0033] For protein transfer membranes with large pore sizes (0.3 μm < R ≤ 1 μm), the permeability of the protein transfer membrane is adjusted to 10–20 s·in. 2 / 100mL, the large pore size combined with the low air permeability can effectively ensure the adsorption capacity of the transfer membrane for proteins.

[0034] Preferably, 12°≤θ1≤24°; θ2≤10°.

[0035] The protein transfer membrane has an adsorption capacity of 30–60 μg / cm³ for BSA protein. 2 .

[0036] In this application, the contact angle θ1 between the transfer membrane and methanol at 0.1 s is in the range of 12–24°, the contact angle θ2 ≤ 10°, and the adsorption capacity of the transfer membrane for BSA protein is 30–60 μg / cm³. 2 This demonstrates that the transfer film provided in this application achieves a balance between wettability, adsorption, and mechanical properties.

[0037] Preferably, the protein transfer film comprises several fibrous portions fused together to form a fibrous aggregate, and the area containing the fibrous aggregate in any square millimeter region of the surface of the protein transfer film is a, where a≤10%.

[0038] Through research, the inventors discovered that by controlling the number of fibers of different thicknesses, and further utilizing a certain amount of high-boiling-point solvent, several fiber parts can be fused together to form a fiber-to-fiber adhesive. The fiber-to-fiber adhesive structure can fix and strengthen the fine fibers, thereby improving the tensile strength of the protein transfer membrane.

[0039] In a second aspect of this application, the method for preparing the above-mentioned PVDF electrospun protein transfer film includes the following steps:

[0040] S1. Prepare a spinning solution, wherein the spinning solution comprises polyvinylidene fluoride, a soluble inorganic salt, and a solvent, wherein the mass fraction of polyvinylidene fluoride in the spinning solution is 7.5-20%, and the concentration of the soluble inorganic salt in the spinning solution is 1-5 mM, wherein the soluble inorganic salt comprises one of sodium chloride, potassium chloride, and magnesium chloride.

[0041] S2. Electrospinning the spinning solution obtained in step S1 using an electrospinning device to form a primary film.

[0042] The spinneret of the electrospinning device has a spinneret hole A and a spinneret hole B, wherein the diameter of the spinneret hole A is 0.5 to 0.8 times the diameter of the spinneret hole B.

[0043] The conditions for electrospinning are as follows: the spinning voltage is 35-70kV, the ambient humidity is 30-70%, the temperature of the fiber receiving area of ​​the negative electrode receiving device is 35-45℃, the temperature of the spinning solution is 5-10℃, and the spinneret rate is 0.02-0.3g / (hole·min).

[0044] S3. The initial film is hot-pressed to obtain a PVDF electrospun protein transfer film.

[0045] This application introduces an appropriate amount of soluble inorganic salt into the spinning solution to disturb the system during electrospinning, inducing the fusion of some fine fibers. This fusion also improves the gloss of the fiber surface. However, excessive inorganic salt addition can lead to two problems: firstly, it can cause the membrane material to harden (due to excessive crystallinity) and increase the hydrophilicity of the membrane surface, resulting in reduced protein adsorption; secondly, it can cause fiber disturbance during spinning, leading to excessive coarse fibers and decreased porosity. Insufficient inorganic salt addition, on the other hand, can result in too few coarse fibers.

[0046] In the electrospinning process, by controlling the ambient humidity and temperature, the temperature of the spinning solution, and the ratio of the diameters of the two spinnerets, the fiber bundles obtained in some spinnerets can be merged, thereby producing the first and second fibers with size deviations.

[0047] Preferably, in step S1, the molecular weight of PVDF in the spinning solution is 50-100K.

[0048] Preferably, the solvent in the spinning solution comprises a miscible low-boiling-point solvent and a high-boiling-point solvent. The low-boiling-point solvent has a boiling point not greater than 100°C and includes at least one of alcohols, ethers, and cyclic ethers. The high-boiling-point solvent has a boiling point not less than 150°C and includes at least one of dimethyl sulfoxide, N,N-dimethylformamide, N-methylpyrrolidone, and toluene. Based on the sum of the volumes of the high-boiling-point solvent and the low-boiling-point solvent, the volume percentage of the high-boiling-point solvent is 0.5% to 3%.

[0049] In a third aspect of this application, this application provides the application of the PVDF electrospun protein transfer membrane as provided in the first aspect in immunoblotting assays.

[0050] Compared with the prior art, the beneficial effects of this application are as follows:

[0051] This application provides an electrospun protein transfer membrane, its preparation method, and its application. In this electrospun protein transfer membrane, two types of fibers with different diameters are randomly stacked. By adjusting the ratio of the number of these two types of fibers with different diameters, the wettability of the protein transfer membrane can be improved while taking into account both its adsorption and mechanical properties. This allows for the control of the contact angle θ1 between the protein transfer membrane and methanol at 0.1 s and the contact angle θ2 between the protein transfer membrane and methanol at 1 s, ensuring that the ratio of θ1 to θ2 is within a suitable range. Consequently, the protein transfer membrane can meet the requirements of immunoblotting assays for the adsorption, wettability, and mechanical properties of the transfer membrane. Attached Figure Description

[0052] To more clearly illustrate the technical solutions in the specific embodiments of this application or the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0053] Figure 1 This is a scanning electron microscope (SEM) schematic diagram of the surface of the protein transfer membrane obtained in Example 1 of this application, and the magnification of the figure is 5000×.

[0054] Figure 2 This is a scanning electron microscope (SEM) schematic diagram of the surface of the protein transfer membrane obtained in Example 2 of this application, and the magnification of the figure is 5000×.

[0055] Figure 3 This is a colorimetric image of the EGRF protein after being transferred using the protein transfer membrane in Example 1. Detailed Implementation

[0056] To better illustrate the purpose, technical solution, and advantages of this application, the following description, in conjunction with specific embodiments and comparative examples, aims to provide a detailed understanding of the content of this application, rather than to limit its scope. All other embodiments obtained by those skilled in the art without inventive effort are within the protection scope of this application.

[0057] Furthermore, the use of terms such as "first," "second," etc., in this application is for descriptive purposes only and should not be construed as indicating or implying their relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of embodiments in this application, unless otherwise stated, "multiple" means two or more. Additionally, the use of "based on" or "according to" implies openness and inclusiveness, because processes, steps, calculations, or other actions "based on" or "according to" one or more of the stated conditions or values ​​may in practice be based on additional conditions or beyond the stated values.

[0058] In this application, the average pore size R of the protein transfer membrane can be measured by a PMI pore size analyzer. Of course, those skilled in the art can also obtain the above parameters by other measurement methods. The above measurement methods are for reference only.

[0059] In this application, the contact angle between the protein transfer film and methanol at 0.1 s and the contact angle between the protein transfer film and methanol at 1 s can be measured using a contact angle tester.

[0060] In this application, the ratio of the number of second fibers to the number of first fibers, the ratio of the number of second medium fibers to the number of second fine fibers, and the area ratio of fiber adhesions in any square millimeter region on the surface of the protein transfer membrane can all be measured and calculated by using computer software (such as Matlab, NIS-Elements, etc.) or manually after the membrane structure has been characterized by scanning electron microscopy.

[0061] In actual measurement, the film surface can be characterized first using an electron microscope to obtain the corresponding SEM image. A certain area can be selected, such as 1 mm². 2 The area size is determined by the actual situation and is calculated as 1mm x 1mm or 100μm x 100μm. Then, the diameter of all fibers in the area is measured using appropriate computer software or manually. The number of first fibers (with a diameter of 1.5R to 5R), second fibers (with a diameter less than 1.5R), second medium fibers (with a diameter ≥ 0.9R and < 1.5R), and second fine fibers (with a diameter < 0.9R) in the area are counted. The ratio of the number of second fibers to first fibers and the ratio of the number of second medium fibers to second fine fibers in the area are then calculated. It is preferable to select at least 3 non-adjacent areas for testing and calculate the average value.

[0062] In actual measurement, the film surface can be characterized first using an electron microscope to obtain the corresponding SEM image. A certain area can be selected, such as 1 mm². 2 (1mm x 1mm) or 100μm x 100μm, the specific area size depends on the actual situation. Then, use the corresponding computer software or manually to measure the area of ​​all fibrous adhesions on this area, and then calculate to obtain the area ratio of fibrous adhesions on the surface of the protein transfer film in this square millimeter area. It is preferable to select at least 3 non-adjacent areas for testing and calculate the average value to obtain the area ratio of fibrous adhesions in any square millimeter area on the surface of the protein transfer film.

[0063] In this application, the air permeability of the protein transfer membrane refers to the time required for 100 mL of air to pass through a 1 square inch protein transfer membrane under a gas pressure of 7 kPa. The test method is as follows: [The text abruptly shifts to a different topic] ...with an effective air permeability area of ​​1 square inch (i.e., 1 in... 2The membrane sample was attached to the surface of a stainless steel fixture with an opening (1.0 mm in diameter). The other end of the stainless steel fixture was connected to a pressure sensor, a gas flow sensor, a gas pressure regulating valve, and a gas source. The gas pressure regulating valve was adjusted so that the pressure sensor reading reached 7 kPa. Timing was started and the reading on the gas flow sensor was read. When the permeation volume reached 100 mL, the gas source was turned off and timing was started again. This yielded the time required for 100 mL of air to pass through a 1 square inch protein transfer membrane under a gas pressure of 7 kPa (i.e., the permeation volume of the protein transfer membrane).

[0064] The average thickness of the protein transfer membrane can be obtained by characterizing the cross-section of the protein transfer membrane using a scanning electron microscope, selecting several measurement points (at least three) evenly on the cross-sectional SEM image of the protein transfer membrane, measuring the radial thickness of the above measurement points along the radial direction of the protein transfer membrane, and then using computer software (such as Matlab, NIS-Elements, etc.) or manual measurement to obtain several measurement values, and taking the average value to obtain the average thickness of the protein transfer membrane.

[0065] In this application, the adsorption capacity of the protein transfer membrane for BSA protein can be obtained by the following method:

[0066] 1. Sample preparation: Take several 1.5ml centrifuge tubes and label them with the membrane type to be tested;

[0067] Cut three 5mm diameter circular membrane pieces from each type of membrane using a die-cutting tool. The membranes must be flat and without any gaps. (Different cutting techniques can be used depending on the shape of the membrane pieces.) Place them into the designated centrifuge tubes.

[0068] The shaker is set to preheat at 27°C.

[0069] 2. Preparation of BSA solution: Weigh the white solid particles of BSA (bovine albumin) and prepare 1 mg / ml of BSA solution. More can be prepared. After preparation, vortex and mix well.

[0070] 3. BSA adsorption: Centrifuge to mix the BSA solution, add 500 μL BSA solution to each centrifuge tube using a pipette, and vortex to mix; place the centrifuge tube holder in a shaker (27°C) for 1 hour, and then preheat the water bath to 37°C.

[0071] 4. Prepare BCA solution: When the shaking time is about to be set, prepare BCA solution (prepare fresh for use), leaving some margin: the volume ratio of solution A to solution B is 50:1. Add solution A first, then add solution B, and vortex to mix.

[0072] 5. BCA adsorption:

[0073] Use several 1.5ml centrifuge tubes and label them with the membrane type;

[0074] Remove the membrane from the centrifuge tube with clean tweezers, wipe it dry with a lint-free cloth, and then transfer it to a new centrifuge tube;

[0075] Vortex the BCA solution, then add 70 μL of PBS solution (1X PBS) + 700 μL of BCA solution to each centrifuge tube using a pipette and vortex to mix.

[0076] Heat in a water bath (37°C) for 0.5 hours;

[0077] After removing it, let it sit at room temperature for 1 hour.

[0078] 6. Test absorbance:

[0079] (1) Ultraviolet spectrophotometer: set wavelength 562 mm, automatic optical path; blank control: PBS solution (1X·PBS);

[0080] (2) Adding test solution to the colorimetric blood: Vortex the solution in the centrifuge tube, and use a pipette (set to 1000 μL) to transfer the solution from the centrifuge tube into the colorimetric blood; wipe the surface of the colorimetric blood, and place it in the instrument for absorbance measurement (A). 562 (Value), record data;

[0081] (3) Pay attention to the cleanliness of the colorimetric blood during the test. After the test, rinse with ultrapure water. Clean the colorimetric blood with anhydrous ethanol every 3 tests. Replace the pipette tip for transferring the solution every time the test is performed.

[0082] (4) Organize the data, output the results, and calculate the adsorption capacity of BSA protein.

[0083] 7. Cleaning: Clean the blood sample with anhydrous ethanol and put it back in its original position; clean the pipette tip and other debris.

[0084] In the above test steps, pay attention to vortexing the solution to ensure a complete reaction;

[0085] The BSA adsorption capacity [μg / cm³] is calculated using the following formula. 2 = (Absorbance [A] - 0.1152A) / 0.0025 [A / μg·mL⁻¹)] × PBS solution volume [μL] / membrane area [cm²] 2 ].

[0086] It should be noted that those skilled in the art can also obtain the above parameters through other measurement methods, and the above measurement methods are for reference only.

[0087] Example 1

[0088] This embodiment provides an electrospun protein transfer film, the preparation method of which includes the following steps:

[0089] S1. A low-boiling-point solvent and a high-boiling-point solvent are mixed in a volume ratio of 99:1 to form a mixed solvent. The low-boiling-point solvent is ethanol, and the high-boiling-point solvent is DMF. A soluble inorganic salt is dissolved in the mixed solvent, and the molar volume ratio of the soluble inorganic salt to the mixed solvent is 1 mM. The soluble inorganic salt is sodium chloride. Polyvinylidene fluoride is dissolved in the solvent containing the soluble inorganic salt to prepare a spinning solution. The mass percentage of polyvinylidene fluoride in the spinning solution is 10%, and the weight-average molecular weight of the polyvinylidene fluoride is 50K.

[0090] S2. Electrospinning the spinning solution obtained in step S1 using an electrospinning device to form a primary film.

[0091] The spinneret of the electrospinning device has a spinneret hole A and a spinneret hole B, wherein the diameter of spinneret hole A is 0.3 mm and the diameter of spinneret hole B is 0.5 mm. The diameter of spinneret hole A is 0.6 times the diameter of spinneret hole B.

[0092] The conditions for electrospinning are as follows: the spinning voltage is 45kV, the ambient temperature is 20±2℃, the ambient humidity is 50±10%, the temperature of the fiber receiving area of ​​the negative electrode receiving device is 35~45℃, the temperature of the spinning solution is 5℃, the spinneret rate is 0.05g / (hole·min), and the receiving distance is 12cm.

[0093] S3. The initial film is hot-pressed using a hot press roller at a temperature of 110°C and a pressure of 0.3 MPa, with the roller passing through at a speed of 0.2 m / min, to obtain a PVDF electrospun protein transfer film.

[0094] Example 2

[0095] This embodiment provides an electrospun protein transfer film, the preparation method of which includes the following steps:

[0096] S1. A low-boiling-point solvent and a high-boiling-point solvent are mixed in a volume ratio of 99:1 to form a mixed solvent. The low-boiling-point solvent is ethanol, and the high-boiling-point solvent is DMF. A soluble inorganic salt is dissolved in the mixed solvent, and the molar volume ratio of the soluble inorganic salt to the mixed solvent is 1 mM. The soluble inorganic salt is sodium chloride. Polyvinylidene fluoride is dissolved in the solvent containing the soluble inorganic salt to prepare a spinning solution. The mass percentage of polyvinylidene fluoride in the spinning solution is 9%, and the weight-average molecular weight of the polyvinylidene fluoride is 50K.

[0097] S2. Electrospinning the spinning solution obtained in step S1 using an electrospinning device to form a primary film.

[0098] The spinneret of the electrospinning device has a spinneret hole A and a spinneret hole B, wherein the diameter of spinneret hole A is 0.3 mm and the diameter of spinneret hole B is 0.6 mm, and the diameter of spinneret hole A is 0.5 times the diameter of spinneret hole B.

[0099] The conditions for electrospinning are as follows: the spinning voltage is 55kV, the ambient temperature is 20±2℃, the ambient humidity is 50±10%, the temperature of the fiber receiving area of ​​the negative electrode receiving device is 35~45℃, the temperature of the spinning solution is 5℃, the spinneret rate is 0.05g / (hole·min), and the receiving distance is 12cm.

[0100] S3. The initial film is hot-pressed using a hot press roller at a temperature of 110°C and a pressure of 0.3 MPa, with the roller passing through at a speed of 0.2 m / min, to obtain a PVDF electrospun protein transfer film.

[0101] Example 3

[0102] This embodiment provides an electrospun protein transfer film, the preparation method of which includes the following steps:

[0103] S1. A low-boiling-point solvent and a high-boiling-point solvent are mixed in a volume ratio of 99:1 to form a mixed solvent. The low-boiling-point solvent is ethanol, and the high-boiling-point solvent is DMF. A soluble inorganic salt is dissolved in the mixed solvent, and the molar volume ratio of the soluble inorganic salt to the mixed solvent is 1 mM. The soluble inorganic salt is sodium chloride. Polyvinylidene fluoride is dissolved in the solvent containing the soluble inorganic salt to prepare a spinning solution. The mass percentage of polyvinylidene fluoride in the spinning solution is 7.5%, and the weight-average molecular weight of the polyvinylidene fluoride is 50K.

[0104] S2. Electrospinning the spinning solution obtained in step S1 using an electrospinning device to form a primary film.

[0105] The spinneret of the electrospinning device has a spinneret hole A and a spinneret hole B, wherein the diameter of spinneret hole A is 0.4 mm and the diameter of spinneret hole B is 0.5 mm. The diameter of spinneret hole A is 0.8 times the diameter of spinneret hole B.

[0106] The conditions for electrospinning are as follows: the spinning voltage is 55kV, the ambient temperature is 20±2℃, the ambient humidity is 50±10%, the temperature of the fiber receiving area of ​​the negative electrode receiving device is 35~45℃, the temperature of the spinning solution is 5℃, the spinneret rate is 0.05g / (hole·min), and the receiving distance is 15cm.

[0107] S3. The initial film is hot-pressed using a hot press roller at a temperature of 110°C and a pressure of 0.3 MPa, with the roller passing through at a speed of 0.2 m / min, to obtain a PVDF electrospun protein transfer film.

[0108] Example 4

[0109] This embodiment provides an electrospun protein transfer film, the preparation method of which includes the following steps:

[0110] S1. A low-boiling-point solvent and a high-boiling-point solvent are mixed in a volume ratio of 99:1 to form a mixed solvent. The low-boiling-point solvent is ethanol, and the high-boiling-point solvent is DMF. A soluble inorganic salt is dissolved in the mixed solvent, and the molar volume ratio of the soluble inorganic salt to the mixed solvent is 2.5 mM. The soluble inorganic salt is sodium chloride. Polyvinylidene fluoride is dissolved in the solvent containing the soluble inorganic salt to prepare a spinning solution. The mass percentage of polyvinylidene fluoride in the spinning solution is 12%, and the weight-average molecular weight of the polyvinylidene fluoride is 50K.

[0111] S2. Electrospinning the spinning solution obtained in step S1 using an electrospinning device to form a primary film.

[0112] The spinneret of the electrospinning device has a spinneret hole A and a spinneret hole B, wherein the diameter of spinneret hole A is 0.3 mm and the diameter of spinneret hole B is 0.5 mm. The diameter of spinneret hole A is 0.6 times the diameter of spinneret hole B.

[0113] The conditions for electrospinning are as follows: the spinning voltage is 45kV, the ambient temperature is 20±2℃, the ambient humidity is 50±10%, the temperature of the fiber receiving area of ​​the negative electrode receiving device is 35~45℃, the temperature of the spinning solution is 5℃, the spinneret rate is 0.05g / (hole·min), and the receiving distance is 12cm.

[0114] S3. The initial film is hot-pressed using a hot press roller at a temperature of 110°C and a pressure of 0.3 MPa, with the roller passing through at a speed of 0.2 m / min, to obtain a PVDF electrospun protein transfer film.

[0115] Example 5

[0116] This embodiment provides an electrospun protein transfer film, the preparation method of which includes the following steps:

[0117] S1. A low-boiling-point solvent and a high-boiling-point solvent are mixed in a volume ratio of 99:1 to form a mixed solvent. The low-boiling-point solvent is ethanol, and the high-boiling-point solvent is DMF. A soluble inorganic salt is dissolved in the mixed solvent, and the molar volume ratio of the soluble inorganic salt to the mixed solvent is 5 mM. The soluble inorganic salt is sodium chloride. Polyvinylidene fluoride is dissolved in the solvent containing the soluble inorganic salt to prepare a spinning solution. The mass percentage of polyvinylidene fluoride in the spinning solution is 15%, and the weight-average molecular weight of the polyvinylidene fluoride is 50K.

[0118] S2. Electrospinning the spinning solution obtained in step S1 using an electrospinning device to form a primary film.

[0119] The spinneret of the electrospinning device has a spinneret hole A and a spinneret hole B, wherein the diameter of spinneret hole A is 0.4 mm and the diameter of spinneret hole B is 0.5 mm. The diameter of spinneret hole A is 0.8 times the diameter of spinneret hole B.

[0120] The conditions for electrospinning are as follows: the spinning voltage is 45kV, the ambient temperature is 20±2℃, the ambient humidity is 50±10%, the temperature of the fiber receiving area of ​​the negative electrode receiving device is 35~45℃, the temperature of the spinning solution is 5℃, the spinneret rate is 0.08g / (hole·min), and the receiving distance is 12cm.

[0121] S3. The initial film is hot-pressed using a hot press roller at a temperature of 110°C and a pressure of 0.3 MPa, with the roller passing through at a speed of 0.2 m / min, to obtain a PVDF electrospun protein transfer film.

[0122] Comparative Example 1

[0123] This comparative example provides an electrospun protein transfer film, the preparation method of which includes the following steps:

[0124] S1. A low-boiling-point solvent and a high-boiling-point solvent are mixed in a volume ratio of 99:1 to form a mixed solvent. The low-boiling-point solvent is ethanol, and the high-boiling-point solvent is DMF. A soluble inorganic salt is dissolved in the mixed solvent, and the molar volume ratio of the soluble inorganic salt to the mixed solvent is 1 mM. The soluble inorganic salt is sodium chloride. Polyvinylidene fluoride is dissolved in the solvent containing the soluble inorganic salt to prepare a spinning solution. The mass percentage of polyvinylidene fluoride in the spinning solution is 10%, and the weight-average molecular weight of the polyvinylidene fluoride is 50K.

[0125] S2. Electrospinning the spinning solution obtained in step S1 using an electrospinning device to form a primary film.

[0126] The spinneret of the electrospinning device has a spinneret hole A and a spinneret hole B, wherein the diameter of spinneret hole A is 0.2 mm and the diameter of spinneret hole B is 0.6 mm. The diameter of spinneret hole A is 0.33 times the diameter of spinneret hole B.

[0127] The conditions for electrospinning are as follows: the spinning voltage is 55kV, the ambient temperature is 20±2℃, the ambient humidity is 50±10%, the temperature of the fiber receiving area of ​​the negative electrode receiving device is 35~45℃, the temperature of the spinning solution is 5℃, the spinneret rate is 0.05g / (hole·min), and the receiving distance is 12cm.

[0128] S3. The initial film is hot-pressed using a hot press roller at a temperature of 110°C and a pressure of 0.3 MPa, with the roller passing through at a speed of 0.2 m / min, to obtain a PVDF electrospun protein transfer film.

[0129] Comparative Example 2

[0130] This comparative example provides an electrospun protein transfer film, the preparation method of which includes the following steps:

[0131] S1. A low-boiling-point solvent and a high-boiling-point solvent are mixed in a volume ratio of 99:1 to form a mixed solvent. The low-boiling-point solvent is ethanol, and the high-boiling-point solvent is DMF. A soluble inorganic salt is dissolved in the mixed solvent, and the molar volume ratio of the soluble inorganic salt to the mixed solvent is 1 mM. The soluble inorganic salt is sodium chloride. Polyvinylidene fluoride is dissolved in the solvent containing the soluble inorganic salt to prepare a spinning solution. The mass percentage of polyvinylidene fluoride in the spinning solution is 10%, and the weight-average molecular weight of the polyvinylidene fluoride is 50K.

[0132] S2. Electrospinning the spinning solution obtained in step S1 using an electrospinning device to form a primary film.

[0133] The spinneret of the electrospinning device has a spinneret hole A and a spinneret hole B, wherein the diameter of spinneret hole A is 0.45 mm and the diameter of spinneret hole B is 0.5 mm. The diameter of spinneret hole A is 0.9 times the diameter of spinneret hole B.

[0134] The conditions for electrospinning are as follows: the spinning voltage is 45kV, the ambient temperature is 20±2℃, the ambient humidity is 50±10%, the temperature of the fiber receiving area of ​​the negative electrode receiving device is 35~45℃, the temperature of the spinning solution is 5℃, the spinneret rate is 0.05g / (hole·min), and the receiving distance is 12cm.

[0135] S3. The initial film is hot-pressed using a hot press roller at a temperature of 110°C and a pressure of 0.3 MPa, with the roller passing through at a speed of 0.2 m / min, to obtain a PVDF electrospun protein transfer film.

[0136] Comparative Example 3

[0137] The difference between this comparative example and Example 1 is that in step S2 of this comparative example, the spinneret of the electrospinning device has a single-diameter spinneret orifice with a diameter of 0.5 mm.

[0138] Comparative Example 4

[0139] This comparative example provides an electrospun protein transfer film, the preparation method of which includes the following steps:

[0140] S1. A low-boiling-point solvent and a high-boiling-point solvent are mixed in a volume ratio of 99:1 to form a mixed solvent. The low-boiling-point solvent is ethanol, and the high-boiling-point solvent is DMF. A soluble inorganic salt is dissolved in the mixed solvent, and the molar volume ratio of the soluble inorganic salt to the mixed solvent is 1 mM. The soluble inorganic salt is sodium chloride. Polyvinylidene fluoride is dissolved in the solvent containing the soluble inorganic salt to prepare a spinning solution. The mass percentage of polyvinylidene fluoride in the spinning solution is 17.5%, and the weight-average molecular weight of the polyvinylidene fluoride is 75K.

[0141] S2. Electrospinning the spinning solution obtained in step S1 using an electrospinning device to form a primary film.

[0142] The spinneret of the electrospinning device has a single-diameter spinneret orifice with a diameter of 0.3 mm.

[0143] The conditions for electrospinning are as follows: the spinning voltage is 45kV, the ambient temperature is 20±2℃, the ambient humidity is 50±10%, the temperature of the fiber receiving area of ​​the negative electrode receiving device is 35~45℃, the temperature of the spinning solution is 5℃, the spinneret rate is 0.1g / (hole·min), and the receiving distance is 12cm.

[0144] S3. The initial film is hot-pressed using a hot press roller at a temperature of 110°C and a pressure of 0.3 MPa, with the roller passing through at a speed of 0.2 m / min, to obtain a PVDF electrospun protein transfer film.

[0145] Comparative Example 5

[0146] The difference between this comparative example and Example 1 is that the temperature of the spinning solution in step S2 of this comparative example is 20°C.

[0147] Comparative Example 6

[0148] The difference between this comparative example and Example 1 is that, in step S1 of this comparative example, the mass percentage of polyvinylidene fluoride in the spinning solution is 8%, and the molar volume ratio of soluble inorganic salt to mixed solvent is 0.5 mM.

[0149] Comparative Example 7

[0150] This comparative example provides an electrospun protein transfer film, the preparation method of which includes the following steps:

[0151] S1. A low-boiling-point solvent and a high-boiling-point solvent are mixed in a volume ratio of 99:1 to form a mixed solvent. The low-boiling-point solvent is ethanol, and the high-boiling-point solvent is DMF. A soluble inorganic salt is dissolved in the mixed solvent, and the molar volume ratio of the soluble inorganic salt to the mixed solvent is 10 mM. The soluble inorganic salt is sodium chloride. Polyvinylidene fluoride is dissolved in the solvent containing the soluble inorganic salt to prepare a spinning solution. The mass percentage of polyvinylidene fluoride in the spinning solution is 20%, and the weight-average molecular weight of the polyvinylidene fluoride is 75K.

[0152] S2. Electrospinning the spinning solution obtained in step S1 using an electrospinning device to form a primary film.

[0153] The spinneret of the electrospinning device has a spinneret hole A and a spinneret hole B, wherein the diameter of spinneret hole A is 0.4 mm and the diameter of spinneret hole B is 0.5 mm. The diameter of spinneret hole A is 0.8 times the diameter of spinneret hole B.

[0154] The conditions for electrospinning are as follows: the spinning voltage is 45kV, the ambient temperature is 20±2℃, the ambient humidity is 50±10%, the temperature of the fiber receiving area of ​​the negative electrode receiving device is 35~45℃, the temperature of the spinning solution is 5℃, the spinneret rate is 0.08g / (hole·min), and the receiving distance is 9cm.

[0155] S3. The initial film is hot-pressed using a hot press roller at a temperature of 110°C and a pressure of 0.3 MPa, with the roller passing through at a speed of 0.2 m / min, to obtain a PVDF electrospun protein transfer film.

[0156] Comparative Example 8

[0157] This comparative example provides an electrospun protein transfer film, the preparation method of which includes the following steps:

[0158] S1. A low-boiling-point solvent and a high-boiling-point solvent are mixed in a volume ratio of 99:1 to form a mixed solvent. The low-boiling-point solvent is ethanol and the high-boiling-point solvent is DMF. Polyvinylidene fluoride is dissolved in the mixed solvent to prepare a spinning solution. The mass percentage of polyvinylidene fluoride in the spinning solution is 8%, and the weight-average molecular weight of polyvinylidene fluoride is 50K.

[0159] S2. Electrospinning the spinning solution obtained in step S1 using an electrospinning device to form a primary film.

[0160] The spinneret of the electrospinning device has a spinneret hole A and a spinneret hole B, wherein the diameter of spinneret hole A is 0.25 mm and the diameter of spinneret hole B is 0.4 mm. The diameter of spinneret hole A is 0.63 times the diameter of spinneret hole B.

[0161] The conditions for electrospinning are as follows: the spinning voltage is 55kV, the ambient temperature is 20±2℃, the ambient humidity is 50±10%, the temperature of the fiber receiving area of ​​the negative electrode receiving device is 35~45℃, the temperature of the spinning solution is 5℃, the spinneret rate is 0.05g / (hole·min), and the receiving distance is 12cm.

[0162] S3. The initial film is hot-pressed using a hot press roller at a temperature of 110°C and a pressure of 0.3 MPa, with the roller passing through at a speed of 0.2 m / min, to obtain a PVDF electrospun protein transfer film.

[0163] Comparative Example 9

[0164] This comparative example provides an electrospun protein transfer film, the preparation method of which includes the following steps:

[0165] S1. A soluble inorganic salt is dissolved in ethanol, the molar volume ratio of the soluble inorganic salt to the ethanol is 1 mM, the soluble inorganic salt is sodium chloride, and polyvinylidene fluoride is dissolved in a solvent containing the soluble inorganic salt to prepare a spinning solution. The mass percentage of polyvinylidene fluoride in the spinning solution is 9%, and the weight average molecular weight of the polyvinylidene fluoride is 50K.

[0166] S2. Electrospinning the spinning solution obtained in step S1 using an electrospinning device to form a primary film.

[0167] The spinneret of the electrospinning device has a spinneret hole A and a spinneret hole B, wherein the diameter of spinneret hole A is 0.4 mm and the diameter of spinneret hole B is 0.5 mm. The diameter of spinneret hole A is 0.8 times the diameter of spinneret hole B.

[0168] The conditions for electrospinning are as follows: the spinning voltage is 55kV, the ambient temperature is 20±2℃, the ambient humidity is 50±10%, the temperature of the fiber receiving area of ​​the negative electrode receiving device is 35~45℃, the temperature of the spinning solution is 5℃, the spinneret rate is 0.05g / (hole·min), and the receiving distance is 15cm.

[0169] S3. The initial film is hot-pressed using a hot press roller at a temperature of 110°C and a pressure of 0.3 MPa, with the roller passing through at a speed of 0.2 m / min, to obtain a PVDF electrospun protein transfer film.

[0170] Example 6

[0171] This embodiment provides an electrospun protein transfer film, the preparation method of which includes the following steps:

[0172] S1. A low-boiling-point solvent and a high-boiling-point solvent are mixed in a volume ratio of 99:1 to form a mixed solvent. The low-boiling-point solvent is ethanol, and the high-boiling-point solvent is DMF. A soluble inorganic salt is dissolved in the mixed solvent. The molar volume ratio of the soluble inorganic salt to the mixed solvent is 1 mM. The soluble inorganic salt is sodium chloride. Polyvinylidene fluoride is dissolved in the solvent containing the soluble inorganic salt to prepare a spinning solution. The mass percentage of polyvinylidene fluoride in the spinning solution is 15%, and the weight-average molecular weight of the polyvinylidene fluoride is 100K.

[0173] S2. Electrospinning the spinning solution obtained in step S1 using an electrospinning device to form a primary film.

[0174] The spinneret of the electrospinning device has a spinneret hole A and a spinneret hole B, wherein the diameter of spinneret hole A is 0.4 mm and the diameter of spinneret hole B is 0.55 mm. The diameter of spinneret hole A is 0.73 times the diameter of spinneret hole B.

[0175] The conditions for electrospinning are as follows: the spinning voltage is 45kV, the ambient temperature is 20±2℃, the ambient humidity is 50±10%, the temperature of the fiber receiving area of ​​the negative electrode receiving device is 35~45℃, the temperature of the spinning solution is 5℃, the spinneret rate is 0.05g / (hole·min), and the receiving distance is 15cm.

[0176] S3. The initial film is hot-pressed using a hot press roller at a temperature of 110°C and a pressure of 0.3 MPa, with the roller passing through at a speed of 0.2 m / min, to obtain a PVDF electrospun protein transfer film.

[0177] Example 7

[0178] This embodiment provides an electrospun protein transfer film, the preparation method of which includes the following steps:

[0179] S1. A low-boiling-point solvent and a high-boiling-point solvent are mixed in a volume ratio of 99:1 to form a mixed solvent. The low-boiling-point solvent is ethanol, and the high-boiling-point solvent is DMF. A soluble inorganic salt is dissolved in the mixed solvent, and the molar volume ratio of the soluble inorganic salt to the mixed solvent is 1 mM. The soluble inorganic salt is sodium chloride. Polyvinylidene fluoride is dissolved in the solvent containing the soluble inorganic salt to prepare a spinning solution. The mass percentage of polyvinylidene fluoride in the spinning solution is 17%, and the weight-average molecular weight of the polyvinylidene fluoride is 100K.

[0180] S2. Electrospinning the spinning solution obtained in step S1 using an electrospinning device to form a primary film.

[0181] The spinneret of the electrospinning device has a spinneret hole A and a spinneret hole B, wherein the diameter of spinneret hole A is 0.4 mm and the diameter of spinneret hole B is 0.5 mm. The diameter of spinneret hole A is 0.8 times the diameter of spinneret hole B.

[0182] The conditions for electrospinning are as follows: the spinning voltage is 45kV, the ambient temperature is 20±2℃, the ambient humidity is 50±10%, the temperature of the fiber receiving area of ​​the negative electrode receiving device is 35~45℃, the temperature of the spinning solution is 5℃, the spinneret rate is 0.05g / (hole·min), and the receiving distance is 12cm.

[0183] S3. The initial film is hot-pressed using a hot press roller at a temperature of 110°C and a pressure of 0.3 MPa, with the roller passing through at a speed of 0.2 m / min, to obtain a PVDF electrospun protein transfer film.

[0184] Example 8

[0185] This embodiment provides an electrospun protein transfer film, the preparation method of which includes the following steps:

[0186] S1. A low-boiling-point solvent and a high-boiling-point solvent are mixed in a volume ratio of 99:1 to form a mixed solvent. The low-boiling-point solvent is ethanol, and the high-boiling-point solvent is DMF. A soluble inorganic salt is dissolved in the mixed solvent. The molar volume ratio of the soluble inorganic salt to the mixed solvent is 1 mM. The soluble inorganic salt is sodium chloride. Polyvinylidene fluoride is dissolved in the solvent containing the soluble inorganic salt to prepare a spinning solution. The mass percentage of polyvinylidene fluoride in the spinning solution is 15%, and the weight-average molecular weight of the polyvinylidene fluoride is 100K.

[0187] S2. Electrospinning the spinning solution obtained in step S1 using an electrospinning device to form a primary film.

[0188] The spinneret of the electrospinning device has a spinneret hole A and a spinneret hole B, wherein the diameter of spinneret hole A is 0.3 mm and the diameter of spinneret hole B is 0.6 mm, and the diameter of spinneret hole A is 0.5 times the diameter of spinneret hole B.

[0189] The conditions for electrospinning are as follows: the spinning voltage is 45kV, the ambient temperature is 20±2℃, the ambient humidity is 50±10%, the temperature of the fiber receiving area of ​​the negative electrode receiving device is 35~45℃, the temperature of the spinning solution is 5℃, the spinneret rate is 0.05g / (hole·min), and the receiving distance is 12cm.

[0190] S3. The initial film is hot-pressed using a hot press roller at a temperature of 110°C and a pressure of 0.3 MPa, with the roller passing through at a speed of 0.2 m / min, to obtain a PVDF electrospun protein transfer film.

[0191] Example 9

[0192] This embodiment provides an electrospun protein transfer film, the preparation method of which includes the following steps:

[0193] S1. A low-boiling-point solvent and a high-boiling-point solvent are mixed in a volume ratio of 99:1 to form a mixed solvent. The low-boiling-point solvent is ethanol, and the high-boiling-point solvent is DMF. A soluble inorganic salt is dissolved in the mixed solvent. The molar volume ratio of the soluble inorganic salt to the mixed solvent is 1 mM. The soluble inorganic salt is sodium chloride. Polyvinylidene fluoride is dissolved in the solvent containing the soluble inorganic salt to prepare a spinning solution. The mass percentage of polyvinylidene fluoride in the spinning solution is 18%, and the weight-average molecular weight of the polyvinylidene fluoride is 100K.

[0194] S2. Electrospinning the spinning solution obtained in step S1 using an electrospinning device to form a primary film.

[0195] The spinneret of the electrospinning device has a spinneret hole A and a spinneret hole B, wherein the diameter of spinneret hole A is 0.4 mm and the diameter of spinneret hole B is 0.6 mm, and the diameter of spinneret hole A is 0.5 times the diameter of spinneret hole B.

[0196] The conditions for electrospinning are as follows: the spinning voltage is 60kV, the ambient temperature is 20±2℃, the ambient humidity is 50±10%, the temperature of the fiber receiving area of ​​the negative electrode receiving device is 35~45℃, the temperature of the spinning solution is 5℃, the spinneret rate is 0.05g / (hole·min), and the receiving distance is 20cm.

[0197] S3. The initial film is hot-pressed using a hot press roller at a temperature of 110°C and a pressure of 0.3 MPa, with the roller passing through at a speed of 0.2 m / min, to obtain a PVDF electrospun protein transfer film.

[0198] Comparative Example 10

[0199] This comparative example provides an electrospun protein transfer film, the preparation method of which includes the following steps:

[0200] S1. A low-boiling-point solvent and a high-boiling-point solvent are mixed in a volume ratio of 99:1 to form a mixed solvent. The low-boiling-point solvent is ethanol, and the high-boiling-point solvent is DMF. A soluble inorganic salt is dissolved in the mixed solvent. The molar volume ratio of the soluble inorganic salt to the mixed solvent is 1 mM. The soluble inorganic salt is sodium chloride. Polyvinylidene fluoride is dissolved in the solvent containing the soluble inorganic salt to prepare a spinning solution. The mass percentage of polyvinylidene fluoride in the spinning solution is 15%, and the weight-average molecular weight of the polyvinylidene fluoride is 100K.

[0201] S2. Electrospinning the spinning solution obtained in step S1 using an electrospinning device to form a primary film.

[0202] The spinneret of the electrospinning device has a spinneret hole A and a spinneret hole B, wherein the diameter of spinneret hole A is 0.3 mm and the diameter of spinneret hole B is 0.7 mm. The diameter of spinneret hole A is 0.43 times the diameter of spinneret hole B.

[0203] The conditions for electrospinning are as follows: the spinning voltage is 45kV, the ambient temperature is 20±2℃, the ambient humidity is 50±10%, the temperature of the fiber receiving area of ​​the negative electrode receiving device is 35~45℃, the temperature of the spinning solution is 5℃, the spinneret rate is 0.05g / (hole·min), and the receiving distance is 15cm.

[0204] S3. The initial film is hot-pressed using a hot-pressing roller at a temperature of 110°C and a pressure of 0.3 MPa.

[0205] A PVDF electrospun protein transfer film was obtained by passing the hot press roller at a speed of 0.2 m / min.

[0206] Comparative Example 11

[0207] This comparative example provides an electrospun protein transfer film, the preparation method of which includes the following steps:

[0208] S1. A low-boiling-point solvent and a high-boiling-point solvent are mixed in a volume ratio of 99:1 to form a mixed solvent. The low-boiling-point solvent is ethanol, and the high-boiling-point solvent is DMF. A soluble inorganic salt is dissolved in the mixed solvent. The molar volume ratio of the soluble inorganic salt to the mixed solvent is 1 mM. The soluble inorganic salt is sodium chloride. Polyvinylidene fluoride is dissolved in the solvent containing the soluble inorganic salt to prepare a spinning solution. The mass percentage of polyvinylidene fluoride in the spinning solution is 10%, and the weight-average molecular weight of the polyvinylidene fluoride is 100K.

[0209] S2. Electrospinning the spinning solution obtained in step S1 using an electrospinning device to form a primary film.

[0210] The spinneret of the electrospinning device has a spinneret hole A and a spinneret hole B, wherein the diameter of spinneret hole A is 0.5 mm and the diameter of spinneret hole B is 0.55 mm. The diameter of spinneret hole A is 0.91 times the diameter of spinneret hole B.

[0211] The conditions for electrospinning are as follows: the spinning voltage is 45kV, the ambient temperature is 20±2℃, the ambient humidity is 50±10%, the temperature of the fiber receiving area of ​​the negative electrode receiving device is 35~45℃, the temperature of the spinning solution is 5℃, the spinneret rate is 0.05g / (hole·min), and the receiving distance is 20cm.

[0212] S3. The initial film is hot-pressed using a hot-pressing roller at a temperature of 110°C and a pressure of 0.3 MPa.

[0213] A PVDF electrospun protein transfer film was obtained by passing the hot press roller at a speed of 0.2 m / min.

[0214] Comparative Example 12

[0215] This comparative example provides an electrospun protein transfer film, the preparation method of which includes the following steps:

[0216] S1. A low-boiling-point solvent and a high-boiling-point solvent are mixed in a volume ratio of 99:1 to form a mixed solvent. The low-boiling-point solvent is ethanol, and the high-boiling-point solvent is DMF. A soluble inorganic salt is dissolved in the mixed solvent, and the molar volume ratio of the soluble inorganic salt to the mixed solvent is 1 mM. The soluble inorganic salt is sodium chloride. Polyvinylidene fluoride is dissolved in the solvent containing the soluble inorganic salt to prepare a spinning solution. The mass percentage of polyvinylidene fluoride in the spinning solution is 12.5%, and the weight-average molecular weight of the polyvinylidene fluoride is 100K.

[0217] S2. Electrospinning the spinning solution obtained in step S1 using an electrospinning device to form a primary film.

[0218] The spinneret of the electrospinning device has a single-diameter spinneret orifice with a diameter of 0.5 mm.

[0219] The conditions for electrospinning are as follows: the spinning voltage is 45kV, the ambient temperature is 20±2℃, the ambient humidity is 50±10%, the temperature of the fiber receiving area of ​​the negative electrode receiving device is 35~45℃, the temperature of the spinning solution is 5℃, the spinneret rate is 0.05g / (hole·min), and the receiving distance is 18cm.

[0220] S3. The initial film is hot-pressed using a hot-pressing roller at a temperature of 110°C and a pressure of 0.3 MPa.

[0221] A PVDF electrospun protein transfer film was obtained by passing the hot press roller at a speed of 0.2 m / min.

[0222] The protein transfer membranes obtained above were structurally characterized using a scanning electron microscope (SEM) (Hitachi S-5500) to characterize the membrane structure of each example sample, and the required data were obtained. Other tests were also conducted to obtain corresponding data. Specific results are shown in Tables 1-2 and... Figures 1-2 As shown.

[0223] Table 1

[0224]

[0225] Table 2

[0226]

[0227]

[0228] Using the protein transfer film obtained above as the test sample, its strength properties were tested. The test method is as follows:

[0229] (1) Bending stiffness: Tested according to the method specified in standard GB / T 18318.1-2009;

[0230] (2) Tensile strength: The test sample was cut into small films 1 cm wide and 10 cm long using a film cutter, and the tensile strength was tested using a microcomputer-controlled electronic universal testing machine.

[0231] Using the protein transfer membrane obtained above as the test sample, its wettability was tested by immersing the PVDF protein transfer membrane in methanol, removing it after 1 second, and then observing whether its surface was sufficiently wetted and whether the color was uniform. Simultaneously, an EGFR protein transfer experiment was performed using the PVDF protein transfer membrane to observe whether the surface bands were uniform and distinct. The color development results of the transfer experiment in Example 1 are as follows: Figure 3 As shown.

[0232] The results of the above effect tests are shown in Table 3.

[0233] Table 3

[0234]

[0235]

[0236] As can be seen from Tables 1-3, the embodiments of this application, by adjusting the ratio of the number of these two fibers with different diameters, control the contact angle θ1 between the protein transfer membrane and methanol at 0.1s and the contact angle θ2 between the protein transfer membrane and methanol at 1s, so that the ratio of θ1 to θ2 is within a suitable range. This can improve the wettability of the protein transfer membrane while taking into account its adsorption and mechanical properties. The test results show that the obtained protein transfer membrane has a bending stiffness ≤3.3mN·cm, tensile strength ≥3.9MPa, good wettability, and excellent color development performance. Thus, the protein transfer membrane can meet the requirements of immunoblotting assay for the adsorption, wettability, and mechanical properties of the transfer membrane.

[0237] For protein transfer membranes with smaller pore sizes (PMI average pore size in the range of 0.1–0.3 μm), compared to Examples 1–5, in Comparative Example 1, the ratio of θ1 to θ2 was too large, resulting in a significant decrease in the protein adsorption capacity of the protein transfer membrane and poor wettability; in Comparative Example 2, the ratio A of the number of second fibers to the number of first fibers was too large, and the ratio of θ1 to θ2 was also too large, resulting in a slight decrease in the protein adsorption capacity of the protein transfer membrane and a significant decrease in tensile strength; in Comparative Example 3, the ratio A of the number of second fibers to the number of first fibers was too small, resulting in a significant decrease in the protein adsorption capacity of the protein transfer membrane, a sharp increase in bending stiffness, insufficient softness, and poor wettability and color development; in Comparative Examples 4–5, the ratio of the number of second fibers to the number of first fibers… When the ratio A is too large, and the ratio of θ1 to θ2 is also too large, the tensile strength decreases, resulting in a slight decrease in the protein adsorption capacity of the protein transfer membrane. In Comparative Example 6, when the ratio A of the number of the second fiber to the number of the first fiber is too large, and the ratio of θ1 to θ2 is also too large, the adsorption of the protein transfer membrane is even worse than that of Comparative Examples 4-5, the bending stiffness increases sharply, the softness is insufficient, and the wettability and color development deteriorate. In Comparative Example 7, when the ratio A of the number of the second fiber to the number of the first fiber is too large, the protein adsorption capacity of the protein transfer membrane decreases, and the wettability and color development deteriorate. In Comparative Example 8, when the ratio A of the number of the second fiber to the number of the first fiber is too large, and the ratio of θ1 to θ2 is also too large, the protein adsorption capacity of the protein transfer membrane decreases significantly, and the tensile strength decreases significantly.

[0238] As can be seen from Examples 1-5 and Comparative Example 9, the formation of the fibrous adhesion structure can effectively improve the tensile strength of the protein transfer film.

[0239] For protein transfer membranes with larger pore sizes (PMI average pore size > 0.3 μm), compared to Examples 6-8, in Comparative Example 10, the ratio A of the number of second fibers to the number of first fibers is too small, and the ratio of θ1 to θ2 is too large, which significantly reduces the protein adsorption capacity of the protein transfer membrane, increases the bending stiffness sharply, reduces the softness, and worsens the wettability; in Comparative Example 11, the ratio A of the number of second fibers to the number of first fibers is too large, which slightly reduces the protein adsorption capacity of the protein transfer membrane and significantly reduces the tensile strength; in Comparative Example 12, the ratio A of the number of second fibers to the number of first fibers is too small, and the ratio of θ1 to θ2 is too large, which significantly reduces the protein adsorption capacity of the protein transfer membrane, increases the bending stiffness sharply, reduces the softness, and worsens the wettability and color development.

[0240] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application and are not intended to limit the scope of protection of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the scope of the claims of this application.

Claims

1. An electrospun protein transfer film, characterized by, The membrane includes a first fiber and a second fiber, which are randomly stacked. The diameter of the first fiber is D1, and the diameter of the second fiber is D2. The average pore size of the protein transfer membrane (PMI) is R. R, D1, and D2 satisfy the following conditions: 0.1 μm ≤ R ≤ 1 μm, 1.5 ≤ D1 / R ≤ 5, and D2 / R < 1.

5. The ratio of the second fiber to the first fiber is A, where A is 2 to 6; The contact angle between the protein transfer membrane and methanol is θ1 when they are in contact for 0.1 seconds, and the contact angle between the protein transfer membrane and methanol is θ2 when they are in contact for 1 second. θ1 and θ2 satisfy the condition: 0.2≤θ2 / θ1≤0.

6.

2. The electrospun protein transfer film of claim 1, wherein, The second fiber comprises a second medium fiber and a second fine fiber, wherein the diameter of the second medium fiber is ≥0.9R and <1.5R, and the diameter of the second fine fiber is <0.9R.

3. The electrospun protein transfer membrane of claim 2, wherein, The ratio of the second medium fiber to the second fine fiber is B, where B is 1 to 2.

4. The electrospun protein transfer film as described in claim 2, characterized in that, A and R satisfy: If 0.1μm≤R≤0.3μm, then 3.3≤A≤6.0; If 0.3μm<R≤1μm, then 2.3≤A≤3.

6.

5. The electrospun protein transfer film as described in claim 2, characterized in that, B and R satisfy: If 0.1μm≤R≤0.3μm, then 1.3≤B≤1.65; If 0.3μm<R≤1μm, then 1.0≤B≤1.

4.

6. The electrospun protein transfer film as described in claim 1, characterized in that, The air permeability of the protein transfer membrane meets the following requirements: If R is 0.1-0.3 μm, the air permeability is 50-70 s-1in 2 / 100 mL; If R is 0.3-1 μm, the air permeability is 10-20 s-1in 2 / 100 mL.

7. The electrospun protein transfer film as described in claim 1, characterized in that, 12°≤θ1≤24°;θ2≤10°; and / or the protein transfer membrane has an adsorption capacity for BSA protein of 30 to 60 ug / cm 2 .

8. The electrospun protein transfer film according to claim 1, characterized in that, The protein transfer membrane contains several fibrous parts that fuse together to form fibrous adhesions. The area ratio of the fibrous adhesions in any square millimeter area on the surface of the protein transfer membrane is a, where a ≤ 10%.

9. A method for preparing a PVDF electrospun protein transfer film as described in any one of claims 1 to 8, characterized in that, Includes the following steps: S1. Prepare a spinning solution, wherein the spinning solution comprises polyvinylidene fluoride, a soluble inorganic salt and a solvent, wherein the mass fraction of polyvinylidene fluoride in the spinning solution is 7.5-20%, and the concentration of the soluble inorganic salt in the spinning solution is 1-5 mM, wherein the soluble inorganic salt comprises one of sodium chloride, potassium chloride and magnesium chloride. S2. Electrospinning the spinning solution obtained in step S1 using an electrospinning device to form a primary film. The spinneret of the electrospinning device has a spinneret hole A and a spinneret hole B, wherein the diameter of the spinneret hole A is 0.5 to 0.8 times the diameter of the spinneret hole B. The conditions for electrospinning are as follows: the spinning voltage is 35~70kV, the ambient humidity is 30~70%, the temperature of the fiber receiving area of ​​the negative electrode receiving device is 35~45℃, the temperature of the spinning solution is 5~10℃, and the spin rate is 0.02~0.3g / (hole·min). S3. The initial film is hot-pressed to obtain a PVDF electrospun protein transfer film.

10. The method for preparing the PVDF electrospun protein transfer film according to claim 8, characterized in that, The weight-average molecular weight of polyvinylidene fluoride in the spinning solution is 50~100K.

11. The method for preparing the PVDF electrospun protein transfer film according to claim 8, characterized in that, The solvent in the spinning solution comprises a miscible low-boiling-point solvent and a high-boiling-point solvent. The low-boiling-point solvent has a boiling point not greater than 100°C and includes at least one of alcohols, ethers, and cyclic ethers. The high-boiling-point solvent has a boiling point not less than 150°C and includes at least one of dimethyl sulfoxide, N,N-dimethylformamide, N-methylpyrrolidone, and toluene. The volume percentage of the high-boiling-point solvent is 0.5% to 3% based on the sum of the volumes of the high-boiling-point solvent and the low-boiling-point solvent.

12. The application of the PVDF electrospun protein transfer membrane as described in any one of claims 1 to 8 in immunoblotting experiments.

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