A low-background, high-protein-binding polyvinylidene fluoride transfer film, its preparation and application

Abstract

This solution provides a low-background, high-protein-binding polyvinylidene fluoride (PVDF) transfer film, its preparation, and application. The steps include: adding an organic solvent and diluent to a stirred tank and mixing, then adding PVDF powder and heating to dissolve it. After the PVDF powder is completely dissolved, adding an ionic liquid and continuing heating to dissolve it yields a mixed casting solution. The ionic liquid is hydrophilic and thermodynamically compatible with PVDF. The mixed casting solution is cast onto a PET substrate using a doctor blade, and the PET substrate with the cast solution is directly immersed in a coagulation bath and held for a period of time. The mixed casting solution undergoes phase separation and forming in the coagulation bath to obtain the PVDF transfer film. The PVDF transfer film is then cleaned and dried to obtain a low-background, high-protein-binding PVDF transfer film, achieving high protein binding capacity while maintaining low luminescence background to ensure excellent transfer results.

Description

Technical Field

[0001] This invention relates to the field of biofilm materials, and particularly to a low-background, high-protein-binding polyvinylidene fluoride transfer film with good crystallization and pore-forming properties, as well as its preparation and application methods. Background Technology

[0002] Western blotting is a molecular biology technique used to detect the presence and expression level of specific proteins in a sample. The process involves: adding protein lysates to a polyacrylamide gel, separating the protein sample by electrophoresis, and then transferring the separated biological sample from the gel to a solid support under an electric field, typically a nitrocellulose (NC) membrane or a polyvinylidene fluoride (PVDF) membrane. A relatively simple method for labeling the target protein is to incubate the transferred protein with an enzyme-labeled primary antibody, allowing the primary antibody to specifically bind to the target protein. Then, a suitable chemiluminescent substrate (such as ECL luminescent solution) is added, forming a complex with the enzyme. This complex generates an optical signal for detection. Finally, the signal is recorded using X-ray film or a chemiluminescence imaging system (such as ChemiDoc), and the protein expression level is analyzed based on the brightness and molecular weight of the target protein band—the presence or absence of the signal indicates the presence of the target protein, while the signal strength reflects, to some extent, the content of the target protein.

[0003] NC membranes were among the first materials used in Western blotting experiments, but their mechanical and chemical stability is inferior to that of PVDF membranes. PVDF transfer membranes maintain stable mechanical properties over a longer period, while NC membranes become brittle with age. Furthermore, PVDF membranes allow for repeated detection by removing antibodies, whereas NC membranes cannot. Therefore, PVDF membranes have a greater advantage in Western blotting experiments. Compared to NC membranes, hydrophobic PVDF membranes have stronger protein binding capacity, allowing them to detect lower sample concentrations under the same conditions, making them particularly suitable for detecting low-abundance proteins. However, currently available PVDF transfer membranes often fail to achieve optimal transfer results due to their relatively poor protein binding capacity.

[0004] PVDF is a hydrophobic material. PVDF transfer membranes can non-specifically adsorb onto biological samples due to hydrophobic interactions. Besides target proteins, they can also adsorb enzyme-labeled antibodies used as probes. During testing, if antibodies non-specifically bind to the PVDF transfer membrane, the optical signal generated by the enzyme-substrate complex will create a deep luminescence background, affecting the observation of the target protein—low concentrations of the target protein may even be completely undetectable. Therefore, after the protein is transferred from the gel to the membrane, the transfer membrane is usually blocked with skim milk powder or bovine serum albumin to prevent non-specific adsorption of subsequent labeled antibodies onto the membrane surface, ensuring the accuracy of the detection results. Our research revealed that PVDF transfer membranes with exceptionally high protein binding capacity possess a higher level of adsorption sites. Even after the blockade process involving bovine serum albumin (BSAP), these adsorption sites cannot be completely blocked due to steric hindrance. Following primary antibody incubation, the primary antibody is non-specifically adsorbed onto the membrane. Subsequent primary antibody incubation also results in non-specific adsorption, leading to a darker background after exposure. This dark background masks low-abundance proteins that should be present, making them easily misjudged as absent in the sample lysis buffer. Conversely, PVDF transfer membranes with lower protein binding capacity struggle to achieve good transfer results. Therefore, there is a trade-off between the protein binding capacity and background depth of PVDF transfer membranes: stronger protein binding capacity leads to better transfer results, but also a greater likelihood of producing a deeper detection background. Throughout the transfer process, the membrane, as the core material, faces high demands regarding its protein binding capacity, porosity, and chemiluminescence background properties.

[0005] In summary, the PVDF transfer films currently available on the market have the drawback of being unable to simultaneously achieve low luminescence background and high protein binding capacity, which limits their application in protein immunoblotting. Summary of the Invention

[0006] The purpose of this invention is to provide a polyvinylidene fluoride (PVDF) transfer film with low background and high protein binding, as well as its preparation and application. By controlling the simultaneous occurrence of liquid-liquid phase separation and liquid-solid phase separation, the PVDF transfer film has a network-loaded spherulite structure, thereby achieving a balance between protein binding capacity and background depth. While ensuring low luminescence background, it achieves high protein binding capacity to ensure excellent transfer effect.

[0007] To achieve the above objectives, firstly, this technical solution provides a method for preparing a low-background, high-protein-binding polyvinylidene fluoride transfer film, comprising the following steps:

[0008] (1) Add organic solvent and diluent to a stirring vessel and mix them. Then add polyvinylidene fluoride powder and heat to dissolve. After the polyvinylidene fluoride powder is completely dissolved, add ionic liquid and continue heating to dissolve to obtain a mixed casting solution. The ionic liquid is hydrophilic and thermodynamically compatible with polyvinylidene fluoride.

[0009] (2) The mixed casting liquid is cast onto the PET substrate by a doctor blade, and the PET substrate with the cast mixed casting liquid is directly immersed into the coagulation bath and kept for a period of time. The mixed casting liquid is separated into phases in the coagulation bath to obtain a polyvinylidene fluoride transfer film.

[0010] (3) The polyvinylidene fluoride transfer film is cleaned and dried to obtain a polyvinylidene fluoride transfer film with low background and high protein binding.

[0011] Polyvinylidene fluoride (PVDF) is a semi-crystalline polymer. The film formation process through phase separation can be divided into liquid-liquid phase separation and liquid-solid phase separation. The pore structure of the film after formation is the result of the competition between liquid-liquid phase separation and liquid-solid phase separation. It is known in this technical field that if the phase separation process of PVDF is mainly liquid-liquid phase separation, the formed film has a dense skin and a finger-like asymmetric structure or a sponge-like symmetric structure. However, if the phase separation process of PVDF is mainly liquid-solid phase separation, the delayed occurrence of phase separation provides conditions for PVDF crystallization, and the surface and interior of the formed film are composed of spherulite particles.

[0012] Our team based our application on the phase separation process of polyvinylidene fluoride (PVDF). By optimizing the formulation and preparation process, we controlled the simultaneous occurrence of liquid-liquid phase separation and liquid-solid phase separation, and finally obtained the structure of PVDF transfer film with a network-supported spherulite structure.

[0013] Specifically, this solution incorporates an ionic liquid that is thermodynamically compatible with polyvinylidene fluoride (PVDF) into the mixed casting solution. On one hand, as a liquid salt, the ionic liquid itself is formed by the ionic bonds between organic cations and anions. Therefore, it can generate an ion-dipole interaction with the -CF2 molecules on the PVDF molecular chain, inducing polar crystallization in PVDF. This provides further conditions for the formation of spherulites through liquid-solid separation of PVDF. Since the specific surface area of ​​the crystals is much larger than that of a completely open network structure, crystallization allows PVDF to generate more adsorption sites, thereby improving its protein binding capacity. On the other hand, because the ionic liquid is hydrophilic, it can also act as a pore-forming agent in the mixed casting solution, causing the developing cellular pores to deform during the liquid-liquid phase separation of PVDF, resulting in a cured PVDF transfer film with an interpenetrating network structure.

[0014] In other words, the ionic liquid in this scheme can not only induce polyvinylidene fluoride (PVDF) crystallization, providing further conditions for PVDF to undergo liquid-solid separation and form spherulites, but also act as a pore-forming agent to promote PVDF liquid-liquid phase separation to generate an interpenetrating network structure, ultimately resulting in a PVDF transfer film with a network-supported spherulite structure. Furthermore, it should be noted that although the ionic liquid is hydrophilic, it is lost during the film formation process, so the final PVDF transfer film remains hydrophobic.

[0015] Preferably, the ionic liquid in step (1) is an imidazole-type ionic liquid. Since the cation of the imidazole-type ionic liquid can generate an "ion-dipole" interaction with the -CF2 on the polyvinylidene fluoride molecular chain, it can fully induce polyvinylidene fluoride to undergo polar crystallization.

[0016] Preferably, the ionic liquid in step (1) is one or more of 1-methyl-3-butylimidazolium chloride, 1-hydroxyethyl-3-methylimidazolium chloride, 1-butyl-3-methylimidazolium sulfate, and 1-ethyl-3-methylimidazolium acetate.

[0017] Preferably, the organic solvent in step (1) is one or more of N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMAc), N-methylpyrrolidone (NMP) or triethyl phosphate (TEP).

[0018] Preferably, the diluent in step (1) is one or more of dibutyl phthalate (DBP), dioctyl phthalate (DOP), glycerin, and acetone.

[0019] Preferably, in step (1), the mass fraction of polyvinylidene fluoride powder in the mixed casting solution is 10-25 wt%, the mass fraction of organic solvent is 35-65 wt%, the mass fraction of diluent is 5-53 wt%, the mass fraction of ionic liquid is 2-5 wt%, and the total mass fraction of polyvinylidene fluoride powder, organic solvent, diluent, and ionic solvent is 100 wt%. It should be noted that when the mass fraction of ionic liquid is too low, it cannot act as a nucleating agent, resulting in poor crystallization induction. The imidazole-type ionic liquid selected in this scheme is a hydrophilic salt. If the mass content of the ionic liquid is high, the pore-forming effect can be better, that is, the liquid-liquid phase separation process is intensified, resulting in a predominantly network-like film with fewer spherulites.

[0020] Preferably, in step (1), the heating temperature for adding polyvinylidene fluoride powder and heating to dissolve it is 30~55℃.

[0021] Preferably, in step (1), the heating temperature during the addition of the ionic liquid and continued heating for dissolution is 30~55℃.

[0022] It is particularly important to emphasize that the ionic liquid must be added only after the polyvinylidene fluoride powder has completely dissolved, as this ensures that the polyvinylidene fluoride powder will dissolve better and faster.

[0023] In addition, this method can be used to determine that the polyvinylidene fluoride powder has been completely dissolved by observing that the ionic liquid solution containing the polyvinylidene fluoride powder is in a clear state.

[0024] Preferably, the room temperature in step (2) is 25°C. This scheme strictly controls the ambient temperature to 25°C and the humidity to below 50%RH to reduce the possibility of liquid-liquid phase separation due to contact with moisture in the environment. Furthermore, the mixed casting solution can be directly immersed in the coagulation bath after being cast by a scraper, thereby avoiding a large amount of volatilization before entering the coagulation bath, which would cause the mixed casting solution to enter the metastable state prematurely. This avoids the possibility of pre-phase separation of the mixed casting solution in the air, providing a sufficient time window for the nucleation and growth of polyvinylidene fluoride microcrystals in the coagulation bath.

[0025] Preferably, the time interval between casting the mixed casting solution onto the PET substrate using a doctor blade and immersing the PET substrate with the cast mixed casting solution directly into the coagulation bath does not exceed 30 seconds. More preferably, the time interval between casting the mixed casting solution onto the PET substrate using a doctor blade and immersing the PET substrate with the cast mixed casting solution directly into the coagulation bath does not exceed 15 seconds.

[0026] Preferably, the coagulation bath is composed of one of methanol, n-butanol, ethanol, and acetone, or an aqueous solution containing methanol, n-butanol, ethanol, and acetone. This scheme selects reagents such as n-butanol, ethanol, and acetone as coagulation bath components, which can slow down the mass transfer rate between the solvent and non-solvent, delaying liquid-liquid phase separation and providing a time window for the nucleation and growth of polyvinylidene fluoride microcrystals, resulting in a spherulitic structure in the cured film.

[0027] Preferably, the phase separation time for obtaining the polyvinylidene fluoride transfer film by phase separation molding of the mixed casting solution in the coagulation bath in step (2) is 3~10 min.

[0028] Preferably, in step (3), the polyvinylidene fluoride transfer film is soaked in running water, rinsed and dried to obtain a polyvinylidene fluoride transfer film with low background and high protein binding.

[0029] Preferably, the temperature of the running water in step (3) is 40-70℃, and the cleaning time is not less than 10 minutes.

[0030] Secondly, this technical solution provides a low-background, high-protein-bonded polyvinylidene fluoride transfer film, which is prepared according to the preparation method of the low-background, high-protein-bonded polyvinylidene fluoride transfer film in the first aspect and has a network-loaded spherulite structure.

[0031] Thirdly, this technical solution provides an application of a low-background, high-protein-binding polyvinylidene fluoride transfer membrane in protein immunoblotting scenarios.

[0032] Compared with existing technologies, this technical solution has the following characteristics and beneficial effects:

[0033] 1. This invention is based on the phase separation mechanism of polyvinylidene fluoride (PVDF), and controls the phase separation process through rational and effective formulation selection and strict and precise process design. First, an imidazole-type ionic liquid, thermodynamically compatible with PVDF, is added to the mixed casting solution. The imidazole cations interact with the -CF2 atoms on the PVDF molecular chain via an "ion-dipole" interaction, inducing polar crystallization of PVDF and providing nucleation conditions for liquid-solid separation and the formation of spherulites. Second, the ionic liquids used in this invention are all hydrophilic. As additives in the mixed casting solution, they act as pore-forming agents, promoting liquid-liquid phase separation. During the PVDF liquid-liquid phase separation stage, they cause deformation of developing cellular pores, resulting in the formation of spherulite particles on the PVDF transfer membrane surface while retaining a network-like open-pore structure, thereby increasing the specific surface area of ​​the membrane and generating more protein binding sites. In addition, selecting a suitable coagulation bath composition slows down the exchange rate between the solvent and the coagulation bath, enhancing the degree of liquid-solid phase separation while simultaneously allowing it to dominate the process and induce PVDF crystallization. The entire phase separation process is a delayed phase separation. Simultaneously, strictly controlling environmental conditions eliminates the possibility of phase separation occurring in the air before the membrane solution enters the coagulation bath, preventing the membrane solution from prematurely entering a metastable state. This creates sufficient time for PVDF microcrystals to nucleate and grow within the coagulation bath, ensuring the occurrence of liquid-solid phase separation.

[0034] 2. The PVDF transfer membrane prepared by this invention achieves a balance between protein binding capacity and background depth. While ensuring low luminescence background, it achieves high protein binding capacity. In particular, it can obtain clear transfer bands for the detection of low-abundance proteins and can be applied to protein immunoblotting to ensure excellent transfer effect. Attached Figure Description

[0035] Figure 1 This is an electron microscope image of the surface structure of the PVDF transfer film in Example 1.

[0036] Figure 2 This is an electron microscope image of the surface structure of the PVDF transfer film in Example 2.

[0037] Figure 3 The image shows an electron microscope (EM) image of the surface structure of the PVDF transfer film in Comparative Example 1.

[0038] Figure 4 The image shows an electron microscope (EM) image of the surface structure of the PVDF transfer film in Comparative Example 2.

[0039] Figure 5 The image shows an electron microscope (EM) image of the surface structure of the PVDF transfer film in Comparative Example 3.

[0040] Figure 6 The image shows an electron microscope (EM) image of the surface structure of the PVDF transfer film in Comparative Example 4.

[0041] Figure 7 This is a reference figure for the protein adsorption amount of the PVDF transfer membranes in Example 1 and Comparative Examples 1 and 2.

[0042] Figure 8 The image shows a comparison of the transfer effects of Example 1 and Comparative Examples 1 and 2. Detailed Implementation

[0043] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention are within the scope of protection of the present invention.

[0044] Example 1:

[0045] (1) Weigh 600g of N,N-dimethylformamide and 225g of acetone in sequence, mix them evenly, add 150g of polyvinylidene fluoride powder (Mw: 300,000 to 500,000), stir at 50°C until completely dissolved, then add 25g of 1-methyl-3-butylimidazolium chloride, let it dissolve and stand for 12 hours to remove bubbles, and obtain a uniform casting solution;

[0046] (2) Under the environment of 25℃ and humidity below 50%RH, the casting solution is coated onto the polyethylene terephthalate base tape, and the liquid film thickness is set to 400μm. The coated liquid film is directly immersed in the coagulation bath (with a time interval controlled within 30s). The coagulation bath composition is 75% V / V acetone aqueous solution. The liquid film is maintained in the coagulation bath for 3min to obtain the nascent PVDF transfer film.

[0047] (3) The nascent transfer film obtained above is immersed in a 60°C hot water tank for 10 minutes and then dried to obtain a PVDF transfer film.

[0048] Figure 1 The image shows a scanning electron microscope (SEM) image of the PVDF transfer film obtained in Example 1. As can be seen from the image, the PVDF transfer film in this invention has a network-like structure with spherulites.

[0049] Bovine serum albumin (BSA) was used as a model to perform static adsorption tests on the PVDF transfer membrane to evaluate its protein-binding capacity. A 5cm x 10cm PVDF transfer membrane was moistened with 20% methanol and then rinsed with purified water. A 0.5 mg / ml BSA solution was prepared, and 100ml of this solution was placed in a glass reagent bottle. The rinsed PVDF transfer membrane was then completely immersed in the BSA solution. The adsorption process was carried out using a shaker for 2 hours. The absorbance of the BSA solution before and after adsorption was measured using a UV spectrophotometer. The protein adsorption capacity of the PVDF transfer membrane was calculated based on this, thus evaluating its protein-binding capacity.

[0050] Example 2

[0051] (1) Weigh 350g of N-methylpyrrolidone and 530g of acetone in sequence, mix them evenly, add 100g of polyvinylidene fluoride powder (Mw: 300,000 to 500,000), stir at 50°C until completely dissolved, then add 20g of 1-hydroxyethyl-3-methylimidazolium chloride, let it dissolve and stand for 12 hours to remove bubbles, and obtain a uniform casting solution;

[0052] (2) Under the environment of 25℃ and humidity below 50%RH, the casting solution is coated onto the polyethylene terephthalate base tape, and the liquid film thickness is set to 400μm. The coated liquid film is directly immersed in the coagulation bath (the time interval is controlled at about 15s). The coagulation bath composition is 60% V / V methanol aqueous solution. The liquid film is maintained in the coagulation bath for 10min to obtain the nascent PVDF transfer film.

[0053] (3) The nascent transfer film obtained above is immersed in a 60°C hot water tank for 10 minutes and then dried to obtain a PVDF transfer film.

[0054] Example 3

[0055] (1) Weigh 350g of N-methylpyrrolidone and 530g of acetone in sequence, mix them evenly, add 100g of polyvinylidene fluoride powder (Mw: 300,000 to 500,000), stir at 50°C until completely dissolved, then add 20g of 1-butyl-3-methylimidazolium sulfate, let it dissolve and stand for 12 hours to remove bubbles, and obtain a uniform casting solution;

[0056] (2) Under the environment of 25℃ and humidity below 50%RH, the casting solution is coated onto the polyethylene terephthalate base tape, and the liquid film thickness is set to 400μm. The coated liquid film is directly immersed in the coagulation bath (the time interval is controlled at about 20s). The coagulation bath composition is 60% V / V n-butanol aqueous solution. The liquid film is maintained in the coagulation bath for 10min to obtain the nascent PVDF transfer film.

[0057] (3) The nascent transfer film obtained above is immersed in a 60°C hot water tank for 10 minutes and then dried to obtain a PVDF transfer film.

[0058] Example 4

[0059] (1) Weigh 350g of N-methylpyrrolidone and 530g of acetone in sequence, mix them evenly, add 100g of polyvinylidene fluoride powder (Mw: 300,000 to 500,000), stir at 50°C until completely dissolved, then add 20g of 1-ethyl-3-methylimidazolium acetate, let it dissolve and stand for 12 hours to remove bubbles, and obtain a uniform casting solution;

[0060] (2) Under the environment of 25℃ and humidity below 50%RH, the casting solution is coated onto the polyethylene terephthalate base tape, and the liquid film thickness is set to 400μm. The coated liquid film is directly immersed in the coagulation bath (the time interval is controlled at about 25s). The coagulation bath composition is 60% V / V ethanol aqueous solution. The liquid film is maintained in the coagulation bath for 10min to obtain the nascent PVDF transfer film.

[0061] (3) The nascent transfer film obtained above is immersed in a 60°C hot water tank for 10 minutes and then dried to obtain a PVDF transfer film.

[0062] Comparative Example 1:

[0063] The difference between this comparative example and Example 1 is that the casting solution underwent pre-phase separation treatment in the air environment after being scraped out by the doctor blade, and the coagulation bath was water.

[0064] (1) Weigh 600g of N,N-dimethylformamide and 225g of acetone in sequence, mix them evenly, add 150g of polyvinylidene fluoride powder (Mw: 300,000 to 500,000), stir at 50°C until completely dissolved, then add 25g of 1-methyl-3-butylimidazolium chloride, let it dissolve and stand for 12 hours to remove bubbles, and obtain a uniform casting solution;

[0065] (2) Under the control of 25℃ and humidity below 50%RH, the casting liquid is coated onto the polyethylene terephthalate base tape, and the liquid film thickness is set to 400μm. The coated liquid film is run in the air environment for 5min for pre-phase separation. The ambient temperature is 25℃ and the humidity is 80%RH. Then it is immersed in the coagulation bath. The coagulation bath component is pure water. The liquid film is maintained in the coagulation bath for 5min to obtain the nascent PVDF transfer film.

[0066] (3) The nascent transfer film obtained above is immersed in a 60°C hot water tank for 10 minutes and then dried to obtain a PVDF transfer film.

[0067] Comparative Example 2:

[0068] The difference between this comparative example and Example 1 is that no ionic liquid was added to the casting solution.

[0069] (1) Weigh 600g of N,N-dimethylformamide and 225g of acetone in sequence, mix them evenly, add 150g of polyvinylidene fluoride powder, stir at 50°C until completely dissolved, let stand for 12 hours to remove bubbles, and obtain a uniform casting solution.

[0070] (2) Under the control of 25℃ and humidity below 50%RH, the casting solution was coated onto the polyethylene terephthalate base tape, and the liquid film thickness was set to 400μm. The coated liquid film was directly immersed in the coagulation bath, which was composed of 75% V / V acetone aqueous solution. The liquid film was maintained in the coagulation bath for 3 minutes to obtain the nascent PVDF transfer film.

[0071] (3) The nascent transfer film obtained above is immersed in a 60°C hot water tank for 10 minutes and then dried to obtain a PVDF transfer film.

[0072] Comparative Example 3:

[0073] The difference between this comparative example and Example 1 is that the mass concentration of the ionic liquid is greater than 5 wt%.

[0074] (1) Weigh 600g of N,N-dimethylformamide and 225g of acetone in sequence, mix them evenly, add 150g of polyvinylidene fluoride powder, stir at 50℃ until completely dissolved, then add 100g of 1-methyl-3-butylimidazolium chloride, let it dissolve and stand for 12 hours to remove bubbles, and obtain a uniform casting solution.

[0075] (2) Under the control of 25℃ and humidity below 50%RH, the casting solution was coated onto the polyethylene terephthalate base tape, and the liquid film thickness was set to 400μm. The coated liquid film was directly immersed in the coagulation bath, which was composed of 75% V / V acetone aqueous solution. The liquid film was maintained in the coagulation bath for 3 minutes to obtain the nascent PVDF transfer film.

[0076] (3) The nascent transfer film obtained above is immersed in a 60°C hot water tank for 10 minutes and then dried to obtain a PVDF transfer film.

[0077] Comparative Example 4:

[0078] The difference between this comparative example and Example 1 is that the mass concentration of the ionic liquid is less than 2 wt%.

[0079] (1) Weigh 600g of N,N-dimethylformamide and 225g of acetone in sequence, mix them evenly, add 150g of polyvinylidene fluoride powder, stir at 50℃ until completely dissolved, then add 10g of 1-methyl-3-butylimidazolium chloride, let it dissolve and stand for 12 hours to remove bubbles, and obtain a uniform casting solution.

[0080] (2) Under the control of 25℃ and humidity below 50%RH, the casting solution was coated onto the polyethylene terephthalate base tape, and the liquid film thickness was set to 400μm. The coated liquid film was directly immersed in the coagulation bath, which was composed of 75% V / V acetone aqueous solution. The liquid film was maintained in the coagulation bath for 3 minutes to obtain the nascent PVDF transfer film.

[0081] (3) The nascent transfer film obtained above is immersed in a 60°C hot water tank for 10 minutes and then dried to obtain a PVDF transfer film.

[0082] Figure 1-4 The images show the microstructures of the PVDF transfer films prepared in Examples 1, 2, and Comparative Examples 1 and 2, respectively. Figure 1 and Figure 2 As can be seen from the above, the PVDF transfer films prepared in Examples 1 and 2 of the present invention have a network-like surface structure with loaded spherulites, and have high porosity and uniform spherulite size.

[0083] In Comparative Example 1, after the casting solution is coated onto the PET substrate with a doctor blade, it first travels a distance in the air. Phase separation is induced by ambient humidity and solvent evaporation. Upon contact with moisture in the environment, liquid-liquid phase separation occurs. After pre-phase separation, the solution is then immersed in a coagulation bath, resulting in delayed phase separation and the formation of a network structure. This structure has a small specific surface area, relatively few adsorption sites on the PVDF transfer membrane surface, and poor protein binding capacity, thus failing to achieve a good transfer effect.

[0084] In Comparative Example 2, the coagulation bath and casting solution contained the same components, resulting in a slower exchange rate between solvent and non-solvent, which provided conditions for delayed phase separation of the liquid film. Furthermore, the absence of hydrophilic ionic liquids in the casting solution further reduced the exchange between the solvent and the coagulation bath, making the phase separation process primarily liquid-solid (i.e., crystallization). The resulting PVDF transfer film lacked a skin layer, with both the surface and interior composed of spherulite particles. This structure endowed the PVDF transfer film with a high specific surface area, numerous adsorption sites, and strong protein binding capacity. However, during protein transfer, the numerous adsorption sites required a longer blocking time, and due to steric hindrance, the adsorption sites were not completely blocked. Subsequent antibody adsorption occurred at the unblocked sites, resulting in a deep luminescent background after exposure, affecting band reading.

[0085] Depend on Figure 5It can be seen that the PVDF transfer film prepared in Example 1 of this invention exhibits a high protein binding capacity, with a higher protein adsorption amount compared to Comparative Example 1, but slightly lower than that of Comparative Example 2. This high level of protein binding capacity is precisely why... Figure 6 As shown, the PVDF transfer membrane prepared by this invention achieves excellent transfer performance in protein transfer detection, especially exhibiting good sensitivity for the detection of low-abundance proteins. Simultaneously, the PVDF transfer membrane prepared by this invention has a low chemiluminescence background, which facilitates clear reading of the transferred bands.

[0086] Comparative Example 3:

[0087] The difference between this comparative example and Example 1 is that the amount of ionic liquid added is higher. Electron microscopy results show that with a higher amount of ionic liquid, the resulting transfer film lacks a spherulitic structure and exhibits a larger pore structure on the surface. Since the ionic liquid used in this application is hydrophilic, it exacerbates the liquid-liquid phase separation of the membrane solution, leading to rapid phase separation and solidification of the membrane solution. This prevents the PVDF crystal nuclei from growing in time, resulting in the absence of a spherulitic structure.

[0088] Comparative Example 4:

[0089] The difference between this comparative example and Example 1 is that the amount of ionic liquid added is lower. Electron microscopy results show that with a lower amount of ionic liquid, the resulting transfer film surface lacks spherulitic structures. The low ionic liquid content means it cannot act as a nucleating agent, resulting in poor crystallization induction.

[0090] Those skilled in the art should understand that the technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments have been described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0091] The above embodiments are merely illustrative of several implementation methods of this application, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of this application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this application should be determined by the appended claims.

Claims

1. A method for preparing a low background high protein binding polyvinylidene fluoride transfer membrane, characterized by, The method comprises the following steps: (1) adding an organic solvent and a diluent into a stirring kettle, mixing, and then adding polyvinylidene fluoride powder for heating and dissolving, adding an ionic liquid for continuous heating and dissolving after the polyvinylidene fluoride powder is completely dissolved, to obtain a mixed casting solution, wherein the ionic liquid is hydrophilic and thermodynamically compatible with the polyvinylidene fluoride, the mass fraction of the polyvinylidene fluoride powder in the mixed casting solution of step (1) is 10-25 wt%, the mass fraction of the ionic liquid is 2-5 wt%, and the ionic liquid is an imidazole type ionic liquid; (2) casting the mixed casting solution on a PET base tape by means of a doctor blade, and directly immersing the PET base tape with the cast mixed casting solution into a coagulation bath to make the mixed casting solution phase separate and form in the coagulation bath to obtain a polyvinylidene fluoride transfer film, the time interval from casting the mixed casting solution on the PET base tape to directly immersing the PET base tape with the cast mixed casting solution into the coagulation bath is not more than 30 seconds, the phase separation time for the mixed casting solution to phase separate and form the polyvinylidene fluoride transfer film in the coagulation bath is 3-10 min, and step (2) is controlled in an environment at room temperature and with a humidity of less than 50% RH, and the coagulation bath comprises one of n-butanol, ethanol, and acetone or an aqueous solution containing n-butanol, ethanol, and acetone; (3) cleaning and drying the polyvinylidene fluoride transfer film to obtain a polyvinylidene fluoride transfer film with low background and high protein binding.

2. The method for preparing a low-background, high-protein-binding polyvinylidene fluoride transfer film according to claim 1, characterized in that, The ionic liquid is one or more of 1-methyl-3-butyl imidazole chlorate, 1-hydroxyethyl-3-methyl imidazole chlorate, 1-butyl-3-methyl imidazole sulfate, and 1-ethyl-3-methyl imidazole acetate.

3. The method for preparing a low-background, high-protein-binding polyvinylidene fluoride transfer film according to claim 1, characterized in that, The mass fraction of the organic solvent in the mixed casting solution of step (1) is 35-65 wt%, and the mass fraction of the diluent is 5-53 wt%.

4. The method of making a low background, high protein binding polyvinylidene fluoride transfer membrane according to claim 1, wherein, The organic solvent in step (1) is one or more of N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, and triethyl phosphate; and the diluent in step (1) is one or more of dibutyl phthalate (DBP), dioctyl phthalate (DOP), glycerol, and acetone.

5. A low background, high protein binding polyvinylidene fluoride transfer membrane characterized by, The polyvinylidene fluoride transfer film with low background and high protein binding is prepared by the method according to any one of claims 1 to 4 and has a reticular loaded spherulitic structure.

Images