Improved protein imprinting film capable of rapidly transferring protein and preparation method of improved protein imprinting film

Abstract

The invention relates to the technical field of protein imprinting membranes, in particular to an improved protein imprinting membrane capable of rapidly transferring protein and a preparation method of the improved protein imprinting membrane. The invention discloses a polyvinylidene fluoride / cellulose nanocrystal composite material, which is prepared from the following components in parts by weight: 10 to 20 parts of polyvinylidene fluoride, 85 to 100 parts of N, N-dimethylformamide solvent, 3 to 5 parts of cellulose nanocrystal reinforcing agent, 1 to 2 parts of 3-glycidoxypropyltrimethoxysilane solution, 6 to 10 parts of ethylenediamine solution, 6 to 10 parts of sodium periodate solution and 12 to 15 parts of EDC / NHS cross-linking agent. Wherein the EDC / NHS cross-linking agent is a mixture of EDC with the concentration being 5 mg / mL and NHS with the concentration being 10 mg / mL, and the molar ratio of EDC to NHS is 1: (1.2-1.5). The EDC / NHS cross-linking agent connects carboxyl and primary amino to form an amido bond, so that non-specific adsorption sites on the surface of the membrane are reduced, and the specificity and sensitivity of an experiment are improved.

Description

Technical Field

[0001] The invention relates to the technical field of protein imprinting membranes, in particular to an improved protein imprinting membrane for rapid protein transfer and a preparation method thereof. Background Art

[0002] Protein blotting is a common technique used to detect, quantify and verify the presence of proteins. In the process of protein blotting, it is usually necessary to transfer proteins from gels to a solid support, which is the so-called "protein blotting membrane". Among them, protein blotting membrane is a solid support used in protein immunoblotting technology to fix and detect proteins transferred from gels. The main function of protein blotting membrane is to provide a stable surface for proteins to attach to it and maintain their spatial structure and biological activity for subsequent detection.

[0003] In high-throughput screening experiments, a large number of samples need to be processed at the same time. In this case, the use of protein imprinting membranes that quickly transfer proteins can greatly improve work efficiency and reduce experimental cycles. However, there are too many nonspecific adsorption sites on the membrane surface, which will reduce the specificity and sensitivity of the detection. In order to ensure the stability of the samples and the accuracy of the experimental results, we propose an improved protein imprinting membrane for quickly transferring proteins and a preparation method thereof. Summary of the invention

[0004] The object of the present invention is to provide an improved protein imprinting membrane for rapid protein transfer and a preparation method thereof, so as to solve the problems raised in the above background technology.

[0005] To achieve the above object, the present invention provides an improved protein imprinting membrane for rapid protein transfer, comprising the following components: 10-20 parts by weight of polyvinylidene fluoride, 85-100 parts by weight of N,N-dimethylformamide solvent, 3-5 parts by weight of cellulose nanocrystal enhancer, 1-2 parts by weight of 3-glycidyloxypropyltrimethoxysilane solution, 6-10 parts by weight of ethylenediamine solution, 6-10 parts by weight of sodium periodate solution and 12-15 parts by weight of EDC / NHS crosslinking agent; The EDC / NHS crosslinking agent is a mixture of 5 mg / mL EDC (1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride) and 10 mg / mL NHS (N-hydroxysuccinimide), and the molar ratio of EDC to NHS is 1:1.2-1.5.

[0006] Preferably, the concentration of the 3-glycidyloxypropyltrimethoxysilane solution is 0.8%-1.2% (v / v).

[0007] Preferably, the concentration of the ethylenediamine solution is 0.6-0.8% (w / v), and the concentration of the sodium periodate solution is 0.5-0.7% (w / v).

[0008] On the other hand, the present invention provides a method for preparing an improved protein imprinting membrane for rapid protein transfer, which is used for any of the improved protein imprinting membranes for rapid protein transfer described above, and the preparation method of the improved protein imprinting membrane is as follows: Preparation of polyvinylidene fluoride membrane: S1.1, respectively weigh 10-20 parts by weight of polyvinylidene fluoride, 85-100 parts by weight of N,N-dimethylformamide solvent and 3-5 parts by weight of cellulose nanocrystal enhancer; and dispersing the cellulose nanocrystal enhancer in a portion of the N,N-dimethylformamide solvent to prepare a cellulose nanocrystal suspension; S1.2, dissolving polyvinylidene fluoride in the remaining N,N-dimethylformamide solvent to prepare a polyvinylidene fluoride solution; S1.3, adding the cellulose nanocrystal suspension to the polyvinylidene fluoride solution and stirring until completely dispersed; S1.4, pouring the mixed solution into a mold to form a film by casting; S1.5, placing the film in an oven for heating and curing until the solvent is completely evaporated to obtain a polyvinylidene fluoride film; Cellulose nanocrystals (CNC) can enhance the strength and stability of the membrane due to their high aspect ratio, large specific surface area and excellent mechanical properties; PVDF is a polymer with high chemical stability and good protein adsorption properties; by mixing CNC with PVDF, polyvinylidene fluoride membranes with better performance can be prepared.

[0009] Surface modification of polyvinylidene fluoride membrane: S2.1, respectively weigh 1-2 parts by weight of 3-glycidyloxypropyltrimethoxysilane solution, 6-10 parts by weight of ethylenediamine solution, 6-10 parts by weight of sodium periodate solution and 12-15 parts by weight of a cross-linking agent; At the same time, the surface of the polyvinylidene fluoride membrane is activated by oxygen plasma treatment; oxygen plasma treatment can introduce polar groups (such as hydroxyl and carboxyl groups) to increase the hydrophilicity and surface energy of the membrane, which is beneficial to subsequent chemical reactions; S2.2, soaking the activated polyvinylidene fluoride membrane in a 3-glycidyloxypropyltrimethoxysilane solution for 18-24 hours, introducing epoxy groups on the surface of the polyvinylidene fluoride membrane to provide reaction sites for subsequent amination and carboxylation reactions; S2.3, immersing the polyvinylidene fluoride membrane functionalized with epoxy groups in step S2.2 in an ethylenediamine solution and soaking it for 12-16 hours at a pH of 9-11, so that the ethylenediamine solution reacts with the epoxy groups to generate primary amine groups (-NH2), thereby increasing the positive charge density of the membrane and facilitating the adsorption of proteins; S2.4, immersing the polyvinylidene fluoride membrane treated in step S2.3 in a sodium periodate solution to oxidize the epoxy groups into carboxyl groups (-COOH). The carboxyl groups have a strong charge density and can form stable ionic bonds with the amino groups of the protein, thereby improving the binding ability of the protein; S2.5, dissolving the EDC / NHS crosslinking agent in N,N-dimethylformamide (DMF) solvent to obtain an EDC / NHS solution, and immersing the polyvinylidene fluoride membrane containing carboxyl groups in the EDC / NHS solution, so that the active ester reacts with the amino groups on the membrane surface to form an amide bond; Use EDC / NHS to activate the carboxyl group, so that it can react with the amino group or other groups containing active hydrogen on the membrane surface to form a stable amide bond, further enhancing the binding force between the membrane and the protein; S2.6. After the reaction is completed, the polyvinylidene fluoride membrane is washed with water to remove unreacted EDC / NHS and other by-products; Finally, the polyvinylidene fluoride membrane is placed in an oven to dry to remove residual solvent and water, thereby obtaining an improved protein imprinting membrane; Carboxyl groups were introduced on the membrane surface and further activated by EDC / NHS to enhance the functionality of the membrane, enabling it to better bind proteins; Specifically, the performance of protein imprinted membranes, including protein adsorption capacity, selectivity and stability, can be effectively improved through the composite of cellulose nanocrystals and PVDF and the functional modification of the membrane surface.

[0010] Preferably, in S1.1, the concentration of the cellulose nanocrystal suspension is 2-3% (w / v).

[0011] Preferably, in S1.2, the concentration of the polyvinylidene fluoride solution is 15-20% (w / v).

[0012] Preferably, in S1.5, curing is continued for 3-4 hours at a temperature of 100-150°C.

[0013] Preferably, in S2.1, the specific steps of activating the surface of the polyvinylidene fluoride film by oxygen plasma treatment are: Place the polyvinylidene fluoride membrane in a plasma reactor, close the reactor, start to evacuate until it reaches 160-240 mTorr, start to introduce high-purity oxygen (oxygen purity is above 99.99%), oxygen flow rate is 10-50 sccm, turn on the plasma generator, set the power to 50-300 watts, ionize the oxygen to generate plasma, set the treatment time to 30-45 minutes, after the treatment is completed, stop the plasma generator, wait for the polyvinylidene fluoride membrane to cool to room temperature, slowly introduce air to restore normal pressure, and take out the treated polyvinylidene fluoride membrane; the surface of the polyvinylidene fluoride membrane should be successfully activated, increasing its surface polarity and reactivity, which is beneficial to the subsequent chemical modification steps.

[0014] Preferably, in S2.5, in the EDC / NHS solution, the mixing ratio of the EDC / NHS crosslinking agent to the N,N-dimethylformamide (DMF) solvent is 2:1.

[0015] Preferably, in S2.6, the polyvinylidene fluoride film is dried at 40-60° C. for 16-18 hours.

[0016] Compared with the prior art, the present invention has the following beneficial effects: 1. In the improved protein imprinting membrane for rapid protein transfer and its preparation method, when preparing the polyvinylidene fluoride membrane, cellulose nanocrystal enhancer is added to improve the strength and stability of the membrane; and 3-glycidyloxypropyltrimethoxysilane solution is used to modify the surface of the polyvinylidene fluoride membrane, and amine groups (-NH2) and carboxyl groups (-COOH) are introduced into the polyvinylidene fluoride membrane. These groups form electrostatic interactions with oppositely charged groups on protein molecules, thereby enhancing the adsorption capacity of proteins.

[0017] 2. In the improved protein imprinting membrane for rapid protein transfer and its preparation method, ethylenediamine is used to react with epoxy groups under alkaline conditions to generate primary amine groups (-NH2), and the epoxy groups are oxidized to carboxyl groups (-COOH) by sodium iodate. EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) are used together to connect the carboxyl groups (-COOH) and the primary amine groups (-NH2) to form amide bonds, thereby reducing nonspecific adsorption sites on the membrane surface and improving the specificity and sensitivity of the experiment; Among them, EDC is used to activate the carboxyl group to make it a good leaving group. NHS, as an auxiliary reagent, reacts with the carboxyl group activated by EDC to form an unstable NHS ester intermediate. The NHS ester intermediate reacts with the primary amine group to form a stable amide bond, which can reduce nonspecific adsorption and improve the specificity and sensitivity of the experiment. DETAILED DESCRIPTION

[0018] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0019] The improved protein imprinting membrane for rapid protein transfer of the present invention comprises the following components: 10-20 parts by weight of polyvinylidene fluoride, 85-100 parts by weight of N,N-dimethylformamide solvent, 3-5 parts by weight of cellulose nanocrystal enhancer, 1-2 parts by weight of 3-glycidyloxypropyltrimethoxysilane solution, 6-10 parts by weight of ethylenediamine solution, 6-10 parts by weight of sodium periodate solution and 12-15 parts by weight of EDC / NHS crosslinking agent; The EDC / NHS crosslinking agent is a mixture of 5 mg / mL EDC (1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride) and 10 mg / mL NHS (N-hydroxysuccinimide), and the molar ratio of EDC to NHS is 1:1.2-1.5.

[0020] The concentration of the 3-glycidyloxypropyltrimethoxysilane solution is 0.8%-1.2% (v / v); The concentration of the ethylenediamine solution is 0.6-0.8% (w / v), and the concentration of the sodium periodate solution is 0.5-0.7% (w / v).

[0021] Example 1: A method for preparing an improved protein imprinting membrane for rapid protein transfer, comprising the following steps: The preparation method of the improved protein imprinting membrane is as follows: Preparation of polyvinylidene fluoride membrane: S1.1, weigh 20 parts by weight of polyvinylidene fluoride, 90 parts by weight of N,N-dimethylformamide solvent and 3 parts by weight of cellulose nanocrystal enhancer respectively; and dispersing the cellulose nanocrystal enhancer in a portion of the N,N-dimethylformamide solvent to prepare a cellulose nanocrystal suspension, wherein the concentration of the cellulose nanocrystal suspension is 2.6% (w / v); S1.2, dissolving polyvinylidene fluoride in the remaining N,N-dimethylformamide solvent to prepare a polyvinylidene fluoride solution, wherein the concentration of the polyvinylidene fluoride solution is 18% (w / v); S1.3, adding the cellulose nanocrystal suspension to the polyvinylidene fluoride solution and stirring until completely dispersed; S1.4, pouring the mixed solution into a mold to form a film by casting; S1.5, placing the film in an oven for heating and curing at 130°C for 3 hours until the solvent is completely evaporated to obtain a polyvinylidene fluoride film; Surface modification of polyvinylidene fluoride membrane: S2.1, respectively weigh 2 parts by weight of 1.2% (v / v) 3-glycidyloxypropyltrimethoxysilane solution, 8 parts by weight of 0.6% (w / v) ethylenediamine solution, 8 parts by weight of 0.7% sodium periodate solution and 12 parts by weight of a cross-linking agent; Meanwhile, the surface of the polyvinylidene fluoride membrane was activated by oxygen plasma treatment; The specific steps for activating the surface of the polyvinylidene fluoride membrane by oxygen plasma treatment are: Place the polyvinylidene fluoride membrane in a plasma reactor, close the reactor, start to evacuate until it reaches 160 mTorr, start to introduce high-purity oxygen with an oxygen flow rate of 18 sccm, start the plasma generator, set the power to 180 watts, ionize the oxygen to generate plasma, set the treatment time to 30 minutes, and after the treatment is completed, stop the plasma generator, wait for the polyvinylidene fluoride membrane to cool to room temperature, slowly introduce air to restore normal pressure, and take out the treated polyvinylidene fluoride membrane; S2.2, immersing the activated polyvinylidene fluoride membrane in a 3-glycidyloxypropyltrimethoxysilane solution, soaking and reacting for 18 hours, and introducing epoxy groups on the surface of the polyvinylidene fluoride membrane; S2.3, immersing the polyvinylidene fluoride membrane functionalized with epoxy groups in step S2.2 in an ethylenediamine solution, and immersing for 12 hours at a pH of 9, so that the ethylenediamine solution reacts with the epoxy groups to generate primary amine groups (-NH2); S2.4, immersing the polyvinylidene fluoride membrane treated in step S2.3 into a sodium periodate solution to oxidize the epoxy groups into carboxyl groups (-COOH); S2.5, dissolving the EDC / NHS crosslinking agent in N,N-dimethylformamide (DMF) solvent to obtain an EDC / NHS solution, wherein in the EDC / NHS solution, the mixing ratio of the EDC / NHS crosslinking agent to the N,N-dimethylformamide (DMF) solvent is 2:1; The EDC / NHS crosslinker was a mixture of 5 mg / mL EDC (1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride) and 10 mg / mL NHS (N-hydroxysuccinimide), and the molar ratio of EDC to NHS was 1:1.2; The polyvinylidene fluoride membrane containing carboxyl groups is immersed in an EDC / NHS solution, and the active ester reacts with the amino groups on the membrane surface to form an amide bond; S2.6. After the reaction is completed, the polyvinylidene fluoride membrane is washed with water to remove unreacted EDC / NHS and other by-products; Finally, the polyvinylidene fluoride membrane was placed in an oven and dried at 50°C for 16 hours to remove residual solvent and moisture, thereby obtaining an improved protein imprinting membrane.

[0022] Example 2: A method for preparing an improved protein imprinting membrane for rapid protein transfer, comprising the following steps: The preparation method of the improved protein imprinting membrane is as follows: Preparation of polyvinylidene fluoride membrane: S1.1, weigh 20 parts by weight of polyvinylidene fluoride, 90 parts by weight of N,N-dimethylformamide solvent and 4 parts by weight of cellulose nanocrystal enhancer respectively; and dispersing the cellulose nanocrystal enhancer in a portion of the N,N-dimethylformamide solvent to prepare a cellulose nanocrystal suspension, wherein the concentration of the cellulose nanocrystal suspension is 2.6% (w / v); S1.2, dissolving polyvinylidene fluoride in the remaining N,N-dimethylformamide solvent to prepare a polyvinylidene fluoride solution, wherein the concentration of the polyvinylidene fluoride solution is 18% (w / v); S1.3, adding the cellulose nanocrystal suspension to the polyvinylidene fluoride solution and stirring until completely dispersed; S1.4, pouring the mixed solution into a mold to form a film by casting; S1.5, placing the film in an oven for heating and curing at 130°C for 3 hours until the solvent is completely evaporated to obtain a polyvinylidene fluoride film; Surface modification of polyvinylidene fluoride membrane: S2.1, respectively weigh 2 parts by weight of 1.2% (v / v) 3-glycidyloxypropyltrimethoxysilane solution, 8 parts by weight of 0.6% (w / v) ethylenediamine solution, 8 parts by weight of 0.7% sodium periodate solution and 12 parts by weight of a cross-linking agent; Meanwhile, the surface of the polyvinylidene fluoride membrane was activated by oxygen plasma treatment; The specific steps for activating the surface of the polyvinylidene fluoride membrane by oxygen plasma treatment are: Place the polyvinylidene fluoride membrane in a plasma reactor, close the reactor, start to evacuate until it reaches 160 mTorr, start to introduce high-purity oxygen with an oxygen flow rate of 18 sccm, start the plasma generator, set the power to 180 watts, ionize the oxygen to generate plasma, set the treatment time to 30 minutes, and after the treatment is completed, stop the plasma generator, wait for the polyvinylidene fluoride membrane to cool to room temperature, slowly introduce air to restore normal pressure, and take out the treated polyvinylidene fluoride membrane; S2.2, immersing the activated polyvinylidene fluoride membrane in a 3-glycidyloxypropyltrimethoxysilane solution, soaking and reacting for 18 hours, and introducing epoxy groups on the surface of the polyvinylidene fluoride membrane; S2.3, immersing the polyvinylidene fluoride membrane functionalized with epoxy groups in step S2.2 in an ethylenediamine solution, and immersing for 12 hours at a pH of 9, so that the ethylenediamine solution reacts with the epoxy groups to generate primary amine groups (-NH2); S2.4, immersing the polyvinylidene fluoride membrane treated in step S2.3 into a sodium periodate solution to oxidize the epoxy groups into carboxyl groups (-COOH); S2.5, dissolving the EDC / NHS crosslinking agent in N,N-dimethylformamide (DMF) solvent to obtain an EDC / NHS solution, wherein in the EDC / NHS solution, the mixing ratio of the EDC / NHS crosslinking agent to the N,N-dimethylformamide (DMF) solvent is 2:1; The EDC / NHS crosslinker was a mixture of 5 mg / mL EDC (1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride) and 10 mg / mL NHS (N-hydroxysuccinimide), and the molar ratio of EDC to NHS was 1:1.2; The polyvinylidene fluoride membrane containing carboxyl groups is immersed in EDC / NHS solution, and the active ester reacts with the amino groups on the membrane surface to form amide bonds; Use EDC / NHS to activate the carboxyl group so that it can react with the amino group or other groups containing active hydrogen on the membrane surface to form a stable amide bond; S2.6. After the reaction is completed, the polyvinylidene fluoride membrane is washed with water to remove unreacted EDC / NHS and other by-products; Finally, the polyvinylidene fluoride membrane was placed in an oven and dried at 50°C for 16 hours to remove residual solvent and moisture, thereby obtaining an improved protein imprinting membrane.

[0023] Example 3: A method for preparing an improved protein imprinting membrane for rapid protein transfer, comprising the following steps: The preparation method of the improved protein imprinting membrane is as follows: Preparation of polyvinylidene fluoride membrane: S1.1, weigh 20 parts by weight of polyvinylidene fluoride, 90 parts by weight of N,N-dimethylformamide solvent and 5 parts by weight of cellulose nanocrystal enhancer respectively; and dispersing the cellulose nanocrystal enhancer in a portion of the N,N-dimethylformamide solvent to prepare a cellulose nanocrystal suspension, wherein the concentration of the cellulose nanocrystal suspension is 2.6% (w / v); S1.2, dissolving polyvinylidene fluoride in the remaining N,N-dimethylformamide solvent to prepare a polyvinylidene fluoride solution, wherein the concentration of the polyvinylidene fluoride solution is 18% (w / v); S1.3, adding the cellulose nanocrystal suspension to the polyvinylidene fluoride solution and stirring until completely dispersed; S1.4, pouring the mixed solution into a mold to form a film by casting; S1.5, placing the film in an oven for heating and curing at 130°C for 3 hours until the solvent is completely evaporated to obtain a polyvinylidene fluoride film; Surface modification of polyvinylidene fluoride membrane: S2.1, respectively weigh 2 parts by weight of 1.2% (v / v) 3-glycidyloxypropyltrimethoxysilane solution, 8 parts by weight of 0.6% (w / v) ethylenediamine solution, 8 parts by weight of 0.7% sodium periodate solution and 12 parts by weight of a cross-linking agent; Meanwhile, the surface of the polyvinylidene fluoride membrane was activated by oxygen plasma treatment; The specific steps for activating the surface of the polyvinylidene fluoride membrane by oxygen plasma treatment are: Place the polyvinylidene fluoride membrane in a plasma reactor, close the reactor, start to evacuate until it reaches 160 mTorr, start to introduce high-purity oxygen with an oxygen flow rate of 18 sccm, start the plasma generator, set the power to 180 watts, ionize the oxygen to generate plasma, set the treatment time to 30 minutes, and after the treatment is completed, stop the plasma generator, wait for the polyvinylidene fluoride membrane to cool to room temperature, slowly introduce air to restore normal pressure, and take out the treated polyvinylidene fluoride membrane; S2.2, immersing the activated polyvinylidene fluoride membrane in a 3-glycidyloxypropyltrimethoxysilane solution, soaking and reacting for 18 hours, and introducing epoxy groups on the surface of the polyvinylidene fluoride membrane; S2.3, immersing the polyvinylidene fluoride membrane functionalized with epoxy groups in step S2.2 in an ethylenediamine solution, and immersing for 12 hours at a pH of 9, so that the ethylenediamine solution reacts with the epoxy groups to generate primary amine groups (-NH2); S2.4, immersing the polyvinylidene fluoride membrane treated in step S2.3 into a sodium periodate solution to oxidize the epoxy groups into carboxyl groups (-COOH); S2.5, dissolving the EDC / NHS crosslinking agent in N,N-dimethylformamide (DMF) solvent to obtain an EDC / NHS solution, wherein in the EDC / NHS solution, the mixing ratio of the EDC / NHS crosslinking agent to the N,N-dimethylformamide (DMF) solvent is 2:1; The EDC / NHS crosslinker was a mixture of 5 mg / mL EDC (1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride) and 10 mg / mL NHS (N-hydroxysuccinimide), and the molar ratio of EDC to NHS was 1:1.2; The polyvinylidene fluoride membrane containing carboxyl groups is immersed in EDC / NHS solution, and the active ester reacts with the amino groups on the membrane surface to form amide bonds; Use EDC / NHS to activate the carboxyl group so that it can react with the amino group or other groups containing active hydrogen on the membrane surface to form a stable amide bond; S2.6. After the reaction is completed, the polyvinylidene fluoride membrane is washed with water to remove unreacted EDC / NHS and other by-products; Finally, the polyvinylidene fluoride membrane was placed in an oven and dried at 50°C for 16 hours to remove residual solvent and moisture, thereby obtaining an improved protein imprinting membrane.

[0024] Example 4: A method for preparing an improved protein imprinting membrane for rapid protein transfer, comprising the following steps: The preparation method of the improved protein imprinting membrane is as follows: Preparation of polyvinylidene fluoride membrane: S1.1, weigh 20 parts by weight of polyvinylidene fluoride, 90 parts by weight of N,N-dimethylformamide solvent and 5 parts by weight of cellulose nanocrystal enhancer respectively; and dispersing the cellulose nanocrystal enhancer in a portion of the N,N-dimethylformamide solvent to prepare a cellulose nanocrystal suspension, wherein the concentration of the cellulose nanocrystal suspension is 2.6% (w / v); S1.2, dissolving polyvinylidene fluoride in the remaining N,N-dimethylformamide solvent to prepare a polyvinylidene fluoride solution, wherein the concentration of the polyvinylidene fluoride solution is 18% (w / v); S1.3, adding the cellulose nanocrystal suspension to the polyvinylidene fluoride solution and stirring until completely dispersed; S1.4, pouring the mixed solution into a mold to form a film by casting; S1.5, placing the film in an oven for heating and curing at 130°C for 3 hours until the solvent is completely evaporated to obtain a polyvinylidene fluoride film; Surface modification of polyvinylidene fluoride membrane: S2.1, respectively weighing 1 part by weight of 1.2% (v / v) 3-glycidyloxypropyltrimethoxysilane solution, 8 parts by weight of 0.6% (w / v) ethylenediamine solution, 8 parts by weight of 0.7% sodium periodate solution and 12 parts by weight of a cross-linking agent; Meanwhile, the surface of the polyvinylidene fluoride membrane was activated by oxygen plasma treatment; The specific steps for activating the surface of the polyvinylidene fluoride membrane by oxygen plasma treatment are: Place the polyvinylidene fluoride membrane in a plasma reactor, close the reactor, start to evacuate until it reaches 160 mTorr, start to introduce high-purity oxygen with an oxygen flow rate of 18 sccm, start the plasma generator, set the power to 180 watts, ionize the oxygen to generate plasma, set the treatment time to 30 minutes, and after the treatment is completed, stop the plasma generator, wait for the polyvinylidene fluoride membrane to cool to room temperature, slowly introduce air to restore normal pressure, and take out the treated polyvinylidene fluoride membrane; S2.2, immersing the activated polyvinylidene fluoride membrane in a 3-glycidyloxypropyltrimethoxysilane solution, soaking and reacting for 18 hours, and introducing epoxy groups on the surface of the polyvinylidene fluoride membrane; S2.3, immersing the polyvinylidene fluoride membrane functionalized with epoxy groups in step S2.2 in an ethylenediamine solution, and immersing for 12 hours at a pH of 9, so that the ethylenediamine solution reacts with the epoxy groups to generate primary amine groups (-NH2); S2.4, immersing the polyvinylidene fluoride membrane treated in step S2.3 into a sodium periodate solution to oxidize the epoxy groups into carboxyl groups (-COOH); S2.5, dissolving the EDC / NHS crosslinking agent in N,N-dimethylformamide (DMF) solvent to obtain an EDC / NHS solution, wherein in the EDC / NHS solution, the mixing ratio of the EDC / NHS crosslinking agent to the N,N-dimethylformamide (DMF) solvent is 2:1; The EDC / NHS crosslinker was a mixture of 5 mg / mL EDC (1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride) and 10 mg / mL NHS (N-hydroxysuccinimide), and the molar ratio of EDC to NHS was 1:1.2; The polyvinylidene fluoride membrane containing carboxyl groups is immersed in an EDC / NHS solution, and the active ester reacts with the amino groups on the membrane surface to form an amide bond; Use EDC / NHS to activate the carboxyl group so that it can react with the amino group or other groups containing active hydrogen on the membrane surface to form a stable amide bond; S2.6. After the reaction is completed, the polyvinylidene fluoride membrane is washed with water to remove unreacted EDC / NHS and other by-products; Finally, the polyvinylidene fluoride membrane was placed in an oven and dried at 50°C for 16 hours to remove residual solvent and moisture, thereby obtaining an improved protein imprinting membrane.

[0025] Example 5: A method for preparing an improved protein imprinting membrane for rapid protein transfer, comprising the following steps: The preparation method of the improved protein imprinting membrane is as follows: Preparation of polyvinylidene fluoride membrane: S1.1, weigh 20 parts by weight of polyvinylidene fluoride, 90 parts by weight of N,N-dimethylformamide solvent and 5 parts by weight of cellulose nanocrystal enhancer respectively; and dispersing the cellulose nanocrystal enhancer in a portion of the N,N-dimethylformamide solvent to prepare a cellulose nanocrystal suspension, wherein the concentration of the cellulose nanocrystal suspension is 2.6% (w / v); S1.2, dissolving polyvinylidene fluoride in the remaining N,N-dimethylformamide solvent to prepare a polyvinylidene fluoride solution, wherein the concentration of the polyvinylidene fluoride solution is 18% (w / v); S1.3, adding the cellulose nanocrystal suspension to the polyvinylidene fluoride solution and stirring until completely dispersed; S1.4, pouring the mixed solution into a mold to form a film by casting; S1.5, placing the film in an oven for heating and curing at 130°C for 3 hours until the solvent is completely evaporated to obtain a polyvinylidene fluoride film; Surface modification of polyvinylidene fluoride membrane: S2.1, respectively weigh 2 parts by weight of 1.2% (v / v) 3-glycidyloxypropyltrimethoxysilane solution, 8 parts by weight of 0.6% (w / v) ethylenediamine solution, 8 parts by weight of 0.7% sodium periodate solution and 15 parts by weight of a cross-linking agent; Meanwhile, the surface of the polyvinylidene fluoride membrane was activated by oxygen plasma treatment; The specific steps for activating the surface of the polyvinylidene fluoride membrane by oxygen plasma treatment are: Place the polyvinylidene fluoride membrane in a plasma reactor, close the reactor, start to evacuate until it reaches 160 mTorr, start to introduce high-purity oxygen with an oxygen flow rate of 18 sccm, start the plasma generator, set the power to 180 watts, ionize the oxygen to generate plasma, set the treatment time to 30 minutes, and after the treatment is completed, stop the plasma generator, wait for the polyvinylidene fluoride membrane to cool to room temperature, slowly introduce air to restore normal pressure, and take out the treated polyvinylidene fluoride membrane; S2.2, immersing the activated polyvinylidene fluoride membrane in a 3-glycidyloxypropyltrimethoxysilane solution, soaking and reacting for 18 hours, and introducing epoxy groups on the surface of the polyvinylidene fluoride membrane; S2.3, immersing the polyvinylidene fluoride membrane functionalized with epoxy groups in step S2.2 in an ethylenediamine solution, and immersing for 12 hours at a pH of 9, so that the ethylenediamine solution reacts with the epoxy groups to generate primary amine groups (-NH2); S2.4, immersing the polyvinylidene fluoride membrane treated in step S2.3 into a sodium periodate solution to oxidize the epoxy groups into carboxyl groups (-COOH); S2.5, dissolving the EDC / NHS crosslinking agent in N,N-dimethylformamide (DMF) solvent to obtain an EDC / NHS solution, wherein in the EDC / NHS solution, the mixing ratio of the EDC / NHS crosslinking agent to the N,N-dimethylformamide (DMF) solvent is 2:1; The EDC / NHS crosslinker was a mixture of 5 mg / mL EDC (1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride) and 10 mg / mL NHS (N-hydroxysuccinimide), and the molar ratio of EDC to NHS was 1:1.2; The polyvinylidene fluoride membrane containing carboxyl groups is immersed in an EDC / NHS solution, and the active ester reacts with the amino groups on the membrane surface to form an amide bond; Use EDC / NHS to activate the carboxyl group so that it can react with the amino group or other groups containing active hydrogen on the membrane surface to form a stable amide bond; S2.6. After the reaction is completed, the polyvinylidene fluoride membrane is washed with water to remove unreacted EDC / NHS and other by-products; Finally, the polyvinylidene fluoride membrane was placed in an oven and dried at 50°C for 16 hours to remove residual solvent and moisture, thereby obtaining an improved protein imprinting membrane.

[0026] Example 6: A method for preparing an improved protein imprinting membrane for rapid protein transfer, comprising the following steps: The preparation method of the improved protein imprinting membrane is as follows: Preparation of polyvinylidene fluoride membrane: S1.1, weigh 20 parts by weight of polyvinylidene fluoride, 90 parts by weight of N,N-dimethylformamide solvent and 5 parts by weight of cellulose nanocrystal enhancer respectively; and dispersing the cellulose nanocrystal enhancer in a portion of the N,N-dimethylformamide solvent to prepare a cellulose nanocrystal suspension, wherein the concentration of the cellulose nanocrystal suspension is 2.6% (w / v); S1.2, dissolving polyvinylidene fluoride in the remaining N,N-dimethylformamide solvent to prepare a polyvinylidene fluoride solution, wherein the concentration of the polyvinylidene fluoride solution is 18% (w / v); S1.3, adding the cellulose nanocrystal suspension to the polyvinylidene fluoride solution and stirring until completely dispersed; S1.4, pouring the mixed solution into a mold to form a film by casting; S1.5, placing the film in an oven for heating and curing at 130°C for 3 hours until the solvent is completely evaporated to obtain a polyvinylidene fluoride film; Surface modification of polyvinylidene fluoride membrane: S2.1, respectively weigh 2 parts by weight of 1.2% (v / v) 3-glycidyloxypropyltrimethoxysilane solution, 8 parts by weight of 0.6% (w / v) ethylenediamine solution, 8 parts by weight of 0.7% sodium periodate solution and 15 parts by weight of a cross-linking agent; Meanwhile, the surface of the polyvinylidene fluoride membrane was activated by oxygen plasma treatment; The specific steps for activating the surface of the polyvinylidene fluoride membrane by oxygen plasma treatment are: Place the polyvinylidene fluoride membrane in a plasma reactor, close the reactor, start to evacuate until it reaches 160 mTorr, start to introduce high-purity oxygen with an oxygen flow rate of 18 sccm, start the plasma generator, set the power to 180 watts, ionize the oxygen to generate plasma, set the treatment time to 30 minutes, and after the treatment is completed, stop the plasma generator, wait for the polyvinylidene fluoride membrane to cool to room temperature, slowly introduce air to restore normal pressure, and take out the treated polyvinylidene fluoride membrane; S2.2, immersing the activated polyvinylidene fluoride membrane in a 3-glycidyloxypropyltrimethoxysilane solution, soaking and reacting for 18 hours, and introducing epoxy groups on the surface of the polyvinylidene fluoride membrane; S2.3, immersing the polyvinylidene fluoride membrane functionalized with epoxy groups in step S2.2 in an ethylenediamine solution, and immersing for 12 hours at a pH of 9, so that the ethylenediamine solution reacts with the epoxy groups to generate primary amine groups (-NH2); S2.4, immersing the polyvinylidene fluoride membrane treated in step S2.3 into a sodium periodate solution to oxidize the epoxy groups into carboxyl groups (-COOH); S2.5, dissolving the EDC / NHS crosslinking agent in N,N-dimethylformamide (DMF) solvent to obtain an EDC / NHS solution, wherein in the EDC / NHS solution, the mixing ratio of the EDC / NHS crosslinking agent to the N,N-dimethylformamide (DMF) solvent is 2:1; The EDC / NHS crosslinker was a mixture of 5 mg / mL EDC (1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride) and 10 mg / mL NHS (N-hydroxysuccinimide), and the molar ratio of EDC to NHS was 1:1.5; The polyvinylidene fluoride membrane containing carboxyl groups is immersed in EDC / NHS solution, and the active ester reacts with the amino groups on the membrane surface to form amide bonds; Use EDC / NHS to activate the carboxyl group so that it can react with the amino group or other groups containing active hydrogen on the membrane surface to form a stable amide bond; S2.6. After the reaction is completed, the polyvinylidene fluoride membrane is washed with water to remove unreacted EDC / NHS and other by-products; Finally, the polyvinylidene fluoride membrane was placed in an oven and dried at 50°C for 16 hours to remove residual solvent and moisture, thereby obtaining an improved protein imprinting membrane.

[0027] Comparative Example 1: The method of Example 5 was adopted to prepare the polyvinylidene fluoride membrane without adding the cellulose nanocrystal reinforcing agent.

[0028] Comparative Example 2: The method of Example 5 was used, but during the surface modification of the polyvinylidene fluoride membrane, the membrane was not activated by oxygen plasma treatment.

[0029] Comparative Example 3: The method of Example 5 was adopted, but no EDC / NHS crosslinking agent was added during the surface modification of the polyvinylidene fluoride membrane.

[0030] Comparative Example 4: The method of Example 5 was adopted, and in the process of surface modification of polyvinylidene fluoride membrane, DCC / WSC crosslinking agent was selected to replace EDC / NHS crosslinking agent.

[0031] Comparative Example 5: The method of Example 5 was adopted without adding 3-glycidyloxypropyltrimethoxysilane solution.

[0032] The improved protein imprinting membrane for rapid protein transfer prepared by the present invention, wherein the performance index inspection items and inspection standards of the improved protein imprinting membrane for rapid protein transfer are as follows: (1) Mechanical strength test: The prepared improved protein imprinted membrane was fixed on the testing device, tensile force was applied, the stress-strain curve of the membrane was recorded, and the fracture strength and toughness of the improved protein imprinted membrane were calculated; (2) Thermal stability test: The prepared improved protein imprinted membrane was placed in an oven, and different temperature points were selected for testing. The improved protein imprinted membrane sample was placed in an oven at a preset temperature and heated at each temperature point for 1 hour. After heating at each temperature point, the sample was taken out and the changes in its physical and chemical properties were observed. The color and morphology of the membrane were observed, and the thermal stability of the membrane was evaluated based on the observed changes. Specifically, by weighing the mass of the sample before and after heating, the percentage of mass loss can be calculated to quantify the thermal stability, and the thermal stability can be evaluated by the mass loss rate (%). (3) Hydrophilicity test: The water absorption test is used to evaluate the ability of the material to absorb water. First, the mass of the dry sample of the improved protein imprinted membrane is weighed, and the prepared improved protein imprinted membrane is immersed in water for a period of time. The sample is taken out, and the excess water on the surface is gently removed, and then it is weighed immediately. The water absorption is calculated based on the difference between the initial mass of the sample and the mass after immersion. The greater the water absorption, the stronger the hydrophilicity of the material. The percentage of water absorption is calculated to more intuitively represent the hydrophilicity of the material. The percentage of water absorption ;in, It is the wet weight of the sample after immersion to remove excess water. Indicates the original mass of the sample when dry; (4) Adsorption capacity test: Place the prepared membrane into a protein solution to allow it to adsorb protein; take out the membrane after adsorption and gently absorb the excess protein solution on the surface with filter paper; place the membrane into a quantitative bottle, add washing solution, and shake to wash; determine the protein concentration in the washing solution and calculate the adsorption capacity of the membrane.

[0033] The improved protein imprinting membranes prepared in the above Examples 1-6 and Comparative Examples 1-5 were tested according to the above standards, and the obtained data are shown in Table 1: Table 1 Performance data of the improved protein imprinting membranes of Examples 1-6 and Comparative Examples 1-5

[0034] The above data fully show that compared with Comparative Examples 1-5, Examples 1-6 show that the cellulose nanocrystal enhancer, 3-glycidyloxypropyltrimethoxysilane and EDC / NHS crosslinker have a positive effect on the mechanical properties, thermal stability, hydrophilicity and protein adsorption capacity of the improved protein imprinting membrane, as follows: It can be seen from Examples 1-3 that: With the continuous increase in the weight of the cellulose nanocrystal reinforcing agent, the fracture strength and toughness of the improved protein imprinted membrane are significantly increased, and at the same time, the mass loss rate of the improved protein imprinted membrane is also continuously reduced. Compared with Comparative Example 1, it can be seen that when preparing the polyvinylidene fluoride membrane without adding the cellulose nanocrystal reinforcing agent, the fracture strength and toughness of the improved protein imprinted membrane are significantly reduced, and the mass loss rate is significantly increased. Therefore, it can be explained that cellulose nanocrystals as a reinforcing agent can significantly improve the mechanical strength and toughness of the membrane, and at the same time, it is also helpful to improve the thermal stability of the membrane; at the same time, because cellulose nanocrystals have a certain hydrophilicity, at the same time, the overall hydrophilicity of the membrane can be improved, and proteins can be more effectively adsorbed, and high stability and selectivity can be maintained; It can be seen from Examples 3-4 that when the weight of other components remains unchanged, as the weight of 3-glycidyloxypropyltrimethoxysilane (GPTMS) solution decreases, the water absorption percentage decreases significantly. By comparing Examples 3-4 with Comparative Example 5, it can be seen that when no 3-glycidyloxypropyltrimethoxysilane solution is added, the water absorption percentage decreases significantly. It can be seen that the content of 3-glycidyloxypropyltrimethoxysilane solution has a significant effect on the hydrophilicity of the improved protein imprinting membrane. GPTMS itself contains polar groups, which can improve the hydrophilicity of the membrane, which is very beneficial for protein adsorption. The improvement of hydrophilicity is conducive to the uniform distribution of proteins on the membrane and better adsorption efficiency. At the same time, GPTMS introduces epoxy groups (-CH2CH(OH)CH2-) to the surface of polyvinylidene fluoride (PVDF) membrane by condensation reaction with hydroxyl groups (-OH) on the surface of improved protein imprinting membrane; the reaction occurs on polar groups such as hydroxyl and carboxyl groups introduced after oxygen plasma treatment, which increases the reactivity of the membrane surface; the carboxyl groups form stable amide bonds with the amino groups of proteins through EDC / NHS activation. The presence of carboxyl groups increases the negative charge density of the membrane, can form electrostatic interactions with the positively charged groups of proteins, and enhance the binding ability of proteins.

[0035] At the same time, combined with Examples 1-4, it can be seen that with the continuous increase in the weight of the cellulose nanocrystal enhancer and the GPTMS solution, the adsorption capacity of the improved protein imprinting membrane for proteins is significantly improved: Among them, the surface of cellulose nanocrystals is rich in hydroxyl groups, which can improve the hydrophilicity of the membrane and thus increase the membrane's ability to adsorb proteins; The GPTMS solution is connected to the membrane surface through covalent bonds, and epoxy groups are introduced on the surface. By introducing new chemical groups, more reaction sites are provided for the membrane surface, thereby enhancing the adsorption capacity of proteins. The protein adsorption capacity of the membrane was significantly improved by the addition of CNC and surface modification of GPTMS. CNC enhanced the mechanical properties of the membrane and increased the number of reaction sites, while GPTMS introduced new chemical functional groups and increased the hydrophilicity and reactivity of the membrane. The combined effect of CNC and GPTMS not only increases the amount of protein adsorption, but also improves the stability of adsorption. For example, the amine and carboxyl groups introduced by GPTMS can form stable ionic bonds or hydrogen bonds with the corresponding groups on the protein.

[0036] It can be seen from Examples 3 and 5 that: as the weight of the EDC / NHS cross-linking agent increases, the adsorption capacity of the prepared improved protein imprinting membrane for proteins is significantly improved. This is because when EDC (water-soluble carbodiimide) is used together with NHS (N-hydroxysuccinimide), the carboxyl group can be activated and converted into an active ester (O-acyl-N-hydroxysuccinimide ester), which can react with a primary amine group (-NH2); through the activation of EDC / NHS, the carboxyl group on the membrane surface reacts with the primary amine group on the protein to form a stable amide bond, which can not only increase the adsorption amount of the protein, but also improve the stability of the binding; by forming an amide bond, the functionality of the membrane surface is enhanced, thereby improving the selectivity of the membrane and the binding capacity of the protein.

[0037] It can be seen from Examples 5 and 6 that when the molar ratio of NHS (N-hydroxysuccinimide) in the EDC / NHS crosslinker is increased, the improved protein imprinting membrane has no significant changes except for a slight improvement in thermal stability. Since the proportion of NHS will indirectly improve thermal stability by promoting the formation of more amide bonds, the main function of the EDC / NHS crosslinker is to enhance the functionality of the membrane, especially to improve the binding capacity of proteins. Increasing the proportion of NHS can ensure more efficient amide bond formation, but this has little effect on the basic physical properties of the membrane (such as breaking strength and toughness), but may have a positive effect on the chemical stability of the membrane (such as thermal stability and hydrophilicity).

[0038] According to the above test experiments, the improved protein imprinting membrane for rapid protein transfer prepared according to Example 5 has the best performance, so Example 5 is taken as the best example; By comparing Example 5 with Comparative Example 3, it can be seen that: during the surface modification of the polyvinylidene fluoride membrane, when the EDC / NHS cross-linking agent is not added, there is no EDC / NHS to activate the carboxyl groups, and the carboxyl groups on the membrane surface cannot effectively react with the amino groups on the protein to form amide bonds, resulting in a significant decrease in the protein adsorption capacity. At the same time, without the amide bonds as additional binding force, the stability of the formed protein-membrane complex will be reduced.

[0039] By comparing Example 5 with Comparative Example 4, it can be seen that when DCC / WSC crosslinking agent is used to replace EDC / NHS crosslinking agent in the process of surface modification of polyvinylidene fluoride membrane, the performance data of the prepared improved protein imprinting membrane are all lower than those of EDC / NHS crosslinking agent. It can be seen that EDC / NHS crosslinker has better protein adsorption capacity, stability and other performance indicators.

[0040] By comparing Example 5 with Comparative Example 2, it can be seen that: in the process of surface modification of the polyvinylidene fluoride membrane, the membrane was not activated by oxygen plasma treatment, and the water absorption percentage of the prepared improved protein imprinting membrane was significantly reduced, and the protein adsorption capacity of the membrane was significantly reduced; since the membrane surface has not been activated, it may lead to reduced interaction with subsequent modification reagents, and without oxygen plasma treatment, the membrane surface lacks sufficient polar groups, resulting in poor hydrophilicity of the membrane, affecting the adsorption efficiency of proteins thereon.

[0041] In summary, oxygen plasma treatment is used to activate the membrane surface and increase the surface polarity and hydrophilicity, which is conducive to the subsequent chemical reactions; Use compounds containing epoxy groups (such as 3-glycidyloxypropyltrimethoxysilane, GPTMS) to react with the activated membrane, which can introduce epoxy groups on the membrane surface. These epoxy groups can be further converted into other functional groups; Using ethylenediamine, it reacts with epoxy groups under alkaline conditions to generate primary amine groups (-NH2); Sodium periodate is used to oxidize the epoxy group to a carboxyl group (-COOH). When EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) are used together, the carboxyl group (-COOH) and the primary amine group (-NH2) can be effectively connected to form an amide bond. Among them, EDC is used to activate the carboxyl group to make it a good leaving group. NHS is used as an auxiliary reagent to react with the carboxyl group activated by EDC to form an unstable NHS ester intermediate. The NHS ester intermediate reacts with the primary amine group to form a stable amide bond, which can reduce nonspecific adsorption and improve the specificity and sensitivity of the experiment.

[0042] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. The above embodiments and descriptions are only preferred examples of the present invention and are not intended to limit the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. An improved protein imprinting membrane for rapid protein transfer, characterized in that: The invention comprises the following components: 10-20 parts by weight of polyvinylidene fluoride, 85-100 parts by weight of N,N-dimethylformamide solvent, 3-5 parts by weight of cellulose nanocrystal reinforcing agent, 1-2 parts by weight of 3-glycidyloxypropyltrimethoxysilane solution, 6-10 parts by weight of ethylenediamine solution, 6-10 parts by weight of sodium periodate solution and 12-15 parts by weight of EDC / NHS crosslinking agent; The EDC / NHS cross-linking agent is a mixture of 5 mg / mL EDC and 10 mg / mL NHS, and the molar ratio of EDC to NHS is 1:1.2-1.

5.

2. The improved protein imprinting membrane for rapid protein transfer according to claim 1, characterized in that: The concentration of the 3-glycidyloxypropyltrimethoxysilane solution is 0.8%-1.2% (v / v).

3. The improved protein imprinting membrane for rapid protein transfer according to claim 1, characterized in that: The concentration of the ethylenediamine solution is 0.6-0.8% (w / v), and the concentration of the sodium periodate solution is 0.5-0.7% (w / v).

4. A method for preparing an improved protein imprinting membrane for rapid protein transfer, which is used to prepare the improved protein imprinting membrane for rapid protein transfer as claimed in any one of claims 1 to 3, characterized in that: The preparation method of the improved protein imprinting membrane is as follows: Preparation of polyvinylidene fluoride membrane: S1.1, respectively weigh 10-20 parts by weight of polyvinylidene fluoride, 85-100 parts by weight of N,N-dimethylformamide solvent and 3-5 parts by weight of cellulose nanocrystal enhancer; and dispersing the cellulose nanocrystal enhancer in a portion of the N,N-dimethylformamide solvent to prepare a cellulose nanocrystal suspension; S1.2, dissolving polyvinylidene fluoride in the remaining N,N-dimethylformamide solvent to prepare a polyvinylidene fluoride solution; S1.3, adding the cellulose nanocrystal suspension to the polyvinylidene fluoride solution and stirring until completely dispersed; S1.4, pouring the mixed solution into a mold to form a film by casting; S1.5, placing the film in an oven for heating and curing until the solvent is completely evaporated to obtain a polyvinylidene fluoride film; Surface modification of polyvinylidene fluoride membrane: S2.1, respectively weigh 1-2 parts by weight of 3-glycidyloxypropyltrimethoxysilane solution, 6-10 parts by weight of ethylenediamine solution, 6-10 parts by weight of sodium periodate solution and 12-15 parts by weight of a cross-linking agent; Meanwhile, the surface of the polyvinylidene fluoride membrane was activated by oxygen plasma treatment; S2.2, immersing the activated polyvinylidene fluoride membrane in a 3-glycidyloxypropyltrimethoxysilane solution, soaking and reacting for 18-24 hours, and introducing epoxy groups on the surface of the polyvinylidene fluoride membrane; S2.3, immersing the polyvinylidene fluoride membrane functionalized with epoxy groups in step S2.2 in an ethylenediamine solution, and soaking for 12-16 hours at a pH of 9-11, so that the ethylenediamine solution reacts with the epoxy groups to generate primary amine groups; S2.4, immersing the polyvinylidene fluoride membrane treated in step S2.3 into a sodium periodate solution to oxidize the epoxy groups into carboxyl groups; S2.5, dissolving an EDC / NHS crosslinking agent in an N,N-dimethylformamide solvent to obtain an EDC / NHS solution, and immersing a polyvinylidene fluoride membrane containing a carboxyl group in the EDC / NHS solution to form an amide bond; S2.

6. After the reaction is completed, the polyvinylidene fluoride membrane is washed with water to remove unreacted EDC / NHS and other by-products; Finally, the polyvinylidene fluoride membrane is placed in an oven to dry to remove residual solvent and moisture, thereby obtaining an improved protein imprinting membrane.

5. The method for preparing the improved protein imprinting membrane for rapid protein transfer according to claim 4, characterized in that: In S1.1, the concentration of the cellulose nanocrystal suspension is 2-3% (w / v).

6. The method for preparing the improved protein imprinting membrane for rapid protein transfer according to claim 4, characterized in that: In S1.2, the concentration of the polyvinylidene fluoride solution is 15-20% (w / v).

7. The method for preparing the improved protein imprinting membrane for rapid protein transfer according to claim 4, characterized in that: In the S1.5, curing is continued for 3-4 hours at a temperature of 100-150°C.

8. The method for preparing the improved protein imprinting membrane for rapid protein transfer according to claim 4, characterized in that: In S2.1, the specific steps of activating the surface of the polyvinylidene fluoride film by oxygen plasma treatment are: Place the polyvinylidene fluoride membrane in a plasma reactor, close the reactor, start to evacuate until it reaches 160-240 mTorr, start to introduce high-purity oxygen with an oxygen flow rate of 10-50 sccm, turn on the plasma generator, set the power to 50-300 watts, ionize the oxygen to generate plasma, set the treatment time to 30-45 minutes, after the treatment is completed, stop the plasma generator, wait for the polyvinylidene fluoride membrane to cool to room temperature, slowly introduce air to restore normal pressure, and take out the treated polyvinylidene fluoride membrane.

9. The method for preparing the improved protein imprinting membrane for rapid protein transfer according to claim 4, characterized in that: In S2.5, in the EDC / NHS solution, the mixing ratio of the EDC / NHS crosslinking agent and the N,N-dimethylformamide solvent is 2:

1.

10. The method for preparing the improved protein imprinting membrane for rapid protein transfer according to claim 4, characterized in that: In the above S2.6, the polyvinylidene fluoride film is dried at 40-60° C. for 16-18 hours.

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