Dual-channel separation membrane and its preparation method, multi-chamber electrophoretic separation device

Abstract

This invention relates to a dual-channel separation membrane. The membrane is modified with a hydrophobic solution, giving its upper and lower surfaces and pore surfaces a low surface free energy similar to that of the hydrophobic substance. This reduces protein adsorption on the membrane surface and pores, thereby reducing protein residue and improving protein permeability and migration. Furthermore, the low protein residue results in good reusability of the membrane. The separation performance of the membrane is also reproducible. This invention also relates to a method for preparing the dual-channel separation membrane, which yields a membrane with low protein residue. This method is simple, low-cost, and suitable for large-scale industrial production. Finally, this invention relates to a multi-chamber electrophoresis separation device capable of effectively separating proteins.

Description

Technical Field

[0001] This invention relates to the field of separation membrane technology, specifically to a dual-channel separation membrane and its preparation method, and a multi-chamber electrophoretic separation device. Background Technology

[0002] Separation membranes generally have functions such as separation, concentration, and purification, and are widely used in fields such as biology, medicine, food, and chemical engineering. The permeability of the membrane to the separated substances and its selective permeability to multiple substances are the two most important evaluation criteria for separation membranes. The former indicates the separation speed, and the latter indicates the separation quality. Currently, commercially available nitrocellulose membranes, cellulose acetate membranes, nylon membranes, and polyethersulfone membranes can all be used to separate proteins. However, most products have multi-layered, overlapping pore structures with varying pore sizes, making it difficult for proteins to cross the membrane, clogging channels, and resulting in high residue levels, thus affecting experimental accuracy.

[0003] Multicompartment electrophoresis utilizes separation membranes to selectively separate free small molecules and charged protein-small molecule complexes. The principle is as follows: Figure 1 As shown, protein-small molecule complexes migrate through a separation membrane between the sample chamber and the receiving chamber, thus separating them from free small molecules and measuring protein migration rate. However, the problem of protein residue on the membrane also hinders accuracy.

[0004] Therefore, how to reduce the amount of protein residue in the separation membrane has become an important research topic. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a dual-pass separation membrane that can separate proteins, reduce the amount of protein residue on the membrane, and improve protein permeability.

[0006] Another objective of this invention is to provide a method for preparing the dual-pass separation membrane, wherein the separation membrane obtained by the method has a low protein residue content, and the method is simple to operate, low in cost, and suitable for large-scale industrial production.

[0007] Another object of the present invention is to provide a multi-chamber electrophoretic separation apparatus including the dual-pass separation membrane.

[0008] To achieve the above objectives, the present invention provides the following technical solution.

[0009] In a first aspect, the present invention provides a dual-channel separation membrane. The dual-channel separation membrane comprises a dual-channel base membrane and a hydrophobic material. The channels of the dual-channel base membrane have a through-pore structure. The hydrophobic material is modified on the upper and lower surfaces and the surface of the channels of the dual-channel base membrane.

[0010] This invention utilizes a hydrophobic solution to modify a dual-channel membrane, giving its upper and lower surfaces and pore surfaces a low surface free energy similar to that of the hydrophobic substance. This reduces protein adsorption on the membrane surface and pores, thereby lowering protein residue and improving protein permeability and migration. Furthermore, the low protein residue results in excellent reusability of the dual-channel separation membrane. Hydrophobicity is the most crucial factor contributing to the membrane's low residue and high reusability.

[0011] The dual-channel base film of this application is a dual-channel film, characterized by: short-range highly ordered pore arrangement, all pores being through-hole structures, non-intersecting internal pores with uniform diameter, adjustable from tens to hundreds of nanometers.

[0012] In some embodiments, the dual-passage base film may be a porous alumina (AAO) dual-passage film. The porous alumina dual-passage film is made of Al2O3, possessing high-temperature resistance and hydrophilic properties. The pore size of the porous alumina film can be 200-300 nm (e.g., 200 nm, 210 nm, 220 nm, 230 nm, 240 nm, 250 nm, 260 nm, 270 nm, 280 nm, 290 nm, or 300 nm), the pore spacing can be 400-500 nm (e.g., 400 nm, 410 nm, 420 nm, 430 nm, 440 nm, 450 nm, 460 nm, 470 nm, 480 nm, 490 nm, or 500 nm), and the thickness can be 40-60 μm (e.g., 40 μm, 45 μm, 50 μm, 55 μm, or 60 μm). Compared with existing cellulose acetate membranes used in multi-chamber electrophoresis technology (such as...) Figure 2 Compared to (as shown), the AAO membrane pore structure (such as...) Figure 3 (As shown) makes it easier for molecules to pass through. Multi-compartment electrophoresis using an AAO double-pass membrane as the protein separation membrane can efficiently reduce the amount of protein-drug small molecule complexes remaining on the membrane, thereby improving the accuracy of protein migration measurement.

[0013] When using cellulose acetate membranes as separation membranes to determine protein migration, the membranes suffer from high protein residue due to their multilayered, interwoven arrangement, varying pore sizes, and hydrophilicity, hindering migration improvement. Furthermore, cellulose acetate membranes are limited by pH and temperature, preventing reuse. In contrast, this invention utilizes a hydrophobic AAO dual-channel membrane with a short-range, highly ordered, through-pore structure. The pores are tightly packed in a hexagonal pattern without internal cross-linking, resulting in uniform pore size adjustable from tens to hundreds of nanometers. Compared to cellulose acetate membranes, the straight-channel structure reduces molecular resistance, facilitating protein passage, reducing membrane residue, and improving protein migration. It eliminates the need for soaking, saving time, and is unaffected by experimental temperature and pH, allowing for reuse.

[0014] In some embodiments, the hydrophobic material may be one or more of polytetrafluoroethylene (PTFE), polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA), polystyrene, polypropylene (PP), and polyvinyl chloride (PVC). PTFE has a very low surface free energy, resulting in low adhesion and a low coefficient of friction, making it a commonly used hydrophobic modification material. This invention combines the ease of surface modification of PTFE and AAO double-pass membranes, and evaluates the hydrophobic state and separation performance of the modified AAO membrane, laying a solid theoretical foundation for improving membrane separation performance.

[0015] In some embodiments, the contact angle of the dual-channel separation membrane can be 50°-120°, for example, 50°, 60°, 70°, 80°, 90°, 100°, 110°, or 120°, preferably 90°-120°. By controlling the contact angle of the dual-channel separation membrane within the above range, this invention helps to reduce the amount of protein residue on the membrane and improve protein migration. When the contact angle is too small, the hydrophobicity of the dual-channel separation membrane is poor, resulting in strong protein adsorption and a high amount of protein residue on the membrane. When the contact angle is too large, the hydrophobicity of the dual-channel separation membrane is too high, causing the solution to mainly form spherical shapes in the chamber, affecting the uniformity of protein stress and making it difficult for the solution to uniformly fill the chamber, which is detrimental to protein migration.

[0016] Secondly, the present invention provides a method for preparing the above-mentioned dual-pass separation membrane, comprising the following steps:

[0017] The double-channel base membrane is immersed in a hydrophobic substance solution, wherein the channels of the double-channel base membrane are through-pore structures;

[0018] After removing the double-channel base film, the hydrophobic substance solution is dropped onto it and allowed to flow through the channels to reach the bottom of the double-channel base film, thereby making full contact with the channel surface;

[0019] By heating, the hydrophobic material is fixed on the upper and lower surfaces and the pore surface of the double-channel base membrane to obtain a hydrophobic double-channel separation membrane.

[0020] The separation membrane obtained by the method of the present invention has a low protein residue, and the method is simple to operate, low in cost, and suitable for large-scale industrial production.

[0021] In some embodiments, the soaking includes: immersing the dual-pass base membrane in the hydrophobic substance solution for 10-20 minutes (e.g., 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, 16 min, 17 min, 18 min, 19 min, or 20 min), then turning it over and soaking it again for 10-20 minutes (e.g., 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, 16 min, 17 min, 18 min, 19 min, or 20 min). Optionally, during the soaking process, the hydrophobic substance solution is stirred every few minutes (e.g., every 4-6 minutes, e.g., 5 minutes) to ensure uniform solute distribution in the solution.

[0022] In some embodiments, the hydrophobic substance solution is a polytetrafluoroethylene (PTFE) solution. The preparation of the PTFE solution includes: shaking the purchased PTFE solution well and diluting it with water until it reaches a non-viscous state, avoiding clogging the pores of the dual-channel base film.

[0023] In some embodiments, the mass percentage concentration of the hydrophobic substance solution can be 0.004%-0.04%, for example, 0.004%, 0.006%, 0.008%, 0.01%, 0.015%, 0.02%, 0.025%, 0.03%, 0.035%, or 0.04%, preferably 0.01%-0.04%. Controlling the concentration of the hydrophobic substance solution within the above range is beneficial for optimizing the contact angle of the dual-channel separation membrane. If the concentration is too low, the improvement in the contact angle of the dual-channel base membrane is limited, resulting in poor hydrophobicity of the dual-channel base membrane. If the concentration is too high, there is significant polymer on the surface and cross-section of the dual-channel base membrane, the polymer distribution is uneven, and soaking the dual-channel base membrane at high concentrations can also cause polymer blockage of the pores, affecting protein passage.

[0024] In some embodiments, the heating temperature is greater than the melting point of the hydrophobic material and less than its thermal decomposition temperature. At the heating temperature, the hydrophobic material melts and solidifies, fixing itself onto the upper and lower surfaces and the pore surfaces of the dual-channel base film. Excessively high temperatures can cause thermal decomposition of the hydrophobic material, while excessively low temperatures prevent it from fully melting and solidifying, resulting in poor fixation.

[0025] In some specific embodiments, the heating temperature can be 350-400℃, for example, 350℃, 355℃, 360℃, 365℃, 370℃, 375℃, 380℃, 385℃, 390℃, 395℃, or 400℃. The heating time can be 20-40 minutes, for example, 20 minutes, 22 minutes, 24 minutes, 26 minutes, 28 minutes, 30 minutes, 32 minutes, 34 minutes, 36 minutes, 38 minutes, or 40 minutes.

[0026] In some specific embodiments, the heating is performed in a muffle furnace. Specifically, after the hydrophobic substance solution flows through the channels to the area below the double-pass base film, the double-pass base film is placed on a tin foil support and positioned horizontally to prevent uneven modification.

[0027] In some embodiments, the hydrophobic solution can be drawn through the pores and reach the underside of the double-pass membrane by vacuum extraction or filtration, ensuring that the pores are modified with solution. Since the pores of the double-pass membrane are very narrow, it is difficult for the hydrophobic solution to flow through the pores using only gravity. This invention utilizes vacuum extraction and filtration to effectively draw the hydrophobic solution through the pores and reach the underside of the double-pass membrane. During this process, it can be clearly observed that the liquid flows from above the membrane through the pores to below the membrane.

[0028] In some specific embodiments, the filtration time can be 3-5 minutes, for example, 3 minutes, 4 minutes or 5 minutes.

[0029] In some specific embodiments, the dual-channel base film is placed in a vacuum oven for vacuum extraction. The vacuum extraction time can be 10-20 minutes, for example, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 16 minutes, 17 minutes, 18 minutes, 19 minutes, or 20 minutes.

[0030] In some embodiments, after the vacuum extraction or filtration is completed, the double-pass membrane is flipped over, and the hydrophobic substance solution is repeatedly dropped onto the double-pass membrane. Then, vacuum extraction or filtration is performed to allow the hydrophobic substance solution to flow through the pores, thereby ensuring sufficient contact with the pore surface. Repeatedly flowing the hydrophobic substance solution through the pores helps to better modify the pore surface, improve its hydrophobicity, and reduce protein adsorption on the pore surface.

[0031] In some embodiments, the volume of the hydrophobic substance solution dropped onto the double-channel base membrane can be determined according to the size of the double-channel base membrane, as long as it can cover the entire upper surface of the double-channel base membrane to ensure that the solution flows through all channels.

[0032] Thirdly, the present invention provides a multi-chamber electrophoretic separation device, comprising a dual-channel separation membrane according to the first aspect of the present invention or a dual-channel separation membrane obtained by the preparation method according to the second aspect of the present invention.

[0033] refer to Figure 4-5A multi-chamber electrophoresis separation apparatus typically includes a buffer solution circulation system 1, a separation system 3, a condensation system 4 for cooling the separation system, and a power supply system 5. The separation system 3 consists of five chambers in sequence: a positive electrode chamber 7, a positive receiving chamber 8, a sample chamber 9, a negative receiving chamber 10, and a negative electrode chamber 11. Sample chamber 9 is separated from positive receiving chamber 8 by a double-pass separation membrane 12, and from negative receiving chamber 10 by a retention membrane 13. A buffer solution inlet 15 is located on the lower side of each of the positive and negative electrode chambers 7 and 11, and a buffer solution outlet 16 is located on the upper side of the corresponding side for buffer solution circulation. A platinum positive electrode 14a and a platinum negative electrode 14b, connected to the power supply system, are respectively placed in the positive electrode chamber 7 and negative electrode chamber 11. Each of the positive electrode chamber 7 and the negative electrode chamber 11 has an organic glass clamp 6 on its outer side, and the two organic glass clamps 6 are connected by screws 17 on both sides and fixed by nuts 18. The buffer solution circulation system 1 consists of a container containing buffer solution, circulation pipes, and a pump 2 installed on the pipes, and is connected to the buffer solution inlet 15 and buffer solution outlet 16 of the separation system 3 to form a loop. The multi-chamber electrophoretic separation device used in this invention is described in detail in the applicant's previous patent application CN201410611855.8, and the disclosure of the multi-chamber electrophoretic separation device in patent CN201410611855.8 can be referred to.

[0034] Retention membranes (ultrafiltration membranes) can be used to retain biomolecules and small molecules, allowing them to remain in the positive and negative receiving chambers. Dual-pass separation membranes allow charged proteins to pass through. By adjusting the pH of the system, the applied voltage, the electrode distance, the membrane thickness, and the pore size, transmembrane migration of biomolecules can be achieved, while simultaneously carrying small molecules to migrate.

[0035] Because the dual-channel separation membrane of the first aspect of the present invention or the dual-channel separation membrane obtained by the preparation method of the second aspect of the present invention is used, the multi-chamber electrophoresis separation device of the present invention can effectively separate proteins, with high protein permeability and low protein residue on the membrane, resulting in high accuracy of protein migration rate measurement.

[0036] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0037] 1. This invention provides a dual-channel separation membrane. The membrane is modified with a hydrophobic solution, giving its upper and lower surfaces and pore surfaces a low surface free energy similar to that of the hydrophobic substance. This reduces protein adsorption on the membrane surface and pores, thereby reducing protein residue and improving protein permeability and migration. Furthermore, due to the low protein residue, the dual-channel separation membrane exhibits good reusability. Additionally, the separation performance of the dual-channel separation membrane is reproducible.

[0038] 2. The present invention also provides a method for preparing a dual-pass separation membrane. The separation membrane obtained by the method has a low protein residue content, and the method is simple to operate, low in cost, and suitable for large-scale industrial production.

[0039] 3. The present invention also provides a multi-chamber electrophoresis separation device, which can effectively separate proteins, with high protein permeability and low protein residue on the membrane, thus making the protein migration rate measurement highly accurate. Attached Figure Description

[0040] Figure 1 This is a schematic diagram illustrating the principle of multi-chamber electrophoresis.

[0041] Figure 2 This is a SEM image of a cellulose acetate membrane.

[0042] Figure 3 This is a SEM image of the AAO dual-channel base film.

[0043] Figure 4 This is a schematic diagram of a multi-chamber electrophoretic separation device.

[0044] Figure 5 This is a schematic diagram of the separation system in a multi-chamber electrophoretic separation device.

[0045] Figure 6 This is an experimental flowchart of the AAO double-pass membrane based on the hydrophobic modification of polytetrafluoroethylene solution in Example 1.

[0046] Figure 7 SEM images of the surface and cross-section of the double-pass base film before and after modification with different concentrations of PTFE (scale bar in the figure is 3 μm): (a) Unmodified; (b) 40% PTFE modified (Comparative Example 1); (c) 4% PTFE modified (Comparative Example 2); (d) 0.04% PTFE modified (Example 2); (e) 0.02% PTFE modified (Example 1); (f) 0.004% PTFE modified (Example 3).

[0047] Figure 8SEM images of the surface and cross-section of the double-pass base film before and after modification with different concentrations of PTFE (scale bar in the figure is 10 μm): (a) Unmodified; (b) 40% PTFE modified (Comparative Example 1); (c) 4% PTFE modified (Comparative Example 2); (d) 0.04% PTFE modified (Example 2); (e) 0.02% PTFE modified (Example 1); (f) 0.004% PTFE modified (Example 3).

[0048] Figure 9 Infrared spectra of double-pass base films before and after modification with different concentrations of PTFE.

[0049] Figure 10 Surface mapping diagrams of the double-pass base film before and after modification with different concentrations of PTFE: (a, a') unmodified; (b, b') 40% PTFE modification (Comparative Example 1); (c, c') 4% PTFE modification (Comparative Example 2); (d, d') 0.04% PTFE modification (Example 2); (e, e') 0.02% PTFE modification (Example 1); (f, f') 0.004% PTFE modification (Example 3).

[0050] Figure 11 Cross-sectional mapping diagrams of the double-pass base film before and after modification with different concentrations of PTFE: (a, a') unmodified; (b, b') 40% PTFE modification (Comparative Example 1); (c, c') 4% PTFE modification (Comparative Example 2); (d, d') 0.04% PTFE modification (Example 2); (e, e') 0.02% PTFE modification (Example 1); (f, f') 0.004% PTFE modification (Example 3).

[0051] Figure 12 The contact angles of the double-pass base film before and after modification with different concentrations of PTFE are shown: (a) unmodified; (b) 0.02% PTFE modified (Example 1); (c) 0.004% PTFE modified (Example 3); (d) 0.04% PTFE modified (Example 2).

[0052] Figure 13 The protein migration rates of the double-pass membrane before and after modification with different concentrations of PTFE are shown.

[0053] Figure 14 The images show the hydrophobic state of the AAO double-channel base film before and after hydrophobic modification, in a water droplet experiment (left) and an immersion experiment (right).

[0054] Figure 15 SEM images of the surface and cross-section of the dual-channel separation membrane of Example 1 before and after use (scale bar is 3 μm): (a) before use; (b) after use.

[0055] Figure 16 Mapping diagrams of the surface and cross-section of the dual-channel separation membrane in Example 1 before and after use.

[0056] Figure 17 To characterize the contact angle of the AAO dual-channel base membrane before and after hydrophobic modification in a dry state: (a) AAO dual-channel base membrane before modification, (b) dual-channel separation membrane of Example 1.

[0057] Figure 18 To characterize the contact angle of the AAO dual-channel base membrane before and after hydrophobic modification in a wetted state: (a) AAO dual-channel base membrane before modification, (b) dual-channel separation membrane of Example 1.

[0058] Figure 19 This is a comparison chart showing the residual amounts of protein on the cellulose acetate membrane and the double-pass separation membrane of Example 1 after electrophoresis.

[0059] Figure 20 RSD comparison diagrams of electrophoresis performed using the same cellulose acetate membrane and the same double-pass separation membrane from Example 1.

[0060] Figure 21 RSD comparison diagrams of electrophoresis using different cellulose acetate membranes and different double-pass separation membranes of Example 1.

[0061] Explanation of reference numerals in the attached figures

[0062] 1. Buffer solution circulation system; 2. Pump; 3. Separation system; 4. Condensation system; 5. Power supply system; 6. Acrylic glass clamp; 7. Positive electrode chamber; 8. Positive receiving chamber; 9. Sample chamber; 10. Negative receiving chamber; 11. Negative electrode chamber; 12. Dual-pass separation membrane; 13. Retention membrane; 14a. Platinum sheet positive electrode; 14b. Platinum sheet negative electrode; 15. Buffer solution inlet; 16. Buffer solution outlet; 17. Screw; 18. Nut. Detailed Implementation

[0063] To make the technical problem to be solved, the technical solution, and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely for explaining the present invention and are not intended to limit the present invention.

[0064] Unless otherwise defined, the technical terms used in the following embodiments have the same meaning as commonly understood by those skilled in the art. Unless otherwise specified, the experimental reagents used in the following embodiments are all conventional biochemical reagents; the raw materials, instruments, and equipment used in the following embodiments can all be obtained commercially or through existing methods; unless otherwise specified, the amounts of experimental reagents used are the amounts used in conventional experimental operations; unless otherwise specified, the experimental methods are all conventional methods.

[0065] In this embodiment, the migration rate can be calculated using the formula: Protein migration rate = Solution peak area in the protein receiving chamber after electrophoresis ÷ Solution peak area in the sample chamber before electrophoresis. Proteins carrying a positive charge will migrate to the negative receiving chamber under the influence of an electric field; proteins carrying a negative charge will migrate to the positive receiving chamber under the influence of an electric field.

[0066] Preparation of dual-pass separation membrane

[0067] Example 1

[0068] The experimental flowchart based on the hydrophobic modification of AAO double-pass membrane with polytetrafluoroethylene solution is as follows: Figure 6 As shown.

[0069] First, shake the purchased polytetrafluoroethylene (PTFE) solution well and dilute it with ultrapure water in a gradient to 30%, 15%, 5%, 0.5%, and 0.02% (mass percentage concentration) to dilute the solution to a non-viscous state and avoid clogging the pores. Add 20 mL of 0.02% PTFE solution to a petri dish. Use tweezers to place the purchased AAO double-pass membrane (i.e., double-pass base membrane) into the petri dish containing the 0.02% PTFE solution. Immerse for 15 minutes, then turn it over and immerse it again for 15 minutes to ensure that the membrane surface is fully in contact with the PTFE solution. During the immersion process, stir the liquid in the petri dish every 5 minutes to prevent uneven distribution of solute in the solution. The soaked AAO double-pass membrane was removed and placed on a support in a vacuum oven, allowing it to float vertically. 400 μL of polytetrafluoroethylene (PTFE) solution was dropped onto the membrane, and vacuum was applied for 15 minutes. This vacuum process allowed the liquid to flow through the pores, ensuring full contact between the membrane pores and the PTFE solution. After 15 minutes, the membrane was flipped over, and another 400 μL of PTFE solution was dropped onto it. Vacuum was then applied for another 15 minutes. During this process, the liquid was clearly observed flowing from the top of the membrane through the pores to the bottom. After vacuuming, the AAO double-pass membrane was removed and placed on a horizontal support to prevent uneven modification. It was then placed in a muffle furnace for high-temperature curing at 380°C for 30 minutes to fix the PTFE solution onto the membrane and pore surfaces, thus obtaining the hydrophobic AAO double-pass separation membrane (Hydrophobic-AAO).

[0070] Example 2

[0071] The hydrophobic AAO dual-channel separation membrane was prepared according to the method described in Example 1, except that the concentration of the polytetrafluoroethylene solution added to the petri dish was 0.04%.

[0072] Example 3

[0073] The hydrophobic AAO dual-pass separation membrane was prepared according to the method described in Example 1, except that the concentration of the polytetrafluoroethylene solution added to the petri dish was 0.004%.

[0074] Example 4

[0075] The hydrophobic AAO dual-channel separation membrane was prepared according to the method described in Example 1, except that the concentration of the polytetrafluoroethylene solution added to the petri dish was 0.01%.

[0076] Example 5

[0077] The hydrophobic AAO dual-channel separation membrane was prepared according to the method described in Example 1, except that the high-temperature curing temperature was 350°C.

[0078] Example 6

[0079] The hydrophobic AAO dual-channel separation membrane was prepared according to the method described in Example 1, except that the high-temperature curing temperature was 400°C.

[0080] Comparative Example 1

[0081] The hydrophobic AAO dual-channel separation membrane was prepared according to the method described in Example 1, except that the concentration of the polytetrafluoroethylene solution added to the petri dish was 40%.

[0082] Comparative Example 2

[0083] The hydrophobic AAO dual-pass separation membrane was prepared according to the method described in Example 1, except that the concentration of the polytetrafluoroethylene solution added to the petri dish was 4%.

[0084] Comparative Example 3

[0085] The hydrophobic AAO dual-channel separation membrane was prepared according to the method described in Example 1, except that the high-temperature curing temperature was 300°C.

[0086] Comparative Example 4

[0087] The hydrophobic AAO dual-channel separation membrane was prepared according to the method described in Example 1, except that the high-temperature curing temperature was 500°C.

[0088] Characterization analysis of dual-pass separation membrane

[0089] 1. Characterization of double-pass base films before and after modification with different concentrations of PTFE

[0090] The surfaces and cross sections of the unmodified double-pass base membrane and the double-pass separation membranes prepared in Examples 1-3 and Comparative Examples 1-2 were characterized by scanning electron microscopy (SEM), X-ray energy dispersive spectroscopy (EDS), Fourier transform infrared spectroscopy (FT-IR), and contact angle.

[0091] (1) SEM characterization

[0092] From the 3μm scale ( Figure 7 It was clearly observed that, compared with the unmodified double-pass membrane, the double-pass membrane modified with high concentrations (40%, 4% PTFE) showed obvious polymers on both the surface and cross-section. Furthermore, at higher concentrations, the polymers easily blocked the pores, affecting protein passage. In contrast, the double-pass membranes modified with low concentrations (0.04%, 0.02%, 0.004% PTFE) showed no obvious polymers on the surface. Compared with the unmodified double-pass membrane, the surface smoothness was slightly altered, but no obvious polymers were observed, and the cross-sectional pores remained unobstructed without significant blockage.

[0093] From a 10μm scale ( Figure 8 Observing the surface and cross-section of the membrane, compared with the unmodified dual-channel base membrane, the polymer distribution on the surface and cross-section of the AAO membrane modified with high concentration (40%, 4% PTFE) was uneven, while no obvious polymer was observed on the surface of the dual-channel base membrane modified with low concentration (0.04%, 0.02%, 0.004% PTFE).

[0094] (2) FT-IR characterization

[0095] Compared with the unmodified double-pass film, the infrared spectrum of the modified double-pass film ( Figure 9 ) at 1215cm -1 and 1155cm -1 The presence of an absorption peak at this location indicates the -CF2 stretching vibration peak, confirming successful modification of polytetrafluoroethylene (PTFE). As the modification concentration decreases from high (40%, 4%) PTFE to low (0.04%, 0.02%, 0.004%) PTFE, the two characteristic absorption peaks gradually become rounded and turn into single peaks. This may be because as the concentration of PTFE decreases, the vibrational freedom of PTFE molecules is significantly reduced due to the influence of the double-pass membrane substrate.

[0096] (3) EDS characterization

[0097] The changes in F and Al elemental content on the membrane surface and cross-section were analyzed, and the results are as follows: Figure 10 , Figure 11 As shown.

[0098] from Figure 10 It is evident that as the modification concentration decreases, the content of F and Al elements on the membrane surface gradually increases.

[0099] Figure 11 The results showed that as the modification concentration decreased, the Al content in the membrane cross section gradually increased, while the F content gradually decreased. Moreover, the F content in the cross section was higher than that on the membrane surface, proving that the modification effect inside the pores was better than that on the membrane surface. The hydrophobicity inside the membrane pores was greater than that on the membrane surface, making it less likely for proteins to remain inside the pores.

[0100] according to Figure 10 and 11 and combined with characterization Figure 7-8 Under high concentration (40%, 4% PTFE) modification, the distribution of fluorine element is uneven, and a large number of pores are blocked, making it difficult for substances to pass through. However, under low concentration (0.04%, 0.02%, 0.004% PTFE) modification, the distribution of fluorine element is more uniform. Therefore, modification is preferably carried out under low concentration (0.04%, 0.02%, 0.004% PTFE).

[0101] (4) Contact angle characterization

[0102] In a dry state, the contact angles of the double-pass base film before and after modification with different concentrations of PTFE are as follows: Figure 12 As shown.

[0103] Hydrophobic modification of the double-pass membrane was performed using PTFE solutions at concentrations of 0.004%, 0.01%, 0.02%, and 0.04%. Under dry conditions, the contact angles of the resulting double-pass membranes were 51.2°, 100.3°, 111.1°, and 117.3°, respectively. Compared to the contact angle of the unmodified double-pass membrane (29.3°), these angles increased by 21.9°, 71°, 81.8°, and 88°, respectively. The difference in hydrophobicity resulted in different protein migration rates, consistent with the following values ​​at 0.004%, 0.01%, 0.02%, and 0.04% PTFE: 28.08%, 50.60%, 61.43%, and 52.80%, respectively. It can be seen that as the contact angle increases, the protein migration rate first increases and then decreases, meaning the residual protein on the membrane first decreases and then increases.

[0104] 2. Optimization of the concentration of hydrophobic modification of polytetrafluoroethylene

[0105] The protein migration rates of the double-pass membrane before and after modification with polytetrafluoroethylene (PTFE) solutions of different concentrations were measured, and the results are as follows: Figure 13 As shown.

[0106] Compared to the unmodified dual-channel membrane, the protein migration rate of the dual-channel separation membrane of the present invention is significantly improved. Both excessively high and low concentrations of the modification solution have a significant impact on protein migration rate. When the modification solution concentration is too low, polytetrafluoroethylene (PTFE) is not uniformly modified onto the membrane surface and within the pores, resulting in poor hydrophobicity. When the modification solution concentration is too high, excessive PTFE deposits on the membrane surface and within the pores, causing blockage and hindering material passage. Therefore, both excessively high and low modification concentrations affect the improvement of protein migration rate. When the PTFE solution concentration is 0.02%, the protein migration rate reaches a maximum of 61.43%. The protein migration rates of the unmodified dual-channel membrane and the dual-channel membranes modified with 0.004% PTFE, 0.01% PTFE, and 0.04% PTFE are 23.53%, 28.08%, 50.60%, and 52.80%, respectively. Therefore, the most preferred modification concentration is 0.02%.

[0107] 3. Temperature optimization during high-temperature curing

[0108] The contact angle and migration rate of the dual-channel separation membrane after different curing temperatures were measured, as shown in Table 1 below.

[0109] Table 1

[0110] Example 1 380℃ 111.1° 61.43％ Example 5 350℃ 105° 58％ Example 6 400℃ 95° 60％ Comparative Example 3 300℃ 80° 35％ Comparative Example 4 500℃ 45° 30％

[0111] As can be seen from Table 1, both excessively high and excessively low curing temperatures are detrimental to improving the migration rate.

[0112] Verification of the hydrophobic effect of the dual-channel separation membrane

[0113] To further verify the hydrophobic effect of the AAO dual-channel base membrane modification and ensure low protein residue, drop and immersion experiments were conducted before and after the AAO dual-channel base membrane modification, and the dual-channel separation membrane was characterized by SEM and EDS before and after use.

[0114] (1) A water droplet test was performed on the AAO double-channel base film before and after hydrophobic modification. Figure 14 (Left) and immersion test ( Figure 14 (Right) Observe the hydrophobic state of the membrane.

[0115] Droplet test: After adding buffer solution to the membrane surface and letting it stand for 30 minutes, it can be clearly observed that, compared with before modification, the droplets on the surface of the dual-channel separation membrane in Example 1 are spherical, while the droplets on the surface of the unmodified AAO dual-channel base membrane are scattered.

[0116] Hydrophobicity test: Unmodified and modified AAO dual-channel membranes were placed in a buffer solution and allowed to stand for 30 minutes. The dual-channel separation membrane of Example 1 floated on the liquid surface, while the unmodified AAO dual-channel membrane sank to the bottom of the liquid. This is because polytetrafluoroethylene has a smaller surface free energy, while water has a larger surface free energy and cannot remain on a surface with a smaller surface free energy, thus exhibiting hydrophobic properties.

[0117] (2) SEM characterization of the dual-channel separation membrane before and after use

[0118] The surface and cross-section of the dual-channel separation membrane of Example 1 before and after use were characterized by SEM. Figure 15 It can be observed that after 20 uses, the dual-channel separation membrane showed no obvious pore blockage, no significant change in pore cross-section, and the pores were smooth and unobstructed with no residue. This proves that the dual-channel separation membrane of the present invention can be reused multiple times.

[0119] (3) EDS characterization of the dual-channel separation membrane before and after use

[0120] The surface and cross-section of the dual-channel separation membrane from Example 1 before and after use were characterized by EDS to investigate the changes in F and Al elemental content on the membrane surface and cross-section. Figure 16 Compared with the original dual-channel separation membrane, the F element content on the surface and cross-section of the dual-channel separation membrane after 20 uses was slightly reduced, while the Al element content did not change significantly, proving that the dual-channel separation membrane of the present invention can be reused.

[0121] (4) Contact angle characterization experiment:

[0122] The contact angles of the AAO dual-channel base film surface before and after hydrophobic modification were measured under both dry and wet conditions. Measurement parameters: measurement range 0–180°, measurement accuracy ±0.1°, resolution ±0.01°.

[0123] In a dry state ( Figure 17 The contact angle of the unmodified AAO dual-channel membrane was 29.3°, while the contact angle of the dual-channel separation membrane in Example 1 was 111.1°. This is because the surface free energy of the AAO dual-channel membrane was reduced after polytetrafluoroethylene was applied to the membrane surface, preventing the solution from spreading on the surface with low surface energy, thus exhibiting hydrophobic properties. This proves that the hydrophobic modification effect of the dual-channel separation membrane in Example 1 is good.

[0124] Since the experiment requires the dual-channel separation membrane to be immersed in solution to separate protein-drug molecule complexes, the unmodified and hydrophobically modified AAO dual-channel base membranes were first soaked in distilled water for 5 minutes before contact angle characterization was performed. Figure 18Compared to the dry state, the surface contact angle of the dual-channel separation membrane of Example 1 in the wet state decreased, but was still greater than 90°, further demonstrating that the AAO dual-channel base membrane was transformed from hydrophilic to hydrophobic by polytetrafluoroethylene modification.

[0125] The above experiments have all demonstrated that the modified AAO double-pass membrane has good hydrophobicity, low protein residue, and can be reused.

[0126] Application of dual-pass separation membrane

[0127] The dual-pass separation membrane prepared in Example 1 was used as the separation membrane in a multi-chamber electrophoresis separation device to determine protein migration.

[0128] The separation performance of the dual-channel separation membrane of the present invention was evaluated based on protein migration rate, considering protein residue, reusability, and reproducibility, as well as the protein mobility, of the dual-channel separation membrane and cellulose acetate membrane used in Example 1. The specific experimental steps are as follows:

[0129] Solution preparation:

[0130] Buffer solution: First prepare 10 mmol / L -1 The pH of the ammonium acetate solution was adjusted to 6.80 by adding sodium hydroxide solution.

[0131] Protein solution: Weigh a certain amount of human serum albumin powder using an analytical balance, dissolve it in the prepared buffer solution, and the protein concentration is 5 mg / mL. -1 Mix at a low speed to prevent the formation of air bubbles.

[0132] Electrophoresis: Add 100 μL of the prepared buffer solution to the positive and negative receiving chambers of the multi-chamber electrophoresis apparatus, respectively. Add 100 μL of the prepared protein solution to the sample chamber. Turn on the instrument's condenser circulation system at a flow rate of 5 mL / min. -1 A voltage of 12V was applied, and electrophoresis was performed for 10 minutes. After 10 minutes, the samples from each chamber were removed into centrifuge tubes. Among them, human serum albumin pI = 4.7. In the pH = 6.80 buffer solution, the protein carries a negative charge and migrates to the positive receiving chamber.

[0133] (1) Residual content test

[0134] Protein electrophoresis experiments were performed using a cellulose acetate membrane and the double-pass separation membrane from Example 1, under conditions of 12V voltage, 10min electrophoresis time, and ammonium acetate buffer solution pH=6.80. Each electrophoresis experiment was performed in triplicate. After electrophoresis, samples from each chamber were removed and analyzed using an HPLC-UV instrument (mobile phase: pH=6.80 ammonium acetate buffer solution, injection volume: 5μL, UV detection wavelength: 280nm). The sum of the peak areas of each chamber sample was compared with the protein solution detection results before electrophoresis to obtain the residual protein amount.

[0135] (2) Reusability test

[0136] Reusability refers to the ability to reuse the same double-pass separation membrane multiple times, and the reusability of the double-pass separation membrane is evaluated based on protein migration rate.

[0137] Under conditions of 12V voltage, 10min electrophoresis time, and ammonium acetate buffer solution pH=8.00, protein electrophoresis experiments were repeated 5 times using the same cellulose acetate membrane and the same double-pass separation membrane from Example 1. After electrophoresis, samples from each chamber were removed and analyzed using HPLC-UV instrument (mobile phase: pH=8.00 ammonium acetate buffer solution, injection volume: 5μL, UV detection wavelength: 280nm). The relative standard deviation (RSD) of protein migration rates of the two separation membranes was compared; the smaller the RSD, the better the reusability.

[0138] (3) Reproducibility

[0139] Reproducibility is evaluated by conducting parallel experiments using different double-pass separation membranes, with protein migration rate as the basis for assessing the reproducibility of the double-pass separation membrane.

[0140] Protein electrophoresis experiments were conducted using a cellulose acetate membrane and the double-pass separation membrane from Example 1, under conditions of 12V voltage, 10min electrophoresis time, and pH 6.80 ammonium acetate buffer solution. After each electrophoresis, the separation membrane was replaced, and the electrophoresis was performed three times in parallel. After electrophoresis, the samples from each chamber were removed and analyzed using an HPLC-UV instrument (mobile phase: pH 6.80 ammonium acetate buffer solution, injection volume: 5μL, UV detection wavelength: 280nm). The relative standard deviation (RSD) of protein migration rates of the two separation membranes was compared; the smaller the RSD, the better the reproducibility.

[0141] Test results:

[0142] Compared to the cellulose acetate membrane, the protein residue of the dual-pass separation membrane in Example 1 was significantly reduced, from 35.17% (cellulose acetate membrane) to 5.80% (dual-pass separation membrane of Example 1). Figure 19 ).

[0143] Compared to the cellulose acetate membrane, the average protein migration rate of the dual-pass separation membrane in Example 1 was significantly improved, increasing from 39.14% (cellulose acetate membrane) to 66.51% (dual-pass separation membrane of Example 1). Furthermore, the reusability of the dual-pass separation membrane of Example 1 (RSD = 1.90%) was superior to that of the cellulose acetate membrane (RSD = 19.81%). Figure 20 This further demonstrates that the dual-channel separation membrane of the present invention has good reusability.

[0144] Compared to cellulose acetate membranes, protein electrophoresis experiments using the double-pass separation membranes of Example 1 with different tensions showed significantly improved protein migration reproducibility, with the RSD decreasing from 11.87% (cellulose acetate membrane) to 1.92% (double-pass separation membrane of Example 1). Figure 21 This demonstrates that the individual differences between the dual-channel separation membranes in Example 1 are small, further proving that the separation performance of the dual-channel separation membrane of the present invention has good reproducibility.

[0145] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A membrane for multi-chamber electrophoretic separation, characterized in that, The membrane is a dual-channel separation membrane, which includes a dual-channel base membrane and a hydrophobic material. The channels of the dual-channel base membrane have a through-hole structure, and the hydrophobic material is modified on the upper and lower surfaces and the surface of the channels of the dual-channel base membrane. The dual-pass base membrane is a porous alumina dual-pass membrane; The hydrophobic material is polytetrafluoroethylene; The contact angle of the dual-channel separation membrane is 90°-120°; The dual-pass separation membrane is prepared by a method comprising the following steps: A double-channel base membrane is immersed in a hydrophobic substance solution, wherein the channels of the double-channel base membrane are through-pore structures; wherein the mass percentage concentration of the hydrophobic substance solution is 0.004%-0.04%; After removing the double-channel base film, the hydrophobic substance solution is dropped onto it and allowed to flow through the channels to reach the bottom of the double-channel base film, thereby making full contact with the channel surface; By heating, the hydrophobic material is fixed on the upper and lower surfaces and pore surfaces of the dual-channel base membrane to obtain a hydrophobic dual-channel separation membrane; wherein the heating temperature is 350-400℃. The dual-channel separation membrane reduces protein adsorption on the membrane surface and pores, reduces the amount of protein residue on the membrane, and improves protein permeability.

2. A method for preparing a membrane for multi-chamber electrophoretic separation, characterized in that, The membrane is a dual-pass separation membrane, and the process includes the following steps: A double-channel base membrane is immersed in a hydrophobic substance solution, wherein the channels of the double-channel base membrane are through-pore structures; wherein the hydrophobic substance is polytetrafluoroethylene, and the mass percentage concentration of the hydrophobic substance solution is 0.004%-0.04%; After removing the double-channel base film, the hydrophobic substance solution is dropped onto it and allowed to flow through the channels to reach the bottom of the double-channel base film, thereby making full contact with the channel surface; By heating, the hydrophobic material is fixed on the upper and lower surfaces and pore surfaces of the double-channel base membrane to obtain a hydrophobic double-channel separation membrane; the heating temperature is 350-400℃. The dual-channel separation membrane reduces protein adsorption on the membrane surface and pores, reduces the amount of protein residue on the membrane, and improves protein permeability.

3. The preparation method according to claim 2, characterized in that, The mass percentage concentration of the hydrophobic substance solution is 0.01%-0.04%.

4. The preparation method according to claim 2 or 3, characterized in that, The hydrophobic solution is made to flow through the channels to the bottom of the dual-channel base membrane by vacuum extraction or filtration.

5. The preparation method according to claim 4, characterized in that, After the vacuum extraction or filtration is completed, the double-pass base membrane is flipped over, and the hydrophobic substance solution is repeatedly dropped onto the double-pass base membrane. Then, the hydrophobic substance solution is made to flow through the inside of the pores by vacuum extraction or filtration, so as to make full contact with the surface of the pores. The filtration time is 3-5 minutes; or, the double-pass base membrane is placed in a vacuum oven for vacuum extraction, and the vacuum extraction time is 10-20 minutes.

6. The preparation method according to claim 2 or 3, characterized in that, The soaking process includes: immersing the double-pass base membrane in the hydrophobic substance solution for 10-20 minutes, turning it over, and soaking it again for 10-20 minutes; during the soaking process, the hydrophobic substance solution is mixed every few minutes to ensure that the solute in the solution is evenly distributed.

7. A multi-chamber electrophoretic separation device, characterized in that, Includes the dual-pass separation membrane as described in claim 1 or the dual-pass separation membrane obtained by the preparation method according to any one of claims 2-6.

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