Fiber-based porous affinity separation material with periodic necking channel structure and preparation method thereof

Abstract

The present invention provides a fiber-based porous affinity separation material with a periodic necking channel structure and a preparation method thereof. The method obtains a fiber-based porous affinity separation material with a periodic necking channel structure by freeze-drying and graft-modifying a nanofiber foam liquid modified by secondary pre-crosslinking of different viscosities. Molecules containing amino groups and aldehyde groups are selected, and a flow chemistry method is adopted to complete the preparation of the porous separation material during the crosslinking and grafting modification process. This process is not only simple, efficient, but also environmentally friendly. The fiber-based porous affinity separation material with a periodic necking channel structure is prepared. Because it has a structure with small pore diameters at both ends and large pore diameter in the middle, or large pore diameters at both ends and small pore diameter in the middle, the contact probability between the liquid and the channel wall can be regulated by means of the change in the size of the pore diameter in the flow direction of the liquid, which not only improves the efficiency of the affinity modification, but also improves the adsorption efficiency of the substance to be separated in the liquid, thereby achieving excellent implementation effects in the preparation and separation processes.

Description

Technical Field

[0001] The present invention relates to the technical field of filtration separation materials, and in particular to a fiber-based porous affinity separation material with a periodic necking pore structure and a preparation method thereof. Background Art

[0002] Affinity separation materials are essential core materials for the biopharmaceutical industry, used to separate and purify drug solutions, extract key biological substances such as proteins and nucleic acids, and provide a strong foundation for the development of high-value-added bioproducts and advanced biotherapeutic technologies. Common affinity separation materials include polymer filler microspheres and porous materials such as aerogels. Both types of materials primarily utilize chemical functional groups with affinity adsorption properties fixed to the surface of their porous aerogels to adsorb and separate biomolecules. Furthermore, these two materials have high porosity, which ensures the smooth passage of drug solutions, demonstrating significant application value in the field of bioaffinity separation.

[0003] Patent publication number CN110498419A discloses nano-silica particles for purifying histidine-tagged proteins and their preparation method. This method involves amminating and then aldehyde-forming the surface of 50-100 nm nano-SiO2 particles. After attaching metal chelating groups, sodium cyanoborohydride is used to reduce the carbon-nitrogen double bond, enhancing the stability of the connection between the metal chelating groups and the nano-SiO2. Although the nano-silica particles prepared in this invention are small and have a high specific surface area, making them simple and easy to use as affinity separation materials for purifying histidine-tagged proteins, actual application requires column packing. The accumulation of small particles creates small pores, resulting in slow flow rates and high resistance for the drug solution, severely impacting separation efficiency.

[0004] To address the slow separation speed associated with filler microsphere materials, the invention patent with publication number CN112194816A proposes a method for preparing modified cellulose aerogels for separating glycoproteins. This method uses cellulose aerogels as a substrate, which, due to their three-dimensional network structure, low density, high specific surface area, and large porosity, can provide more binding sites for glycoproteins. The dendritic macromolecule polyethyleneimine (PEI) has abundant functional groups and is easily modified. By modifying it, the density of phenylboronic acid ligands can be increased, thereby enhancing their affinity for glycoproteins. The large pore size of this material ensures rapid passage of glycoprotein solutions during the adsorption process, but it is difficult to ensure sufficient adsorption of glycoproteins in the solution, and the conflict between affinity adsorption capacity and separation speed remains difficult to resolve.

[0005] The invention patent with publication number CN114245757A proposes a structure-controllable ion-exchange nanofiber skeleton three-dimensional separation material and its preparation method. The method prepares nanofibers by melt spinning, pre-disperses nanofibers, prepares a pre-crosslinked nanofiber suspension, and freeze-dry cross-links to obtain a nanofiber skeleton three-dimensional separation material with a stable structure, a high specific surface area, and a large adsorption capacity. By regulating the composition and freezing method of the pre-crosslinked nanofiber suspension, the microstructure of the nanofiber skeleton three-dimensional separation material is regulated. When different amounts of polyelectrolytes are added to the pre-crosslinked nanofiber suspension, a high-strength and high-adsorption ion-exchange nanofiber skeleton three-dimensional separation material with diversified structures can be obtained, which can effectively improve the separation efficiency. However, the pores are all straight up and down structures, and the contact efficiency of the liquid with the pore wall when passing through is still low. There is still a lot of room for structural optimization and separation performance improvement.

[0006] In view of this, it is necessary to design a fiber-based porous affinity separation material with a periodic necking pore structure and a preparation method thereof to solve the above problems. Summary of the Invention

[0007] In view of the defects of the above-mentioned prior art, the purpose of the present invention is to provide a fiber-based porous affinity separation material with a periodic necking pore structure and a preparation method thereof. By constructing a fiber-based porous affinity separation material in which the pore walls are composed of periodically repeated necking structural units, the problem that the porous pores are difficult to achieve high-efficiency contact and adsorption with the liquid when the liquid to be separated passes through rapidly is solved, that is, the contradiction between permeability and adsorption efficiency is solved.

[0008] To achieve the above object, the present invention provides a fiber-based porous affinity separation material having a periodic necking channel structure and a preparation method thereof, comprising the following steps:

[0009] S1. Mixing two secondary pre-crosslinked modified nanofiber foam solutions with different viscosities and then freeze-drying them to prepare a nanofiber aerogel with a periodic necked pore structure;

[0010] S2, performing a first grafting modification on the nanofiber aerogel prepared in step S1 using a spacer arm aqueous solution to obtain a spacer arm-modified nanofiber aerogel;

[0011] S3. Using an aptamer aqueous solution to perform a second grafting modification on the spacer-arm modified nanofiber aerogel prepared in step S2 to obtain the fiber-based porous affinity separation material having a periodic necking pore structure.

[0012] Furthermore, in step S1, the two secondary pre-crosslinked modified nanofiber foam liquids with different viscosities include a secondary pre-crosslinked modified nanofiber foam liquid 1 with a viscosity of 1mPa·s≤X<10mPa·s and a secondary pre-crosslinked modified nanofiber foam liquid 2 with a viscosity of 10mPa·s≤X≤10000mPa·s.

[0013] Furthermore, in step S1, the preparation process of the secondary pre-crosslinked modified nanofiber foam liquid is as follows:

[0014] 1) Mixing and dispersing the copolymer nanofibers with a first crosslinking agent to obtain a pre-crosslinked modified nanofiber foam liquid;

[0015] 2) The pre-crosslinked modified nanofiber foam liquid is mixed and stirred with a second crosslinking agent, a chitosan aqueous solution, and a polyethyleneimine aqueous solution to obtain the secondary pre-crosslinked modified nanofiber foam liquid.

[0016] Furthermore, in step S1, the chitosan concentration in the second pre-crosslinked modified nanofiber foam liquid is 0.05-0.5wt.%, and the concentration of polyethyleneimine is 0.1-0.2wt.%; the chitosan concentration in the second pre-crosslinked modified nanofiber foam liquid is 0.5-5wt.%, and the concentration of polyethyleneimine is 0.2-1wt.%.

[0017] Furthermore, in step S2, the process of the first grafting modification is: placing the nanofiber aerogel in a closed container with an inlet and outlet, placing the spacer arm aqueous solution in an open container, entering the closed container through the inlet of the closed container by a peristaltic pump, then passing through the nanofiber aerogel, and then flowing out through the outlet of the closed container, and grafting the surface of the pores of the nanofiber aerogel for 0.5 to 2 hours at room temperature; in step S3, the process of the second grafting modification is: using the aptamer aqueous solution to perform the second grafting modification on the spacer arm modified nanofiber aerogel according to the steps of the first grafting modification in step S2.

[0018] Furthermore, in step S2 and step S3, the concentration range of the spacer arm aqueous solution and the aptamer aqueous solution is 1 to 10 wt.%; the spacer arm molecule in the spacer arm aqueous solution includes one of glutaraldehyde, adipaldehyde, pentamethylenediamine, and hexamethylenediamine; the ligand molecule in the aptamer aqueous solution includes one of the aptamers of CD63, CD9, CD81 with an amino or aldehyde terminal group, protein A, and protein G.

[0019] The present invention also provides a fiber-based porous affinity separation material with a periodic necking pore structure prepared by the preparation method, wherein the pore wall of the fiber-based porous affinity separation material is composed of periodically repeated necking structural units; the necking structural units include a fiber network skeleton and a polymer functional layer with affinity adsorption function attached to the surface of the fiber network skeleton.

[0020] Furthermore, the fiber network skeleton is composed of copolymer nanofibers; the copolymer nanofibers include ethylene vinyl alcohol copolymer nanofibers.

[0021] Furthermore, the polymer functional layer is cross-linked by a polymer cross-linked network layer, a spacer arm and an affinity ligand; the polymer cross-linked network layer is cross-linked by chitosan, polyethyleneimine and glutaraldehyde; and the affinity ligand is an aptamer with an amino or aldehyde terminal group.

[0022] Furthermore, the necking structure unit is a structure with small pore diameters at both ends and a large pore diameter in the middle, or with a structure with large pore diameters at both ends and a small pore diameter in the middle.

[0023] The beneficial effects of the present invention are:

[0024] 1. The present invention provides a fiber-based porous affinity separation material with a periodic necking pore structure and a method for preparing the same. Two specially prepared, secondary pre-crosslinked, modified nanofiber foam solutions with different viscosities are mixed and then freeze-dried to produce a nanofiber aerogel with a periodic necking pore structure. The nanofiber aerogel is then grafted twice with a spacer arm aqueous solution and then an aptamer aqueous solution to obtain a fiber-based porous affinity separation material with a periodic necking pore structure. This method, which involves simply preparing foam solutions with different viscosities, allows for the regulation of aerogel pores, resulting in a simple and efficient process.

[0025] 2. The fiber-based porous affinity separation material with a periodic necking channel structure provided by the present invention selects green organic molecules containing amino groups and aldehyde groups, and adopts the flow chemistry method to carry out multiple chemical reactions during multiple cross-linking, spacer arm grafting modification, and ligand grafting modification to complete the preparation of the porous separation material. The whole process has the advantages of being green and pollution-free.

[0026] 3. The fiber-based porous affinity separation material with a periodic necking channel structure provided by the present invention regulates the contact probability between the liquid and the channel wall by means of the change in the size of the pore size in the flow direction of the liquid. On the one hand, it can improve the efficiency and speed of affinity modification, and on the other hand, it can improve the adsorption efficiency of the substance to be separated in the liquid, thereby having a better implementation effect in both the preparation process and the separation process. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 A schematic structural diagram of a fiber-based porous affinity separation material having a periodic necked pore structure provided by the present invention;

[0028] Figure 2 Schematic diagram of stress changes under compressive strain for materials prepared when the chitosan concentration in the nanofiber foam solution 1 used in Example 1 is 0.05 wt.% and the chitosan concentration in the nanofiber foam solution 1 used in Example 2 is 0.5 wt.%;

[0029] Figure 3 This is a microstructure diagram of the fiber-based porous affinity separation material with a periodic necked pore structure prepared in Example 1;

[0030] Figure 4 Schematic diagram of strength change when the polyethyleneimine concentration in the nanofiber foam solution 1 used in Example 3 is 0.15 wt.% and the polyethyleneimine concentration in the nanofiber foam solution 1 used in Comparative Example 4 is 1 wt.%;

[0031] Figure 5 Electron micrographs of the structures corresponding to the materials prepared in Example 1 and Example 4 (top row from left to right) and Comparative Example 5 and Comparative Example 6 (bottom row from left to right);

[0032] Figure 6 This is an electron microscope image of the material structure prepared in Example 5. DETAILED DESCRIPTION

[0033] In order to make the objectives, technical solutions and advantages of the present invention more clear, the present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0034] It should also be noted here that, in order to avoid obscuring the present invention due to unnecessary details, only structures and / or processing steps closely related to the solutions of the present invention are shown in the drawings, while other details that are not closely related to the present invention are omitted.

[0035] In addition, it should be noted that the terms "comprises", "includes" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or apparatus that includes a series of elements includes not only those elements, but also includes other elements not explicitly listed, or also includes elements inherent to such process, method, article or apparatus.

[0036] A method for preparing a fiber-based porous affinity separation material having a periodic necked pore structure comprises the following steps:

[0037] S1. Mixing two secondary pre-crosslinked modified nanofiber foam solutions with different viscosities and then performing directional freeze-drying to prepare a nanofiber aerogel with a periodic necked channel structure; the two secondary pre-crosslinked modified nanofiber foam solutions with different viscosities include a first secondary pre-crosslinked modified nanofiber foam solution with a viscosity of 1 mPa·s≤X<10 mPa·s and a second secondary pre-crosslinked modified nanofiber foam solution with a viscosity of 10 mPa·s≤X≤10000 mPa·s;

[0038] The preparation process of the secondary pre-crosslinked modified nanofiber foam liquid is as follows:

[0039] 1) Melt-extruding the sea-phase polymer to prepare copolymer nanofibers;

[0040] The marine polymer comprises a mixture of a copolymer masterbatch and cellulose acetate butyrate; the copolymer masterbatch comprises polyethylene-vinyl alcohol copolymer particles; the mass ratio of the polyethylene-vinyl alcohol copolymer particles to the cellulose acetate butyrate is 2:8 to 3:7; the average diameter of the copolymer nanofibers ranges from 50 to 1000 nm;

[0041] 2) mixing the copolymer nanofibers with a first crosslinking agent aqueous solution having a concentration of 1 to 5 wt.% at a mass ratio of 1:20 to 1:30, and dispersing the mixture in an emulsifier for 10 to 20 minutes to obtain a pre-crosslinked modified nanofiber foam liquid after the reaction is completed;

[0042] 3) mixing the pre-crosslinked modified nanofiber foam liquid with a second crosslinker aqueous solution having a concentration of 1 to 5 wt.%, a chitosan aqueous solution having a concentration of 0.05 to 5%, and a polyethyleneimine aqueous solution having a concentration of 0.1 to 1% in a volume ratio of 1:0.3:2:0.5 to 1:0.1:1:0.1, and stirring at a constant speed for 18 to 25 minutes to obtain a secondary pre-crosslinked modified nanofiber foam liquid;

[0043] The first cross-linking agent aqueous solution and the second cross-linking agent aqueous solution both include glutaraldehyde solution;

[0044] The directional freeze-drying process is as follows: placing the mixed two secondary pre-crosslinked modified nanofiber foam liquids of different average diameters on a metal block, immersing the metal block in a solution at a temperature of -209°C to -40°C; the solution comprises one of ethanol, isopropanol, and liquid nitrogen; and performing the directional freeze-drying for 46 to 50 hours;

[0045] S2, performing a first grafting modification on the nanofiber aerogel prepared in step S1 using a spacer arm aqueous solution to obtain a spacer arm-modified nanofiber aerogel;

[0046] S3. Using an aptamer aqueous solution to perform a second grafting modification on the spacer-arm modified nanofiber aerogel prepared in step S2 to obtain the fiber-based porous affinity separation material having a periodic necking pore structure.

[0047] In step S2, the first grafting modification process is as follows: placing the nanofiber aerogel in a sealed container with an inlet and an outlet, placing the spacer arm aqueous solution in an open container, entering the sealed container through the inlet of the sealed container by a peristaltic pump, then passing through the nanofiber aerogel, and then flowing out through the outlet of the sealed container, and grafting the pore surface of the nanofiber aerogel at room temperature for 0.5 to 2 hours;

[0048] In step S3, the second grafting modification process is: using the aptamer aqueous solution to perform the second grafting modification on the spacer arm modified nanofiber aerogel according to the first grafting modification steps in step S2.

[0049] In step S2 and step S3, the concentration range of the spacer arm aqueous solution and the aptamer aqueous solution is 1 to 10 wt.%; the spacer arm molecule in the spacer arm aqueous solution includes one of glutaraldehyde, adipaldehyde, pentamethylenediamine, and hexamethylenediamine; the ligand molecule in the aptamer aqueous solution includes one of the aptamers of CD63, CD9, CD81 with an amino or aldehyde terminal group, protein A, and protein G.

[0050] By combining two pre-crosslinked nanofiber foams of varying viscosities, freeze-drying them, and then performing two grafting modifications, a fiber-based porous affinity separation material with a periodic necking pore structure capable of regulated aerogel pores can be obtained. This porous separation material is prepared by selecting green organic molecules containing amino and aldehyde groups and employing flow chemistry methods to perform multiple chemical reactions during multiple crosslinking, spacer arm grafting, and ligand grafting. This process is not only simple and efficient, but also environmentally friendly and pollution-free.

[0051] Specifically, in some embodiments of the present invention, the chitosan concentration in the second pre-crosslinked modified nanofiber foam liquid is 0.05-0.5wt.%, and the concentration of polyethyleneimine is 0.1-0.2wt.%; the chitosan concentration in the second pre-crosslinked modified nanofiber foam liquid is 0.5-5wt.%, and the concentration of polyethyleneimine is 0.2-1wt.%.

[0052] With such a setting, the aerogel prepared by the secondary pre-crosslinked modified nanofiber foam liquid prepared with low concentration of chitosan has a fiber ball-like structure, and the aerogel prepared by the secondary pre-crosslinked modified nanofiber foam liquid prepared with high concentration of chitosan has a sheet-like or honeycomb structure, which paves the way for the subsequent preparation of fiber-based porous affinity separation materials with periodic necking channel structure, in which the thin part is sheet-like or honeycomb-like, and the thick part is a fiber ball-like structure.

[0053] The present invention also provides a fiber-based porous affinity separation material having a periodic necked pore structure prepared by the preparation method, wherein the pore walls of the fiber-based porous affinity separation material are composed of periodically repeated necked structural units; the necked structural units include a fiber network skeleton and a polymer functional layer with affinity adsorption function attached to the surface of the fiber network skeleton;

[0054] The fiber network skeleton is composed of copolymer nanofibers; the copolymer nanofibers include ethylene vinyl alcohol copolymer nanofibers;

[0055] The polymer functional layer is formed by cross-linking a polymer cross-linked network layer, a spacer arm and an affinity ligand; the polymer cross-linked network layer is formed by cross-linking chitosan, polyethyleneimine and glutaraldehyde; the affinity ligand is an aptamer with an amino or aldehyde terminal group;

[0056] The aperture size of the necking structure unit in a direction perpendicular to the axial direction of the channel changes with the axial position, and forms a structure with small apertures at both ends and large aperture in the middle or large apertures at both ends and small aperture in the middle.

[0057] In this way, the prepared fiber-based porous affinity separation material with a periodic necking pore structure with small pore diameters at both ends and a large pore diameter in the middle or large pore diameters at both ends and a small pore diameter in the middle can regulate the contact probability between the liquid and the pore wall by means of the change in the size of the pores in the flow direction of the liquid. On the one hand, it can improve the efficiency and speed of affinity modification, and on the other hand, it can improve the adsorption efficiency of the substance to be separated in the liquid, thereby having a more excellent implementation effect in both the preparation process and the separation process.

[0058] The fiber-based porous affinity separation material with a periodic necking channel structure and its preparation method provided by the present invention are specifically described below with reference to the embodiments:

[0059] Example 1

[0060] This embodiment provides a method for preparing a fiber-based porous affinity separation material having a periodic necked pore structure, which specifically comprises the following steps:

[0061] S1, mixing a second pre-crosslinked modified nanofiber foam liquid 1 with a viscosity of 1 mPa·s and a second pre-crosslinked modified nanofiber foam liquid 2 with a viscosity of 10 mPa·s and then directional freeze-drying them for 48 hours to prepare a nanofiber aerogel with a periodic necked pore structure;

[0062] The directional freeze-drying process is as follows: placing the mixed two secondary pre-crosslinked modified nanofiber foam liquids on a copper block, and immersing the copper block in liquid nitrogen at a temperature of -100°C;

[0063] The preparation process of the secondary pre-crosslinked modified nanofiber foam liquid is as follows:

[0064] 1) Melt-extruding the sea-phase polymer to prepare copolymer nanofibers;

[0065] The marine polymer comprises a mixture of copolymer masterbatch and cellulose acetate butyrate; the copolymer masterbatch comprises polyethylene-vinyl alcohol copolymer particles; the mass ratio of the polyethylene-vinyl alcohol copolymer particles to the cellulose acetate butyrate is 2:8;

[0066] 2) respectively mixing the two copolymer nanofibers having different average diameters with a 1 wt.% glutaraldehyde solution at a mass ratio of 1:20 and dispersing them in an emulsifier for 15 minutes to obtain a pre-crosslinked modified nanofiber foam liquid after the reaction;

[0067] 3) mixing the pre-crosslinked modified nanofiber foam liquid with a 1 wt.% glutaraldehyde solution, a 0.05 wt.% chitosan aqueous solution, and a 0.1 wt.% polyethyleneimine aqueous solution in a volume ratio of 1:0.3:2:0.5, and stirring at a constant speed for 20 minutes to obtain a secondary pre-crosslinked modified nanofiber foam liquid with a viscosity of 1 mPa·s;

[0068] The pre-crosslinked modified nanofiber foam liquid was mixed with a 1 wt.% glutaraldehyde solution, a 0.5 wt.% chitosan aqueous solution, and a 0.2 wt.% polyethyleneimine aqueous solution in a volume ratio of 1:0.3:2:0.5, and stirred at a constant speed for 20 minutes to obtain a secondary pre-crosslinked modified nanofiber foam liquid II with a viscosity of 10 mPa·s;

[0069] S2, the first grafting modification process is as follows: placing the nanofiber aerogel prepared in step S1 in a sealed container with an inlet and an outlet, placing a 1 wt.% glutaraldehyde aqueous solution in an open container, allowing a peristaltic pump to enter the sealed container through the inlet, then pass through the nanofiber aerogel, and then flow out through the outlet of the sealed container, performing grafting modification on the pore surface of the nanofiber aerogel at room temperature for 1 hour to obtain a spacer-arm modified nanofiber aerogel;

[0070] S3. Place the nanofiber aerogel treated in step S2 in a closed container with an inlet and outlet, place an aqueous solution of an aptamer with a ligand molecule of protein A at a concentration of 1 wt.% in an open container, and enter the closed container through the inlet of the closed container via a peristaltic pump, then pass through the nanofiber aerogel treated in step S2, and then flow out through the outlet of the closed container. At room temperature, the pore surface of the nanofiber aerogel treated in step S2 is grafted and modified for 1 hour to obtain the fiber-based porous affinity separation material with a periodic necking pore structure.

[0071] In this embodiment, a fiber-based porous affinity separation material with a periodic necking pore structure prepared by the above-mentioned preparation method is applied to the separation and purification of biomacromolecules. The unique pore structure design of this material not only increases the specific surface area and provides more binding sites, but also effectively regulates the molecular diffusion and screening process through the periodic necking structure, thereby achieving efficient capture and release of target biomacromolecules.

[0072] Examples 2 to 7

[0073] Examples 2 to 7 respectively provide a fiber-based porous affinity separation material having a periodic necking channel structure and a preparation method thereof. The difference from Example 1 is that the concentrations of chitosan and polyethyleneimine in the two secondary pre-crosslinked modified nanofiber foam solutions used in step S1 are different:

[0074] The chitosan concentration in the second pre-crosslinked modified nanofiber foam solution in Example 2 is 0.5wt.%, and the concentration of polyethyleneimine is 0.1wt.%; the chitosan concentration in the second pre-crosslinked modified nanofiber foam solution is 0.5wt.%, and the concentration of polyethyleneimine is 0.2wt.%.

[0075] The chitosan concentration in the second pre-crosslinked modified nanofiber foam solution in Example 3 is 0.05wt.%, and the concentration of polyethyleneimine is 0.15wt.%; the chitosan concentration in the second pre-crosslinked modified nanofiber foam solution is 0.5wt.%, and the concentration of polyethyleneimine is 0.2wt.%.

[0076] The chitosan concentration in the second pre-crosslinked modified nanofiber foam solution in Example 4 is 0.05wt.%, and the concentration of polyethyleneimine is 0.1wt.%; the chitosan concentration in the second pre-crosslinked modified nanofiber foam solution is 5wt.%, and the concentration of polyethyleneimine is 0.2wt.%.

[0077] The chitosan concentration in the second pre-crosslinked modified nanofiber foam solution in Example 5 is 0.05wt.%, and the concentration of polyethyleneimine is 0.1wt.%; the chitosan concentration in the second pre-crosslinked modified nanofiber foam solution is 0.5wt.%, and the concentration of polyethyleneimine is 1wt.%.

[0078] The chitosan concentration in the second pre-crosslinked modified nanofiber foam liquid in Example 6 is 0.4wt.%, and the concentration of polyethyleneimine is 0.1wt.%; the chitosan concentration in the second pre-crosslinked modified nanofiber foam liquid is 5wt.%, and the concentration of polyethyleneimine is 0.2wt.%.

[0079] The chitosan concentration in the second pre-crosslinked modified nanofiber foam solution in Example 7 is 0.4wt.%, and the concentration of polyethyleneimine is 0.1wt.%; the chitosan concentration in the second pre-crosslinked modified nanofiber foam solution is 5wt.%, and the concentration of polyethyleneimine is 1wt.%.

[0080] The remaining steps are consistent with those in Example 1 and will not be repeated here.

[0081] Comparative Examples 1 to 8

[0082] Comparative Examples 1 to 8 respectively provide a fiber-based porous affinity separation material having a periodic necking channel structure and a preparation method thereof. The difference from Example 1 is that the concentrations of chitosan and polyethyleneimine in the two secondary pre-crosslinked modified nanofiber foam solutions used in step S1 are different:

[0083] The chitosan concentration in the second pre-crosslinked modified nanofiber foam solution in Comparative Example 1 is 0.01wt.%, and the concentration of polyethyleneimine is 0.1wt.%; the chitosan concentration in the second pre-crosslinked modified nanofiber foam solution is 0.5wt.%, and the concentration of polyethyleneimine is 0.2wt.%.

[0084] The chitosan concentration in the second pre-crosslinked modified nanofiber foam solution in Comparative Example 2 is 0.8wt.%, and the concentration of polyethyleneimine is 0.1wt.%; the chitosan concentration in the second pre-crosslinked modified nanofiber foam solution is 0.5wt.%, and the concentration of polyethyleneimine is 0.2wt.%.

[0085] The chitosan concentration in the second pre-crosslinked modified nanofiber foam solution in Comparative Example 3 is 0.05wt.%, and the concentration of polyethyleneimine is 0.01wt.%; the chitosan concentration in the second pre-crosslinked modified nanofiber foam solution is 0.5wt.%, and the concentration of polyethyleneimine is 0.2wt.%.

[0086] The chitosan concentration in the second pre-crosslinked modified nanofiber foam liquid in Comparative Example 4 is 0.05wt.%, and the concentration of polyethyleneimine is 1wt.%; the chitosan concentration in the second pre-crosslinked modified nanofiber foam liquid is 0.5wt.%, and the concentration of polyethyleneimine is 0.2wt.%.

[0087] The chitosan concentration in the second pre-crosslinked modified nanofiber foam liquid in Comparative Example 5 is 0.05wt.%, and the concentration of polyethyleneimine is 0.1wt.%; the chitosan concentration in the second pre-crosslinked modified nanofiber foam liquid is 0.1wt.%, and the concentration of polyethyleneimine is 0.2wt.%.

[0088] The chitosan concentration in the second pre-crosslinked modified nanofiber foam liquid in Comparative Example 6 is 0.05wt.%, and the concentration of polyethyleneimine is 0.1wt.%; the chitosan concentration in the second pre-crosslinked modified nanofiber foam liquid is 7wt.%, and the concentration of polyethyleneimine is 0.2wt.%.

[0089] The chitosan concentration in the second pre-crosslinked modified nanofiber foam liquid in Comparative Example 7 is 0.05wt.%, and the concentration of polyethyleneimine is 0.1wt.%; the chitosan concentration in the second pre-crosslinked modified nanofiber foam liquid is 0.5wt.%, and the concentration of polyethyleneimine is 0.1wt.%.

[0090] The chitosan concentration in the second pre-crosslinked modified nanofiber foam liquid in Comparative Example 8 is 0.05wt.%, and the concentration of polyethyleneimine is 0.1wt.%; the chitosan concentration in the second pre-crosslinked modified nanofiber foam liquid is 0.5wt.%, and the concentration of polyethyleneimine is 3wt.%.

[0091] The remaining steps are consistent with those in Example 1 and will not be repeated here.

[0092] The changes in parameters in each step of the embodiment and the comparative example are shown in Table 1. Comparing the experimental results of Examples 1 and 2 and Comparative Examples 1 and 2, it is found that when the chitosan concentration in the prepared secondary pre-crosslinked modified nanofiber foam solution is gradually increased within the range of 0.05 to 0.5 wt.%, the strength of the obtained fiber-based porous affinity separation material with a periodic necking channel structure is significantly improved (e.g. Figure 2As shown in Figure 2, the compression rebound performance gradually increases with the increase of chitosan concentration, which is also beneficial to Figure 3 The formation of the spherical structure shown. However, when the chitosan concentration is lower than this range, the obtained fiber-based porous affinity separation material with a periodic necking channel structure is almost inelastic. This is because when the chitosan concentration is too low, no bridges can be formed between the nanofibers, and the connection is only based on the interaction between the nanofibers. When subjected to external forces, the nanofibers cannot support each other, resulting in the strength of the nanofiber material being too weak, making it unsuitable for experiments and unable to form a spherical structure. When the chitosan concentration exceeds this range, the surface morphology of the obtained fiber-based porous affinity separation material with a periodic necking channel structure will change. This is because when the chitosan solution concentration is high, a cross-linking reaction will occur between the polyethyleneimine solution and glutaraldehyde and chitosan, causing the morphology of the nanofiber material to change from a spherical structure to a sheet-like structure, which is not conducive to the formation of a material with a periodic necking channel structure.

[0093] By comparing the experimental results of Example 1, Example 3 and Comparative Examples 3 to 4, it is found that when the concentration of polyethyleneimine in the prepared secondary pre-crosslinked modified nanofiber foam solution 1 gradually increases within the range of 0.1 to 0.2 wt.%, the obtained fiber-based porous affinity separation material with a periodic necking channel structure has more binding sites, that is, the polyethyleneimine solution contains amino groups. The higher the concentration, the more amino groups there are, which is more conducive to subsequent grafting modification. However, when the polyethyleneimine concentration is lower than this range, the obtained fiber-based porous affinity separation material with a periodic necking channel structure has only weak amino groups on the surface, which will result in a low grafting content, which is not conducive to the subsequent filtration and separation of biomacromolecules. When the polyethyleneimine concentration exceeds this range, the strength of the obtained fiber-based porous affinity separation material with a periodic necking channel structure becomes weaker (such as Figure 4 This is because the glutaraldehyde solution, chitosan solution and polyethyleneimine solution will undergo a cross-linking reaction. When the polyethyleneimine solution is too high, the polyethyleneimine will instantly cross-link with the chitosan solution, while ignoring the existence of nanofibers, which is not conducive to the formation of a spherical structure.

[0094] Comparing the experimental results of Example 1, Example 4 and Comparative Examples 5 to 6, it is found that when the chitosan concentration in the prepared secondary pre-crosslinked modified nanofiber foam solution 2 gradually increases within the range of 0.5 to 5 wt.%, the strength of the obtained fiber-based porous affinity separation material with a periodic necking pore structure gradually increases, and the honeycomb structure becomes more and more obvious (such as Figure 5As shown), it is conducive to the formation of the subsequent periodic necking channel structure. However, when the chitosan concentration is lower than this range, the obtained fiber-based porous affinity separation material with a periodic necking channel structure cannot form a sheet-like and honeycomb structure. When the chitosan concentration exceeds this range, the presence of nanofibers in the obtained fiber-based porous affinity separation material with a periodic necking channel structure will be ignored. This is because when the concentration of the chitosan solution is too high, the nanofibers will be enclosed in the chitosan solution, resulting in the presence of very few nanofibers on the surface of the obtained fiber-based porous affinity separation material with a periodic necking channel structure, which will affect the subsequent grafting and filtration separation of biomacromolecules.

[0095] Comparing the experimental results of Example 1, Example 5 and Comparative Examples 7-8, it is found that when the concentration of polyethyleneimine in the prepared secondary pre-crosslinked modified nanofiber foam solution 2 gradually increases within the range of 0.2-1wt.%, the honeycomb structure of the obtained fiber-based porous affinity separation material with a periodic necking channel structure is more regular (such as Figure 6 As shown in the figure, the grafting rate and adsorption capacity are getting higher and higher. However, when the polyethyleneimine concentration is lower than this range, the fiber-based porous affinity separation material with a periodic necking channel structure obtained has few functional groups on the surface, which will lead to a relatively low grafting rate in the subsequent grafting process, affecting the final biomacromolecule filtration separation effect. When the polyethyleneimine concentration exceeds this range, the strength of the fiber-based porous affinity separation material with a periodic necking channel structure obtained will weaken, and the underwater memory resilience will also weaken. This is because when the polyethyleneimine concentration is too high, it will instantly undergo a cross-linking reaction with the chitosan solution and glutaraldehyde when added to the solution, and instantly solidify, while ignoring the role of nanofibers, which will affect the compression resilience of the material.

[0096] Comparing Example 5 and Comparative Example 8, it can be seen that increasing the concentration of polyethyleneimine does not change the grafting rate much, and the rebound effect is not as good as that of Example 5. At the same time, the cost of the reagents used in Comparative Example 8 is also higher.

[0097] This shows that the effects of the prepared fiber-based porous affinity separation material with a periodic necking channel structure are all the result of the effects of various parameters.

[0098] Table 1 Experimental parameter settings for examples and comparative examples

[0099]

[0100]

[0101] In summary, the present invention provides a method for preparing a fiber-based porous affinity separation material with a periodic necking pore structure. By mixing two secondary pre-crosslinked modified nanofiber foam liquids with different viscosities and performing directional freeze-drying, and then performing two grafting modifications, a fiber-based porous affinity separation material with a periodic necking pore structure that can regulate the aerogel pores can be obtained. Among them, green organic molecules containing amino groups and aldehyde groups are selected, and a flow chemistry method is used to perform multiple chemical reactions during multiple crosslinking, spacer arm grafting modification, and ligand grafting modification to complete the preparation of the porous separation material. This process is not only simple and efficient but also has the advantages of being green and pollution-free. In addition, the aerogel prepared by the secondary pre-crosslinked modified nanofiber foam liquid prepared with a low concentration of chitosan has a fiber ball-like structure, and the aerogel prepared by the secondary pre-crosslinked modified nanofiber foam liquid prepared with a high concentration of chitosan has a sheet-like or honeycomb-like structure, which also paves the way for the subsequent preparation of fiber-based porous affinity separation materials with a periodic necking pore structure. The present invention also provides a fiber-based porous affinity separation material with a periodic necking pore structure, whose pore walls are composed of periodically repeated necking structural units. Because it has a structure with small pore diameters at both ends and large pore diameters in the middle, or large pore diameters at both ends and small pore diameters in the middle, it can regulate the contact probability between the liquid and the pore wall by means of the change in the size of the pores in the flow direction of the liquid. On the one hand, it can improve the efficiency and speed of affinity modification, and on the other hand, it can improve the adsorption efficiency of the substance to be separated in the liquid, thereby having a more excellent implementation effect in both the preparation process and the separation process.

[0102] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for preparing a fiber-based porous affinity separation material having a periodic necking channel structure, characterized in that: The steps include: S1. Mixing two secondary pre-crosslinked modified nanofiber foam solutions with different viscosities and then freeze-drying them to prepare a nanofiber aerogel with a periodic necked pore structure; S2, performing a first grafting modification on the nanofiber aerogel prepared in step S1 using a spacer arm aqueous solution to obtain a spacer arm-modified nanofiber aerogel; S3. Using an aptamer aqueous solution to perform a second grafting modification on the spacer-arm modified nanofiber aerogel prepared in step S2 to obtain the fiber-based porous affinity separation material having a periodic necking pore structure.

2. The method for preparing a fiber-based porous affinity separation material having a periodic necked channel structure according to claim 1, characterized in that: In step S1, the two secondary pre-crosslinked modified nanofiber foam liquids with different viscosities include a first secondary pre-crosslinked modified nanofiber foam liquid with a viscosity of 1 mPa·s≤X<10 mPa·s and a second secondary pre-crosslinked modified nanofiber foam liquid with a viscosity of 10 mPa·s≤X≤10000 mPa·s.

3. The method for preparing a fiber-based porous affinity separation material having a periodic necked channel structure according to claim 2, characterized in that: In step S1, the preparation process of the secondary pre-crosslinked modified nanofiber foam liquid is as follows: 1) Mixing and dispersing the copolymer nanofibers with a first crosslinking agent to obtain a pre-crosslinked modified nanofiber foam liquid; 2) The pre-crosslinked modified nanofiber foam liquid is mixed with a second crosslinking agent, a chitosan aqueous solution, and a polyethyleneimine aqueous solution to obtain the secondary pre-crosslinked modified nanofiber foam liquid.

4. The method for preparing a fiber-based porous affinity separation material having a periodic necked pore structure according to claim 3, characterized in that: The chitosan concentration in the second pre-crosslinked modified nanofiber foam solution is 0.05-0.5wt.%, and the polyethyleneimine concentration is 0.1-0.2wt.%; the chitosan concentration in the second pre-crosslinked modified nanofiber foam solution is 0.5-5wt.%, and the polyethyleneimine concentration is 0.2-1wt.%.

5. The method for preparing a fiber-based porous affinity separation material having a periodic necked channel structure according to claim 1, characterized in that: In step S2, the process of the first grafting modification is: placing the nanofiber aerogel in a closed container with an inlet and outlet, placing the spacer arm aqueous solution in an open container, entering the closed container through the inlet of the closed container by a peristaltic pump, then passing through the nanofiber aerogel, and then flowing out through the outlet of the closed container. At room temperature, the pore surface of the nanofiber aerogel is grafted and modified for 0.5 to 2 hours; in step S3, the process of the second grafting modification is: using the aptamer aqueous solution to perform the second grafting modification on the spacer arm-modified nanofiber aerogel according to the steps of the first grafting modification in step S2.

6. The method for preparing a fiber-based porous affinity separation material having a periodic necked pore structure according to claim 5, characterized in that: In step S2 and step S3, the concentration range of the spacer arm aqueous solution and the aptamer aqueous solution is 1~10wt.%; the spacer arm molecule in the spacer arm aqueous solution includes one of glutaraldehyde, adipaldehyde, pentamethylenediamine, and hexamethylenediamine; the ligand molecule in the aptamer aqueous solution includes one of the aptamers of CD63, CD9, CD81 with an amino or aldehyde terminal group, protein A, and protein G.

7. A fiber-based porous affinity separation material having a periodic necked pore structure, characterized in that: The pore walls of the fiber-based porous affinity separation material are composed of periodically repeated necking structural units; the necking structural units include a fiber network skeleton and a polymer functional layer with affinity adsorption function attached to the surface of the fiber network skeleton.

8. The fiber-based porous affinity separation material having a periodic necked channel structure according to claim 7, characterized in that: The fiber network skeleton is composed of copolymer nanofibers; the copolymer nanofibers include ethylene vinyl alcohol copolymer nanofibers.

9. The fiber-based porous affinity separation material having a periodic necked channel structure according to claim 8, characterized in that: The polymer functional layer is cross-linked by a polymer cross-linked network layer, a spacer arm and an affinity ligand; the polymer cross-linked network layer is cross-linked by chitosan, polyethyleneimine and glutaraldehyde; the affinity ligand is an aptamer with an amino or aldehyde terminal group.

10. The fiber-based porous affinity separation material having a periodic necked channel structure according to claim 8, characterized in that: The necking structure unit is a structure with small pore diameters at both ends and a large pore diameter in the middle, or with a large pore diameter at both ends and a small pore diameter in the middle.

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