Surface blotting membrane applied to separation and purification of bovine serum albumin and preparation method of surface blotting membrane

Abstract

The invention discloses a surface blotting membrane capable of being applied to separation and purification of bovine serum albumin and a preparation method of the surface blotting membrane in the technical field of molecular imprinting. The surface blotting membrane is composed of a surface polymerization blotting layer and a cellulose acetate porous membrane substrate. According to the preparation method, bovine serum albumin is taken as a template molecule, methyl methacrylate and a cellulose acetate fiber porous membrane modified by a silane coupling agent KH570 are reacted to achieve the purpose of imprinting bovine serum albumin, and after a template is eluted, the surface imprinted membrane which can be applied to separation and purification of bovine serum albumin is prepared. The surface imprinted membrane can be used for specific selective separation of bovine serum albumin.

Description

Technical Field

[0001] The invention relates to the technical field of molecular imprinting, and in particular to a surface imprinting membrane used for separating and purifying bovine serum albumin and a preparation method thereof. Background Art

[0002] The application date is: 2014.12.10, the application number is CN201410391048.X, the publication number is: CN104193872A, the publication date is: 2014.12.10, the patent name is: "A preparation method and application of methyl p-hydroxybenzoate molecularly imprinted porous membrane". Methyl p-hydroxybenzoate is used as a template molecule and a methyl p-hydroxybenzoate molecularly imprinted porous membrane is prepared at room temperature using a high internal phase emulsion template method.

[0003] The application date is: 2020.12.10, the application number is CN202011439309.2, the publication number is: CN112403449A, the publication date is: 2021.02.26, and the patent name is: "A preparation and application of phenylethanol glycosides separated by aminated mesoporous silica molecular imprinting hybrid membrane". A preparation method for separating phenylethanol glycosides based on aminated mesoporous silica molecular imprinting hybrid membrane is invented, and the specific recognition effect of phenylethanol glycoside molecular imprinting membrane is used to separate and purify phenylethanol glycosides in Cistanche tubulosa.

[0004] The application date is: 2021.05.26, the application number is: CN202110579671.8, the publication number is: CN113385150A, the publication date is: 2021.09.14, and the patent name is: "Magnetic molecular imprinted porous membrane and its preparation method and application". Iron tetroxide nanoparticles are introduced into the molecular imprinted porous membrane, and an external magnetic field is used to make the nanoparticles change the shape and size of the imprinted cavity to achieve the purpose of desorbing the template molecules.

[0005] Most of the porous membranes prepared by the above inventions use small molecules as template molecules. This type of molecular imprinting membrane has been widely studied. Although the other part uses protein macromolecules as template molecules, there is no stable attachment point when polymer is imprinted on the film surface, and the imprinting cavity is unstable and easy to fall off. The invention uses a silane coupling agent KH570 to modify the deacetylated cellulose acetate porous membrane. The coupling agent reacts with the hydroxyl groups on the deacetylated cellulose acetate film and simultaneously adds double bonds to the surface of the porous membrane. The double bonds react with methyl methacrylate to enable the polymethyl methacrylate imprinting surface layer to be stably attached.

[0006] Molecular imprinting technology has always been a research hotspot in sensing technology, electrochemistry, selective adsorption, etc. According to statistics, most studies focus on molecular imprinting materials with small molecules as templates, and the research on imprinted porous membranes with macromolecules as templates is relatively lagging behind. The surface molecular imprinting porous membrane technology synthesized by this method can provide an effective idea for molecular imprinting polymers with macromolecules as templates. Summary of the invention

[0007] The purpose of the present invention is to provide a surface imprinted membrane for separating and purifying bovine serum albumin and a preparation method thereof, so as to overcome the defects of the prior art. The molecularly imprinted porous membrane uses macromolecules such as bovine serum albumin as template molecules, and uses silane coupling agent KH570 as a "bridge" to connect the cellulose acetate base membrane and the polymethyl methacrylate imprinted layer, so that the molecularly imprinted porous layer can be stably composited on the surface of the cellulose acetate porous membrane.

[0008] To this end, the present invention provides a method for preparing a surface imprinted membrane for separating and purifying bovine serum albumin, comprising the following steps:

[0009] S1. Preparation of porous membrane substrate: dissolving cellulose acetate in N-methylpyrrolidone to prepare a casting solution, scraping the film with a scraper, and then preparing a porous membrane through liquid phase conversion, and then deacetylation modification of the prepared porous membrane to obtain the porous membrane substrate; in the casting solution, the mass percentage of cellulose acetate is 16-20wt%, and the mass percentage of N-methylpyrrolidone is 80-84wt%;

[0010] S2. Aldehydation modification of porous membrane substrate: placing the deacetylated cellulose acetate porous membrane in a sodium periodate solution with a concentration of 7-9 wt % and leaving it in the dark at room temperature for 8±1 h to obtain an aldehydated porous membrane;

[0011] S3. Coupling agent modification: water, ethanol, and silane coupling agent KH570 were mixed in a container at a volume ratio of 20:(18-22):(2.5-3.5), and then the hydroformylation porous membrane was placed therein and reacted at 75±5°C for 24±2h;

[0012] S4. Grafting bovine serum albumin: The porous membrane modified with the coupling agent is placed in a bovine serum albumin solution with a concentration of 2.5±0.2 mg / ml at room temperature for 12±2 h, and the aldehyde group is grafted with the amino group on the protein;

[0013] S5. Surface imprinted porous membrane: Methyl methacrylate, ethylene glycol dimethacrylate, 4.0 mg / ml sodium bisulfite and 4 mg / ml ammonium persulfate were mixed in a volume ratio of 22: (2.7-2.9): (2.9-3.1): (2.9-3.1) at 45±2°C under argon atmosphere, and the porous membrane after step S4 was placed in the mixed solution for reaction for 10±2h;

[0014] S6. Elution of template molecules: The porous membrane prepared in S5 is placed in a 0.1±0.01 M sulfuric acid solution and shaken for 2±0.5 h to completely remove the template protein, thereby obtaining a surface imprinted membrane composed of a porous membrane base membrane and a surface layer with an imprinted cavity for separation and purification of bovine serum albumin.

[0015] Furthermore, the deacetylation modification in step S1 is to immerse the porous membrane obtained by liquid phase conversion preparation in a 0.1±0.01M NaOH alcohol solution for deacetylation modification.

[0016] The thickness of the doctor blade in step S1 is 250 μm.

[0017] In step S4, the bovine serum albumin solution is obtained by taking 250.0±20 mg of bovine serum albumin and dissolving it in 10.0 ml of phosphate buffer solution (PBS) and then making up the volume in a 100.0 ml volumetric flask.

[0018] The surface imprinting membrane used for separation and purification of bovine serum albumin prepared by the above method can be used for specific selective separation of bovine serum albumin.

[0019] The porous membrane was repeatedly rinsed with deionized water to remove the residual dilute sulfuric acid solution to obtain the polymethyl methacrylate imprinted surface layer of the bovine serum albumin imprinted cavity.

[0020] The specific selective adsorption of bovine serum albumin was verified by preparing bovine serum albumin and lysozyme Lys solutions with the same concentration for specific adsorption tests. The prepared surface molecular imprinted porous membrane was immersed in the solution for 3 hours, and then the solution was taken out and its concentration change was analyzed by ultraviolet spectrophotometer to calculate the adsorption amount.

[0021] Preferably, the concentrations of the bovine serum albumin and lysozyme Lys are both 2.5 mg / ml. The surface imprinted membrane used for separation and purification of bovine serum albumin has an adsorption capacity of 7.48 nmol / cm 2 The adsorption capacity of lysozyme Lys was 2.06 nmol / cm 2 .

[0022] The beneficial effects of the present invention are:

[0023] The present invention deacetylates cellulose acetate, which is not easy to directly formaldehyde, to increase the number of hydroxyl active sites on the surface of the porous membrane. On the one hand, these hydroxyl active sites can be selectively oxidized using sodium periodate to increase the amount of bovine serum albumin grafted onto the porous membrane. On the other hand, the active hydroxyl groups can directly react with the silane coupling agent KH570 to introduce double bonds. The introduced double bonds provide attachment sites for the methyl methacrylate imprinting reaction of bovine serum albumin, allowing polymethyl methacrylate to stably attach to the surface of the porous membrane. At the same time, the preparation method of the surface imprinted membrane used for separating and purifying bovine serum albumin prepared by the present invention also provides a new feasible solution for preparing imprinted porous membranes of other proteins. The adsorption capacity of bovine serum albumin by the molecular imprinted porous membrane prepared by the present invention is 7.48 nmol / cm 2 , while the non-imprinted porous membrane is 1.34nmol / cm 2 The molecularly imprinted porous membrane uses macromolecules such as bovine serum albumin as template molecules, and uses silane coupling agent KH570 as a "bridge" to connect the cellulose acetate base membrane and the polymethyl methacrylate imprinting layer, so that the molecularly imprinted porous layer can be stably composited on the surface of the cellulose acetate porous membrane. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of the preparation process of the surface imprinting membrane used for separation and purification of bovine serum albumin in Example 1.

[0025] Figure 2 Scanning electron microscope (SEM) image of the surface of the cellulose acetate porous substrate membrane in Example 1.

[0026] Figure 3 Surface scanning electron microscope (SEM) image of the surface imprinted membrane (MIM) used for separation and purification of bovine serum albumin in Example 1.

[0027] Figure 4 FITR graph of the modified surface imprinting membrane (MIM) prepared in Example 1 for separation and purification of bovine serum albumin at each step.

[0028] Figure 5 In the selective adsorption test in Examples 1 and 2, the adsorption amount of bovine serum albumin was specifically adsorbed. DETAILED DESCRIPTION

[0029] The present invention is described in detail below in conjunction with specific implementation modes.

[0030] Example 1

[0031] A method for preparing a surface imprinted membrane for separating and purifying bovine serum albumin comprises the following steps in sequence:

[0032] 1. Preparation of casting solution: 3.6 g of cellulose acetate (molecular weight 60,000) and 16.4 g of N-methylpyrrolidone were placed in a three-necked flask, mechanically stirred for 12 h in a water bath at 40 °C, and then allowed to stand for 10 h to remove bubbles in the casting solution.

[0033] 2. Preparation of cellulose acetate substrate membrane: The casting liquid was poured on a quartz glass plate and manually scraped using a scraper with a side height of 250 μm. After scraping, the casting liquid was allowed to flow in the air for 60 seconds, and then immersed in deionized water for solution replacement for 24 hours to prepare a cellulose acetate porous substrate membrane (CA). Figure 2 FIG. 1 is a surface morphology diagram of the substrate membrane, which shows that it is a relatively smooth porous membrane before printing.

[0034] 3. Preparation of surface imprinted porous membrane (MIM) containing bovine serum albumin. CA was subjected to deacetylation, selective oxidation, silane coupling agent KH570, protein grafting, surface imprinting treatment and template protein elution treatment in sequence to obtain MIM. The specific steps are as follows: (e.g. Figure 1 As shown, a is a schematic diagram of the MIM preparation process, b is the imprinting layer and bovine serum albumin template molecules in the schematic diagram, and c is a schematic diagram of the selective adsorption principle of the imprinting cavity)

[0035] (1) Deacetylation modification of cellulose acetate porous membrane (CA): CA was immersed in 0.1 M NaOH alcohol solution and reacted at room temperature for 2 h to obtain a porous membrane with a high hydroxyl content. The membrane was then washed with anhydrous ethanol and deionized water for multiple times until the washing solution was neutral.

[0036] (2) Selective oxidation modification: The membrane obtained in (1) was placed in an 8% wt NaIO4 aqueous solution and reacted for 8 h at room temperature in the dark to selectively oxidize the hydroxyl groups to aldehyde groups.

[0037] (3) Modification with silane coupling agent KH570: 40.0 ml H2O, 40.0 ml C2H5OH and 6.0 ml silane coupling agent KH570 were weighed and mixed with the aid of an ultrasonic machine. The membrane obtained in (2) was placed in a three-necked flask with a uniformly mixed mixture and reacted in a water bath at 75°C for 24 h.

[0038] (4) Grafting bovine serum albumin onto the porous membrane surface: Weigh 250.0 mg of bovine serum albumin into 10.0 ml of diluted phosphate buffer solution (0.1 PBS), and dilute it to a 100.0 ml volumetric flask to prepare a 2.5 mg / ml bovine serum albumin solution. Take 30 ml of this protein solution into a container, and place the membrane obtained in (3) into the container. The aldehyde groups obtained by oxidation on the membrane surface are grafted with the amino groups on the lysine in the bovine serum albumin for 12 hours.

[0039] (5) Surface imprinting of bovine serum albumin porous membrane: 22.0 ml of methyl methacrylate, 2.8 ml of ethylene glycol dimethacrylate, 4.0 mg / ml of sodium bisulfite (3.0 ml) and 4.0 mg / ml of ammonium persulfate (3.0 ml) were mixed in a three-necked flask. The mixed solution was deoxygenated with argon for 10 min. The porous membrane described in (4) was placed in the mixed solution and reacted at 45°C for 10 h. The imprinted membrane was washed several times with anhydrous ethanol and deionized water respectively. (The prepared MIM surface is as shown in Figure 3 As shown, with cellulose acetate base film ( Figure 2 ), its surface is rougher and part of the granular polymer can be clearly seen, indicating the successful preparation of the polymer imprint layer)

[0040] (6) Elution of template molecules: Prepare a sulfuric acid solution with a concentration of 0.1 M and take 20 ml into a centrifuge tube; place the porous membrane prepared in (5) in the centrifuge tube and shake it in a shaker for 2 h to completely remove the template protein; repeatedly rinse the porous membrane with deionized water to remove the residual dilute sulfuric acid solution to obtain a polymethyl methacrylate imprinted surface layer of the bovine serum albumin imprinted cavity.

[0041] The infrared structural characterization of each step in this embodiment is as follows: Figure 4 As shown, ae in the figure corresponds to (1)-(5) of the above-mentioned preparation steps respectively. Figure 4 a and b are at a wavelength of 1748 cm -1 The disappearance of the carbonyl group in the acetyl group indicates the success of the deacetylation modification; b and c have a wavelength of 851cm -1 The appearance of the hemiacetal peak at 637 cm indicates that the hydroformylation modification is successful. The changes in the peak values ​​and peak shapes between c and d are obvious, indicating that the silane coupling agent KH570 is successfully modified. Compared with d, e has a new peak at 637 cm -1 , 1246cm -1 , 1718cm -1 The peak of Figure 2 and Figure 3 The results obtained after comparison with the SEM images are consistent.

[0042] The same concentration (2.5 mg / ml) of bovine serum albumin and lysozyme Lys solution were prepared for specific adsorption test. The prepared surface molecular imprinted porous membrane was immersed in the solution for 3 hours, and then the solution was taken out and its concentration change was analyzed by ultraviolet spectrophotometer to calculate the adsorption amount.

[0043] Adsorption results ( Figure 5 ) shows that the surface imprinted membrane prepared in this example for separation and purification of bovine serum albumin has an adsorption capacity of 7.48 nmol / cm 2The adsorption capacity of lysozyme Lys was 2.06 nmol / cm 2 , indicating its selective adsorption effect on the template molecule of bovine serum albumin.

[0044] Comparative Example 1

[0045] Steps 1, 2, and 3 are the same as those in Example 1, except for step 4.

[0046] Preparation of non-imprinted porous membrane (NIM): CA is sequentially subjected to deacetylation, selective oxidation, silane coupling agent KH570, and surface imprinting treatment to prepare NIM. The specific steps are as follows:

[0047] (1) Deacetylation modification of cellulose acetate porous membrane (CA): CA was immersed in 0.1 M NaOH alcohol solution and reacted at room temperature for 2 h to obtain a porous membrane with a high hydroxyl content. The membrane was then washed with anhydrous ethanol and deionized water for multiple times until the washing solution was neutral.

[0048] (2) Selective oxidation modification: The membrane obtained in (1) was placed in an 8% wt NaIO4 aqueous solution and reacted for 8 h at room temperature in the dark to selectively oxidize the hydroxyl groups to aldehyde groups.

[0049] (3) Modification with silane coupling agent KH570: 40.0 ml H2O, 40.0 ml C2H5OH and 6.0 ml silane coupling agent KH570 were weighed and mixed with the aid of an ultrasonic machine. The membrane obtained in (2) was placed in a three-necked flask with a uniformly mixed mixture and reacted in a water bath at 75°C for 24 h.

[0050] (4) Surface imprinting of bovine serum albumin porous membrane: 22.0 ml of methyl methacrylate, 2.8 ml of ethylene glycol dimethacrylate, 4.0 mg / ml of sodium bisulfite (3.0 ml) and 4.0 mg / ml of ammonium persulfate (3.0 ml) were mixed in a three-necked flask. The mixed solution was deoxygenated with argon for 10 min, and then the porous membrane described in (3) was placed in the mixed solution and reacted at 45°C for 10 h. The imprinted membrane was washed multiple times with anhydrous ethanol and deionized water, respectively.

[0051] The same concentration (2.5 mg / ml) of bovine serum albumin and lysozyme Lys solution were prepared for specific adsorption test. The prepared non-imprinted porous membrane was immersed in the solution for 3 hours, and then the solution was taken out and its concentration change was analyzed by ultraviolet spectrophotometer to calculate the adsorption amount.

[0052] The specific adsorption experiment results in this example are ( Figure 5 ) showed that the adsorption capacity of BSA by non-imprinted porous membrane (NIM) was 1.34 nmol / cm 2The adsorption capacity of lysozyme Lys was 1.68 nmol / cm 2 This indicates that if the non-imprinted porous membrane prepared without adding the template molecule (bovine serum albumin) does not have a specific adsorption effect on bovine serum albumin, the purpose of separating and purifying bovine serum albumin cannot be achieved.

[0053] Comparative Example 2

[0054] Steps 1, 2, 3, and 4 are the same as those in Example 1, except for step 5.

[0055] Preparation of surface imprinted cavity porous membrane (MIM) containing bovine serum albumin: CA is sequentially subjected to deacetylation, selective oxidation, silane coupling agent KH570, protein grafting, surface imprinting treatment and template protein elution treatment to prepare MIM. The specific steps are as follows:

[0056] (1) Deacetylation modification of cellulose acetate porous membrane (CA): CA was immersed in 0.1 M NaOH alcohol solution and reacted at room temperature for 2 h to obtain a porous membrane with a high hydroxyl content. The membrane was then washed with anhydrous ethanol and deionized water for multiple times until the washing solution was neutral.

[0057] (2) Selective oxidation modification: The membrane obtained in (1) was placed in an 8% wt NaIO4 aqueous solution and reacted for 8 h at room temperature in the dark to selectively oxidize the hydroxyl groups to aldehyde groups.

[0058] (3) Modification with silane coupling agent KH570: 40.0 ml H2O, 40.0 ml C2H5OH and 6.0 ml silane coupling agent KH570 were weighed and mixed with the aid of an ultrasonic machine. The membrane obtained in (2) was placed in a three-necked flask with a uniformly mixed mixture and reacted in a water bath at 75°C for 24 h.

[0059] (4) Grafting bovine serum albumin onto the porous membrane surface: Weigh 250.0 mg of bovine serum albumin into 10.0 ml of diluted phosphate buffer solution (0.1 PBS), and dilute it to a 100.0 ml volumetric flask to prepare a 2.5 mg / ml bovine serum albumin solution. Take 30 ml of this protein solution into a container, and place the membrane obtained in (3) into the container. The aldehyde groups obtained by oxidation on the membrane surface are grafted with the amino groups on the lysine in the bovine serum albumin for 12 hours.

[0060] (5) Surface imprinting of bovine serum albumin porous membrane: 22.0 ml of methyl methacrylate, 2.8 ml of ethylene glycol dimethacrylate, 4.0 mg / ml of sodium bisulfite (3.0 ml) and 4.0 mg / ml of ammonium persulfate (3.0 ml) were mixed in a three-necked flask. The mixed solution was deoxygenated with argon for 10 min, and then the porous membrane described in (4) was placed in the mixed solution and reacted at 45°C for 5 h. The imprinted membrane was washed multiple times with anhydrous ethanol and deionized water, respectively.

[0061] (6) Elution of template molecules: Prepare a sulfuric acid solution with a concentration of 0.1 M and take 20 ml into a centrifuge tube; place the porous membrane prepared in (5) in the centrifuge tube and shake it in a shaker for 2 h to completely remove the template protein; repeatedly rinse the porous membrane with deionized water to remove the residual dilute sulfuric acid solution to obtain a polymethyl methacrylate imprinted surface layer of the bovine serum albumin imprinted cavity.

[0062] A 2.5 mg / ml bovine serum albumin solution was prepared to conduct an adsorption experiment on the molecular imprinted membrane obtained in this example. After the membrane was immersed in the solution for 3 hours, a small amount of the bovine serum albumin solution was taken and its concentration change was analyzed by ultraviolet spectrophotometer. The adsorption amount was calculated to be 5.54 nmol / cm 2 .

[0063] In this example, when surface imprinting polymerization (5) is performed, the imprinting time is 5 h, while that in Example 1 is 10 h. The adsorption amount of bovine serum albumin in this example is 5.54 nmol / cm 2 Compared with 7.48 nmol / cm in Example 1 2 The lower value is due to the short imprinting time resulting in a thinner imprinting layer, and the selectivity for bovine serum albumin needs to be improved.

[0064] Comparative Example 3

[0065] Steps 1, 2, 3, and 4 are the same as those in Example 1, except for step 5.

[0066] Preparation of surface imprinted porous membrane (MIM) containing bovine serum albumin, CA is sequentially subjected to deacetylation, selective oxidation, silane coupling agent KH570, protein grafting, surface imprinting treatment and template protein elution treatment to prepare MIM, the specific steps are as follows:

[0067] (1) Deacetylation modification of cellulose acetate porous membrane (CA): CA was immersed in 0.1 M NaOH alcohol solution and reacted at room temperature for 2 h to obtain a porous membrane with a high hydroxyl content. The membrane was then washed with anhydrous ethanol and deionized water for multiple times until the washing solution was neutral.

[0068] (2) Selective oxidation modification: The membrane obtained in (1) was placed in an 8% wt NaIO4 aqueous solution and reacted for 8 h at room temperature in the dark to selectively oxidize the hydroxyl groups to aldehyde groups.

[0069] (3) Modification with silane coupling agent KH570: 40.0 ml H2O, 40.0 ml C2H5OH and 6.0 ml silane coupling agent KH570 were weighed and mixed with the aid of an ultrasonic machine. The membrane obtained in (2) was placed in a three-necked flask with a uniformly mixed mixture and reacted in a water bath at 75°C for 24 h.

[0070] (4) Grafting bovine serum albumin onto the porous membrane surface: Weigh 250.0 mg of bovine serum albumin into 10.0 ml of diluted phosphate buffer solution (0.1 PBS), and dilute it to a 100.0 ml volumetric flask to prepare a 2.5 mg / ml bovine serum albumin solution. Take 30 ml of this protein solution into a container, and place the membrane obtained in (3) into the container. The aldehyde groups obtained by oxidation on the membrane surface are grafted with the amino groups on the lysine in the bovine serum albumin for 12 hours.

[0071] (5) Surface imprinting of bovine serum albumin porous membrane: 22.0 ml of methyl methacrylate, 2.8 ml of ethylene glycol dimethacrylate, 4.0 mg / ml of sodium bisulfite (3.0 ml) and 4.0 mg / ml of ammonium persulfate (3.0 ml) were mixed in a three-necked flask. The mixed solution was deoxygenated with argon for 10 min, and then the porous membrane described in (4) was placed in the mixed solution and reacted at 45°C for 15 h. The imprinted membrane was washed multiple times with anhydrous ethanol and deionized water, respectively.

[0072] (6) Elution of template molecules: Prepare a sulfuric acid solution with a concentration of 0.1 M and take 20 ml into a centrifuge tube; place the porous membrane prepared in (5) in the centrifuge tube and shake it in a shaker for 2 h to completely remove the template protein; repeatedly rinse the porous membrane with deionized water to remove the residual dilute sulfuric acid solution to obtain a polymethyl methacrylate imprinted surface layer of the bovine serum albumin imprinted cavity.

[0073] A 2.5 mg / ml bovine serum albumin solution was prepared to conduct an adsorption experiment on the molecular imprinted membrane obtained in this example. After the membrane was immersed in the solution for 3 h, a small amount of the bovine serum albumin solution was taken and its concentration change was analyzed by ultraviolet spectrophotometer. The adsorption amount was calculated to be 3.41 nmol / cm 2 .

[0074] In this example, during the surface imprinting polymerization (5), the imprinting time was 15 h, while that in Example 1 was 10 h. The adsorption amount of bovine serum albumin in this example was 3.41 nmol / cm 2 Compared with 7.48 nmol / cm in Example 1 2The large gap is due to the fact that the imprinting time is too long, resulting in a thick imprinting layer that completely covers the imprinting cavity, leading to a significant decrease in the selectivity for bovine serum albumin.

[0075] Through further experiments on comparative examples 2 and 3, the experimental results are shown in the following table:

[0076] Blotting time (h) 6h 8h 10h 12h 14h <![CDATA[Q(nmol / cm 2 )]]> 5.87 6.83 7.48 6.12 5.21

[0077] As can be seen from the table above, with the extension of imprinting time, the selective adsorption capacity of the molecular imprinted membrane for bovine serum albumin increases first and then decreases, reaching a maximum adsorption capacity of 7.48 nmol / cm at 10 h. 2 , so the best imprinting time is 10h.

[0078] The present invention is not limited to the above-mentioned embodiments. On the basis of the technical solution disclosed in the present invention, technicians in this field can make some substitutions and deformations to some technical features therein according to the disclosed technical content without creative labor, and these substitutions and deformations are all within the protection scope of the present invention.

Claims

1. A method for preparing a surface imprinted membrane for separation and purification of bovine serum albumin, characterized in that: The steps include: S1. Preparation of porous membrane substrate: dissolving cellulose acetate in N-methylpyrrolidone to prepare a casting solution, scraping the film with a scraper, and then preparing a porous membrane through liquid phase conversion, and then deacetylation modification of the prepared porous membrane to obtain the porous membrane substrate; in the casting solution, the mass percentage of cellulose acetate is 16-20wt%, and the mass percentage of N-methylpyrrolidone is 80-84wt%; S2. Aldehydation modification of porous membrane substrate: The deacetylated cellulose acetate porous membrane was placed in a sodium periodate solution with a content of 7-9 wt % and placed in the dark at room temperature for 8±1 h to obtain an aldehyde porous membrane; S3. Coupling agent modification: water, ethanol and silane coupling agent KH570 were mixed in a container at a volume ratio of 20: (18-22): (2.5-3.5), and then the hydroformylation porous membrane was placed therein and reacted at 75±5°C for 24±2h; S4. Grafting bovine serum albumin: The porous membrane modified with the coupling agent was placed in a 2.5±0.2 mg / ml bovine serum albumin solution at room temperature for 12±2 h to perform a grafting reaction between the aldehyde group and the amino group on the protein; S5. Surface imprinted porous membrane: Methyl methacrylate, ethylene glycol dimethacrylate, 4.0 mg / ml sodium bisulfite and 4 mg / ml ammonium persulfate were mixed at a volume ratio of 22: (2.7-2.9): (2.9-3.1): (2.9-3.1) at 45±2°C under an argon atmosphere, and the porous membrane after step S4 was placed in the mixed solution to react for 10±2 hours; S6. Elution of template molecules: The porous membrane prepared in S5 is placed in a 0.1±0.01 M sulfuric acid solution and shaken for 2±0.5 h to completely remove the template protein, thereby obtaining a surface imprinted membrane composed of a porous membrane base membrane and a surface layer with an imprinted cavity for separation and purification of bovine serum albumin.

2. The method for preparing a surface imprinted membrane for separating and purifying bovine serum albumin according to claim 1, characterized in that: The deacetylation modification in step S1 is to immerse the porous membrane obtained by liquid phase conversion preparation in a 0.1±0.01M NaOH alcohol solution for deacetylation modification.

3. The method for preparing a surface imprinted membrane for separating and purifying bovine serum albumin according to claim 1, characterized in that: The thickness of the doctor blade in step S1 is 250 μm.

4. The method for preparing a surface imprinted membrane for separation and purification of bovine serum albumin according to claim 1, characterized in that: In step S4, the bovine serum albumin solution is obtained by taking 250.0±20 mg of bovine serum albumin and dissolving it in 10.0 ml of phosphate buffer solution (PBS), and then making up the volume in a 100.0 ml volumetric flask.

5. A surface imprinting membrane for separation and purification of bovine serum albumin prepared according to the method of any one of claims 1 to 4.

6. The surface imprinting membrane for separation and purification of bovine serum albumin according to claim 5, characterized in that: Used for specific selective separation of bovine serum albumin.

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