A monolithic crystal gel medium embedded with bacterial cellulose and a preparation method thereof

Abstract

The present invention discloses a whole crystal gel medium embedded with bacterial cellulose and a preparation method thereof, wherein the crystal gel medium has an internal pore size of 10 to 300 microns, a porosity of 70 to 88%, and a permeability of 10 ‑12 ~10 ‑13 m 2 ; After chemical modification, the crystal gel has a higher adsorption capacity with bovine serum albumin as a model protein. The crystal gel has hydroxyethyl methacrylate with embedded bacterial cellulose particles as a skeleton and grafted anionic functional groups. The crystal gel medium provided by the present invention has good biocompatibility, wherein the bacterial cellulose is biodegradable, and due to the embedded bacterial cellulose particles, more functional groups are obtained, the specific surface area is increased, and the mechanical strength of the crystal gel medium is further enhanced, which is conducive to repeated use and has a good application prospect in the field of biological separation.

Description

Technical Field

[0001] The invention belongs to the field of biochemical separation, and in particular relates to a bacterial cellulose embedded integral crystal gel medium and a preparation method thereof. Background Art

[0002] Crystal gel is a porous material prepared by freezing crystallization pores and monomer polymerization or cross-linking reaction. It has excellent properties such as good elasticity, ultra-large pores, high specific surface area and high permeability, and can be recycled. There are a large number of ultra-large pores with a size of tens to hundreds of microns in the crystal gel, with large pore space and small mass transfer resistance. The matrix of the crystal gel is mostly natural or biocompatible materials, and has broad application prospects in the field of biological separation. Bacterial cellulose has a unique 3D nanostructure, large specific surface area, high water holding capacity, high chemical purity, degree of polymerization and tensile strength, rich in hydroxyl groups, good biocompatibility and biodegradability, and can be used as a new medium for biological separation and a new carrier for high-density culture of microbial cells.

[0003] Conventional crystal gel media have few adsorption points and low adsorption capacity. Due to the special structural characteristics of bacterial cellulose, it can be embedded in the crystal gel medium to effectively increase the specific surface area, so that the crystal gel contains more hydroxyl groups, which is conducive to grafting reactions, obtains more functional groups, and improves separation efficiency; at the same time, it can increase the mechanical properties of the crystal gel, which is conducive to repeated use, but no related reports have been seen. Summary of the invention

[0004] In view of the above technical problems existing in the prior art, the object of the present invention is to provide a bacterial cellulose embedded integral crystal gel medium and a preparation method thereof.

[0005] The present invention relates to a whole crystal gel medium embedded with bacterial cellulose, wherein the crystal gel medium has hydroxyethyl methacrylate and bacterial cellulose as a composite skeleton, a porosity of 70-85%, a pore size of 10-300 microns, and a water permeability of 10 -12 ~10 -13 m 2 .

[0006] The method for preparing the bacterial cellulose embedded monolithic crystal gel medium comprises the following steps:

[0007] S1: Dispersing hydroxyethyl methacrylate HEMA and polyethylene glycol diacrylate PEG-DA in ultrapure water at a mass ratio of 70-80:30-20 to prepare a monomer solution, wherein the total mass fraction of HEMA and PEG-DA in the monomer solution is 10-15%;

[0008] S2: adding bacterial cellulose particles to the monomer solution in step S1, wherein the amount of bacterial cellulose particles is 0.5-25% of the total mass of HEMA and PEG-DA, and stirring to disperse them uniformly;

[0009] S3: Add initiator and accelerator to the particle solution stirred evenly in step S2, continue stirring evenly, quickly pour the reaction solution into a microtube pre-cooled at -5 to -10°C, and set the cooling system temperature to -20 to -30°C, and continue the low-temperature reaction for 24 to 48 hours to obtain a composite crystal gel column; take out the microtube, thaw at room temperature, and rinse the composite crystal gel column with a large amount of pure water for standby use;

[0010] S4: Under the action of a catalyst, the monomer with a tertiary amino functional group is grafted onto the composite cryogel column obtained in step S3 through a grafting reaction to obtain an anionic embedded bacterial cellulose integral cryogel medium. The preparation is completed.

[0011] Furthermore, in step S1, the mass ratio of HEMA to PEG-DA is 75-77:25-23.

[0012] Furthermore, in step S2, the preparation method of the bacterial cellulose particles is: soak the bacterial cellulose membrane in a 5-10% by mass sodium hydroxide solution for 2-4 hours, rinse with pure water, boil in a 90-100°C water bath for 0.5-1 hour, take out the bacterial cellulose membrane, make particles with a pore size of less than 500 microns, and squeeze out the water to complete the preparation.

[0013] Furthermore, in step S2, the amount of bacterial cellulose particles is 5-15% of the total mass of HEMA and PEG-DA. The particle size of the bacterial cellulose particles is less than 500 microns.

[0014] Furthermore, in step S3, the initiator and the accelerator are ammonium persulfate and tetramethylethylenediamine, respectively, and the amount of both is 0.5-1.5% of the total mass of HEMA and PEG-DA, preferably 1.0%.

[0015] Furthermore, in step S4, the monomer with a tertiary amino functional group in the grafting reaction is dimethylaminoethyl methacrylate.

[0016] Furthermore, in step S4, the specific process of the grafting reaction is: the catalyst solution is loaded and flowed through the prepared crystal gel matrix to make the catalyst solution fully contact with the crystal gel matrix, and then the monomer solution with tertiary amino functional groups is loaded and flowed through the crystal gel matrix to make the monomer with tertiary amino functional groups fully contact with the crystal gel matrix, the temperature is controlled at 45-60°C, and after reacting for 1-3 hours, the unreacted solution is rinsed with pure water to obtain an anionic embedded bacterial cellulose integral crystal gel medium.

[0017] Furthermore, the volume of the monomer solution is 3 to 5 times the volume of the composite crystal gel column, the monomer concentration is 1 to 2 M, and the catalyst solution is Cu 3+Ionic solution, concentration is 0.03~0.05M.

[0018] Compared with the prior art, the beneficial technical effects of the present invention are:

[0019] 1. The present invention introduces bacterial cellulose particles into the composite crystal gel medium. The bacterial cellulose particles have a nanoporous structure, and their addition can effectively increase the specific surface area. At the same time, the bacterial cellulose particles are rich in hydroxyl groups, and hydroxyethyl methacrylate also has hydroxyl groups, which is more conducive to the reaction of grafted anionic functional groups. At the same time, bacterial cellulose can increase the mechanical strength of the crystal gel. Compared with conventional crystal gels, it is less likely to collapse at high flow rates or after repeated use. In short, the composite crystal gel medium provided by the present invention contains more functional groups, which is conducive to improving the separation efficiency.

[0020] 2. The crystal gel medium provided by the present invention has better biocompatibility, wherein the embedded bacterial cellulose is biodegradable, and at the same time the mechanical strength of the crystal gel medium is further enhanced, which is conducive to reuse. DETAILED DESCRIPTION

[0021] The present invention will be further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto.

[0022] When the composite crystal gel medium is filled in the separation column for loading, if the mechanical strength of the composite crystal gel medium is high, the composite crystal gel medium in the column will not collapse even when the flow rate of the loading liquid is high. Therefore, the embodiment of the present invention provides a maximum loading flow rate that can keep the column from collapsing when the loading liquid is stable. By comparing the magnitude of the maximum flow rate, the strength of the mechanical properties of the composite crystal gel medium can be explained.

[0023] In the following examples, the initiator and the accelerator are ammonium persulfate and tetramethylethylenediamine, respectively.

[0024] Example 1

[0025] The bacterial cellulose membrane was soaked in 5% sodium hydroxide solution for 4 hours, rinsed with pure water, boiled in a 90°C water bath for 1 hour, and the bacterial cellulose membrane was taken out to make particles with a pore size of less than 500 microns, and the water was squeezed out. Hydroxyethyl methacrylate and polyethylene glycol diacrylate were added to pure water in a mass ratio of 77:23 to prepare a monomer solution, wherein the total mass concentration of the hydroxyethyl methacrylate and polyethylene glycol diacrylate mixture was 15% (w / w). The bacterial cellulose particles squeezed out of water were added to the uniformly stirred monomer solution in proportion, and the proportion of bacterial cellulose particles to the total mass of (HEMA and PEG-DA) monomers was 0.5%. In the uniformly stirred particle solution, add initiator and accelerator, where each of the initiator and accelerator accounts for 1.0% (w / w) of the total monomer (HEMA and PEG-DA), continue stirring for 1 minute, quickly pour the reaction solution into the microtube that has been pre-cooled at -5°C, and set the cooling system temperature to -20°C, and continue the low-temperature reaction for 48 hours; take out the microtube, thaw at room temperature, and rinse the composite crystal gel column with a large amount of pure water. The composite crystal gel column is grafted, first the catalyst solution is loaded and flowed through the prepared crystal gel matrix, and then the monomer solution is loaded and flowed through the crystal gel matrix. The grafted monomer is dimethylaminoethyl methacrylate, the concentration of the monomer aqueous solution is 1M, the amount of the monomer aqueous solution is 3 times the volume of the composite crystal gel column, and the grafting reaction catalyst is 0.03MCu 3+ The ion solution, the catalyst dosage is 3 times the volume of the composite crystal gel column, the grafting reaction temperature is 45°C, and the reaction time is 3h. The obtained composite crystal gel medium has a porosity of 85%, a pore size of 10 to 300 microns, and a water permeability of 7.1×10 -13 m 2 Bovine serum albumin was taken and deionized water was added to prepare a BSA solution with a concentration of 6 mg / ml, which was used as the loading solution. The adsorption capacity of the composite crystal gel medium for bovine serum albumin was 3.4 mg / mL (wet crystal gel) after testing.

[0026] The composite crystal gel column of Example 1 was loaded with the prepared 6 mg / ml BSA solution, and the maximum flow rate at which the crystal gel could maintain its structure without deformation and collapse was 12 cm / min.

[0027] Example 2

[0028] The bacterial cellulose membrane was soaked in 10% sodium hydroxide solution for 2 hours, rinsed with pure water, boiled in a 100°C water bath for 0.5 hours, and the bacterial cellulose membrane was taken out to make particles with a pore size of less than 500 microns, and the water was squeezed out. Hydroxyethyl methacrylate and polyethylene glycol diacrylate were added to pure water in a ratio of 77:23 to prepare a monomer solution, wherein the total mass concentration of the hydroxyethyl methacrylate and polyethylene glycol diacrylate mixture was 10% (w / w). The dried bacterial cellulose particles were added to the uniformly stirred monomer solution in proportion, and the proportion of bacterial cellulose particles to the total mass of (HEMA and PEG-DA) monomers was 10%. In the uniformly stirred particle solution, add initiator and accelerator, where the initiator and accelerator each account for 1.0% (w / w) of the total monomer (HEMA and PEG-DA), continue stirring for 2 minutes, quickly pour the reaction solution into the microtube that has been pre-cooled at -10°C, and set the cooling system temperature to -30°C, and continue the low-temperature reaction for 24 hours; take out the microtube, thaw at room temperature, and rinse the composite crystal gel column with a large amount of pure water. The composite crystal gel column is grafted, first the catalyst solution is loaded and flowed through the prepared crystal gel matrix, and then the monomer solution is loaded and flowed through the crystal gel matrix. The grafted monomer is dimethylaminoethyl methacrylate, the concentration of the monomer aqueous solution is 1M, the amount of the monomer aqueous solution is 5 times the volume of the composite crystal gel column, and the grafting reaction catalyst is 0.05MCu 3+ The ion solution was used, the catalyst dosage was 5 times the volume of the composite crystal gel column, the grafting reaction temperature was 60°C, and the reaction time was 2h. The obtained composite crystal gel medium had a porosity of 88%, a pore size of 10 to 300 microns, and a water permeability of 5.6×10 -12 m 2 Bovine serum albumin was taken and added with deionized water to prepare a BSA solution with a concentration of 1 mg / ml, which was used as the loading solution. The adsorption capacity of the composite crystal gel medium for bovine serum albumin was 7.4 mg / mL (wet crystal gel) after testing.

[0029] The composite crystal gel column of Example 2 was loaded with the prepared 1 mg / ml BSA solution, and the maximum flow rate at which the crystal gel could maintain its structure without deformation and collapse was 13 cm / min.

[0030] Example 3

[0031] The bacterial cellulose membrane was soaked in 10% sodium hydroxide solution for 2 hours, rinsed with pure water, boiled in a 100°C water bath for 1 hour, and the bacterial cellulose membrane was taken out to make particles with a pore size of less than 500 microns, and the water was squeezed out. Hydroxyethyl methacrylate and polyethylene glycol diacrylate were added to pure water in a ratio of 77:23 to prepare a monomer solution, wherein the total mass concentration of the hydroxyethyl methacrylate and polyethylene glycol diacrylate mixture was 15% (w / w). The bacterial cellulose particles squeezed out of water were added to the uniformly stirred monomer solution in proportion, and the proportion of bacterial cellulose particles to the total mass of (HEMA and PEG-DA) monomers was 6%. In the uniformly stirred particle solution, add initiator and accelerator, where the initiator and accelerator each account for 1.0% (w / w) of the total amount of monomers (HEMA and PEG-DA), continue stirring for 1 minute, quickly pour the reaction solution into the microtube that has been pre-cooled at -5°C, and set the cooling system temperature to -20°C, and continue the low-temperature reaction for 48 hours; take out the microtube, thaw at room temperature, and rinse the composite crystal gel column with a large amount of pure water. The composite crystal gel column is grafted, first the catalyst solution is loaded and flowed through the prepared crystal gel matrix, and then the monomer solution is loaded and flowed through the crystal gel matrix. The grafted monomer is dimethylaminoethyl methacrylate, the concentration of the monomer aqueous solution is 2M, the amount of the monomer aqueous solution is 3 times the volume of the composite crystal gel column, and the grafting reaction catalyst is 0.03MCu 3+ The ion solution, the catalyst dosage is 5 times the volume of the composite crystal gel column, the grafting reaction temperature is 45°C, and the reaction time is 2h. The obtained composite crystal gel medium has a porosity of 75%, a pore size of 10 to 300 microns, and a water permeability of 2.3×10 -13 m 2 Bovine serum albumin was taken and added with deionized water to prepare a BSA solution with a concentration of 8 mg / ml, which was used as the loading solution. The adsorption capacity of the composite crystal gel medium for bovine serum albumin was 5.4 mg / mL (wet crystal gel) after testing.

[0032] The composite crystal gel column of Example 3 was loaded with the prepared 8 mg / ml BSA solution, and the maximum flow rate at which the crystal gel could maintain its structure without deformation and collapse was 18 cm / min.

[0033] Example 4

[0034] The bacterial cellulose membrane was soaked in 10% sodium hydroxide solution for 2 hours, rinsed with pure water, boiled in a 100°C water bath for 2 hours, and the bacterial cellulose membrane was taken out to make particles with a pore size of less than 500 microns, and the water was squeezed out. Hydroxyethyl methacrylate and polyethylene glycol diacrylate were added to pure water in a ratio of 77:23 to prepare a monomer solution, wherein the total mass concentration of the hydroxyethyl methacrylate and polyethylene glycol diacrylate mixture was 15% (w / w). The dried bacterial cellulose particles were added to the uniformly stirred monomer solution in proportion, and the proportion of the bacterial cellulose particles to the total mass of the (HEMA and PEG-DA) monomers was 25%. In the uniformly stirred particle solution, add initiator and accelerator, where the initiator and accelerator each account for 1.0% (w / w) of the total monomer (HEMA and PEG-DA), continue stirring for 2 minutes, quickly pour the reaction solution into the microtube that has been pre-cooled at -10°C, and set the cooling system temperature to -20°C, and continue the low-temperature reaction for 48 hours; take out the microtube, thaw at room temperature, and rinse the composite crystal gel column with a large amount of pure water. The composite crystal gel column is grafted, first the catalyst solution is loaded and flowed through the prepared crystal gel matrix, and then the monomer solution is loaded and flowed through the crystal gel matrix. The grafted monomer is dimethylaminoethyl methacrylate, the concentration of the monomer aqueous solution is 2M, the amount of the monomer aqueous solution is 3 times the volume of the composite crystal gel column, and the grafting reaction catalyst is 0.05MCu 3+ The ion solution, the catalyst dosage is 3 times the volume of the composite crystal gel column, the grafting reaction temperature is 45°C, and the reaction time is 2h. The obtained composite crystal gel medium has a porosity of 70%, a pore size of 10 to 300 microns, and a water permeability of 2.4×10 -13 m 2 Bovine serum albumin was taken and added with deionized water to prepare a BSA solution with a concentration of 6 mg / ml, which was used as the loading solution. The adsorption capacity of the composite crystal gel medium for bovine serum albumin was found to be 2.9 mg / mL (wet crystal gel).

[0035] The composite crystal gel column of Example 4 was loaded with the prepared 6 mg / ml BSA solution, and the maximum flow rate at which the crystal gel could maintain its structure without deformation and collapse was 13 cm / min.

[0036] Example 5

[0037] Hydroxyethyl methacrylate and polyethylene glycol diacrylate were added to pure water in a ratio of 77:23 to prepare a monomer solution, wherein the total mass concentration of the mixture of hydroxyethyl methacrylate and polyethylene glycol diacrylate was 15% (w / w). In the uniformly stirred solution, initiators and accelerators were added, wherein the initiators and accelerators each accounted for 1.0% (w / w) of the total amount of monomers (HEMA and PEG-DA), and stirring was continued for 2 minutes. The reaction solution was quickly poured into a microtube that had been precooled at -10°C, and the cooling system temperature was set to -20°C. The reaction was continued at low temperature for 48 hours; the microtube was taken out, thawed at room temperature, and the crystal gel column was rinsed with a large amount of pure water. The crystal gel column was grafted, and the catalyst solution was first loaded and flowed through the prepared crystal gel matrix, and then the monomer solution was loaded and flowed through the crystal gel matrix. The grafted monomer was dimethylaminoethyl methacrylate, the concentration of the monomer aqueous solution was 1M, the amount of the monomer aqueous solution was 3 times the volume of the composite crystal gel column, and the grafting reaction catalyst was 0.05 MCu 3+ The ion solution, the catalyst dosage is 3 times the volume of the composite crystal gel column, the grafting reaction temperature is 45°C, and the reaction time is 2h. The obtained crystal gel medium has a porosity of 87%, a pore size of 10 to 300 microns, and a water permeability of 5.8×10 -13 m 2 , the maximum flow rate can reach 3cm / min. Take bovine serum albumin, add deionized water to prepare a BSA solution with a concentration of 1mg / ml, as the sample solution, and the adsorption capacity of the composite crystal gel medium for bovine serum albumin is 1.7mg / mL (wet crystal gel) after testing.

[0038] The composite crystal gel column of Example 5 was loaded with the prepared 1 mg / ml BSA solution. The maximum flow rate at which the crystal gel could maintain its structure without deformation and collapse was only 3 cm / min.

[0039] The contents described in this specification are merely an enumeration of implementation forms of the inventive concept, and the protection scope of the present invention should not be regarded as being limited to the specific forms described in the embodiments.

Claims

1. A method for preparing a monolithic crystal gel medium embedded with bacterial cellulose, characterized in that The crystal gel medium has hydroxyethyl methacrylate and bacterial cellulose as a composite skeleton, a porosity of 70-85%, a pore size of 10-300 microns, and a water permeability of 10 -12 ~10 -13 m 2 ; The preparation method comprises the following steps: S1: Disperse hydroxyethyl methacrylate HEMA and polyethylene glycol diacrylate PEG-DA in ultrapure water at a mass ratio of 70-80:30-20 to prepare a monomer solution, wherein the total mass fraction of HEMA and PEG-DA in the monomer solution is 10-15%; S2: Add bacterial cellulose particles to the monomer solution in step S1, wherein the amount of bacterial cellulose particles is 0.5-25% of the total mass of HEMA and PEG-DA, and stir to disperse evenly; S3: Add initiator and accelerator to the particle solution stirred evenly in step S2, continue stirring evenly, quickly pour the reaction solution into a microtube that has been pre-cooled at -5~-10 ℃, and set the cooling system temperature to -20~-30 ℃, and continue low-temperature reaction for 24~48 hours to obtain a composite crystal gel matrix; take out the microtube, thaw at room temperature, and rinse the composite crystal gel matrix with a large amount of pure water for standby use; S4: grafting the monomer with tertiary amino functional group onto the composite cryogel matrix obtained in step S3 through a grafting reaction under the action of a catalyst to obtain an anionic embedded bacterial cellulose overall cryogel medium, and the preparation is completed; In step S4, the monomer with a tertiary amino functional group in the grafting reaction is dimethylaminoethyl methacrylate; The preparation method of the bacterial cellulose particles is as follows: Komagataeibacter hansenii The bacterial cellulose membrane obtained by Y21DM fermentation is soaked in a 5-10% by mass sodium hydroxide solution for 2-4 h, rinsed with pure water, boiled in a 90-100 ℃ water bath for 0.5-1 h, and the bacterial cellulose membrane is taken out and made into particles with a pore size of less than 500 microns. The preparation is completed by squeezing out the water.

2. The method for preparing a bacterial cellulose embedded monolithic crystal gel medium according to claim 1, characterized in that In step S1, the mass ratio of HEMA to PEG-DA is 75-77: 25-23.

3. The method for preparing a bacterial cellulose embedded monolithic crystal gel medium according to claim 1, characterized in that In step S2, the amount of bacterial cellulose particles added is 5-15% of the total mass of HEMA and PEG-DA; the particle size of the bacterial cellulose particles is below 500 microns.

4. The method for preparing a bacterial cellulose embedded monolithic crystal gel medium according to claim 1, characterized in that In step S3, the initiator and the accelerator are ammonium persulfate and tetramethylethylenediamine, respectively, and the amount of both is 0.5-1.5% of the total mass of HEMA and PEG-DA.

5. The method for preparing a bacterial cellulose embedded monolithic crystal gel medium according to claim 4, characterized in that The amount of ammonium persulfate and tetramethylethylenediamine used is 1.0% of the total mass of HEMA and PEG-DA.

6. The method for preparing a bacterial cellulose embedded monolithic crystal gel medium according to claim 1, characterized in that In step S4, the specific process of the grafting reaction is: the catalyst solution is loaded and flowed through the prepared crystal gel matrix to make the catalyst solution fully contact with the crystal gel matrix, and then the monomer solution with tertiary amino functional groups is loaded and flowed through the crystal gel matrix to make the monomer with tertiary amino functional groups fully contact with the crystal gel matrix. After reacting for a certain time at a certain temperature, the unreacted solution is rinsed with pure water to obtain an anionic embedded bacterial cellulose integral crystal gel medium.

7. A method for preparing a bacterial cellulose embedded monolithic crystal gel medium as claimed in claim 6, characterized in that In step S4, the volume of the monomer solution is 3 to 5 times the volume of the composite crystal gel column, the monomer concentration is 1 to 2 M, and the catalyst solution is Cu 3+ The ion solution has a concentration of 0.03~0.05 M in the grafting solution, the reaction temperature is 45~60 ℃, and the reaction time is 1~3h.