Preparation method and application of magnetic carboxylation modified agarose microspheres
By wrapping agarose on Fe3O4 magnetic particles and performing carboxylic functionalization, the problem that existing magnetic microspheres are difficult to achieve superparamagnetic and controllable particle size is solved, and magnetic carboxylicization with high adsorption efficiency and bioaffinity is prepared. Modified agarose microspheres, suitable for biological isolation applications.
Patent Information
- Application Number
- CN202411332707.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-24
- Publication Date
- 2024-12-27
AI Technical Summary
It is difficult for existing magnetic microspheres to achieve the requirements of superparamagnetic, stable dispersion, high surface functional group content, controllable particle size and specific adsorption proteins at the same time. The preparation method is complex and the particle size is uncontrollable.
Using Fe3O4 magnetic particles as the magnetic core, wrap agarose and modify it by carboxymethylation of chloroacetic acid to form magnetic agarose microspheres with carboxyl functional groups on the surface. The preparation method includes the preparation of Fe3O4 magnetic nanoparticles and magnetic agarose microspheres and carboxylic group functionalization treatment.
It has achieved simple preparation of magnetic carboxylated modified agarose microspheres, which have superparamagnetic, stable dispersion, high surface functional groups, controllable particle size and high adsorption efficiency, and is suitable for the specific binding and separation of biological macromolecules such as proteins.
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Figure CN119186506A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of magnetic material preparation, and particularly relates to a preparation method of magnetic carboxyl modified agarose microspheres and application thereof. Background Art
[0002] Magnetic polymer microspheres are magnetic composite microspheres with a special structure formed by combining magnetic inorganic materials and organic polymers in a certain way. By functionalizing the surface of the microspheres, they can be coupled with a variety of biochemical substances and separated from the sample to be tested under the action of an external magnetic field. Generally speaking, the application of functionalized magnetic microspheres in the biological field needs to meet several conditions: ① high magnetic responsiveness; ② high surface functional group content; ③ controllable particle size to meet different needs; ④ able to specifically bind to biological macromolecules such as proteins and nucleic acids, and good biocompatibility.
[0003] There are many functionalized magnetic microspheres reported at present, but they cannot achieve superparamagnetism, stable dispersion in the medium, high surface functional group content, particle size regulation between nanometer and micrometer levels, and specific adsorption of proteins and other requirements at the same time. Magnetic microspheres based on agarose, a natural polysaccharide, can better achieve biocompatibility and have great advantages and prospects. The patent application with the prior art publication number CN 104587977 A discloses a method for preparing agarose magnetic microspheres with surface carboxylation modification, by preparing agarose microspheres without magnetic core, activating and cross-linking the magnetic core agarose microspheres, preparing a mixed solution of agarose microspheres and iron ions, preparing agarose magnetic microspheres, amino modification of the microsphere surface, carboxyl modification of the microsphere surface, and obtaining agarose magnetic microspheres with surface carboxylation modification. However, it still has the defects of complex preparation method and uncontrollable particle size. Summary of the invention
[0004] In view of the defects in the above-mentioned prior art, the purpose of the present invention is to design and provide a preparation method and application of magnetic carboxyl modified agarose microspheres. 3 O 4 The magnetic particles are used as magnetic cores, and a layer of agarose is coated on the surface of the magnetic microspheres. The agarose on the surface of the magnetic microspheres is carboxymethylated by using chloroacetic acid to grow -COOH functional groups on the surface, thereby obtaining magnetic agarose microspheres with carboxyl functional groups on the surface.
[0005] In order to achieve the above object, the present invention adopts the following technical solutions:
[0006] In one aspect, the present invention provides a method for preparing magnetic carboxyl-modified agarose microspheres, comprising the following steps:
[0007] (1)FeCl 3 6H 2 O and FeCl2 4H 2 O was dissolved in water, stirred evenly, and then heated and sodium dodecyl sulfate (SDS) was added for stirring reaction. The pH was adjusted, and the reaction continued after heating. Stirring was stopped, and after cooling to room temperature, magnetic separation was performed and repeated washing was performed to obtain Fe 3 O 4 Magnetic nanoparticles;
[0008] (2) Weigh the Fe prepared in step (1) above 3 O 4 The magnetic nanoparticles are stirred evenly, the oil phase solution is added, the mixture is heated, the agarose solution is added, the mixture is stirred until the droplets are evenly dispersed, the reaction is carried out, the mixture is cooled to room temperature, the magnetic separation is carried out, and the mixture is repeatedly washed with anhydrous ethanol and ultrapure water to obtain magnetic agarose microspheres;
[0009] (3) isopropanol and water are weighed and mixed, heated and stirred evenly, the magnetic agarose microspheres obtained in the above step (2) and the alkaline compound are added in sequence, swelling and alkalization reaction is carried out at a constant temperature, an isopropanol solution of chloroacetic acid is added dropwise, the reaction is continued at a constant temperature, an ethanol solution is added to terminate the reaction, and after cooling to room temperature, magnetic separation is carried out, and the magnetic agarose microspheres functionalized with surface carboxyl groups are obtained by repeated washing with an ethanol solution.
[0010] The preparation method, in step (1), heating to a temperature of 50-60° C.;
[0011] The reaction time is 10 to 15 minutes;
[0012] The temperature is raised to 60-80° C.;
[0013] The time for the continued reaction is 15 to 30 minutes.
[0014] The preparation method, in step (1), the FeCl 3 6H 2 O, FeCl 2 ·4H 2 The mass ratio of O and sodium dodecyl sulfate is 2:2.5:0.2 to 2:3:0.5.
[0015] The preparation method, wherein the heating to 92-95° C. is performed in step (2);
[0016] The reaction time is 1 to 1.5 hours;
[0017] The Fe 3 O 4 The mass volume ratio of the magnetic nanoparticles, the oil phase solution and the agarose solution is 1.5 g:150 mL:20 mL to 1.5 g:200 mL:30 mL.
[0018] In the preparation method, the mass fraction of agarose in the agarose solution in step (2) is 3-6%;
[0019] The preparation process of the agarose solution is as follows: weigh agarose, dissolve it in water, and stir at high temperature to obtain the agarose solution;
[0020] Preferably, the high temperature stirring conditions are: temperature 92-95° C., time 30-60 min.
[0021] In the preparation method, the oil phase solution in step (2) comprises a long-chain alkane compound, a stearate, an alkylphenol and an ethylene oxide condensate;
[0022] The mass volume ratio of the long-chain alkane compound, stearate, alkylphenol and ethylene oxide condensate is 150mL:0.8g:1mL to 180mL:1.2g:2mL;
[0023] The volume ratio of the agarose solution to the long-chain alkane compound is 1:5-25;
[0024] Preferably, the long-chain alkane compound is liquid paraffin or cyclohexane; the stearate is magnesium stearate or zinc stearate; and the condensate of alkylphenol and ethylene oxide is OP-10, OP-4, OP-7 or OP-9.
[0025] The preparation method, in step (3), the heating is raised to 60-65° C.;
[0026] The swelling and alkalization reaction time is 1 to 4 hours;
[0027] The constant temperature reaction time is 6-8h.
[0028] In the preparation method, the alkaline compound in step (3) is ammonia water, sodium hydroxide or sodium bicarbonate;
[0029] The mass ratio of the magnetic agarose microspheres to the alkaline compound is 1:(2-10);
[0030] The volume ratio of isopropanol to water is (1-10):1;
[0031] The concentration of the isopropanol solution of chloroacetic acid is 0.5-1 g / mL.
[0032] In a second aspect, the present invention provides a magnetic carboxyl-modified agarose microsphere obtained by any one of the preparation methods described above.
[0033] In a third aspect, the present invention provides use of any one of the preparation methods described in preparing magnetic carboxyl-modified agarose microspheres.
[0034] Compared with the prior art, the present invention has the following beneficial effects:
[0035] The preparation method of the magnetic carboxyl modified agarose microspheres of the present invention is simple, and the prepared magnetic carboxyl modified agarose microspheres can achieve the advantages of having superparamagnetism, being stably dispersed in a medium, having a high content of surface functional groups, having a particle size regulated between nanometer and micrometer levels, and specifically adsorbing proteins.
[0036] The preparation process of the present invention is relatively simple and has strong controllability. By controlling the experimental conditions, the particle size of the microspheres can be achieved to be around 200 nanometers; the magnetic content is high and the adsorption efficiency is high; the biological affinity is high and can specifically bind to proteins, and can be further used for the separation of biological macromolecules such as proteins. BRIEF DESCRIPTION OF THE DRAWINGS
[0037] Figure 1 This is a schematic diagram of a scanning electron microscope of agarose magnetic microspheres prepared in Example 1;
[0038] Figure 2 This is a schematic diagram of a scanning electron microscope of agarose magnetic microspheres prepared in Example 1;
[0039] Figure 3 This is a transmission electron microscopic diagram of the agarose magnetic microspheres prepared in Example 1. DETAILED DESCRIPTION
[0040] The present invention will be described in detail below in conjunction with the accompanying drawings and examples, but it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. If specific conditions are not specified in the examples, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.
[0041] Embodiment 1:
[0042] This embodiment 1 provides a magnetic carboxyl modified agarose microsphere, and the preparation method thereof comprises:
[0043] (1)Fe 3 O 4 Preparation of magnetic particles: weigh 2 g FeCl 3 6H 2 O and 2.5 g FeCl 2 ·4H 2O was dissolved in 50 mL of water, heated to 56 °C, 0.2 g of SDS was added, stirred at 330 rpm for 10 min, 30 mL of ammonia water was added, the temperature was raised to 80 °C, the reaction was continued for 15 min, stirring was stopped, and the reaction was allowed to stand for 1.5 h. After the reaction was completed, a magnet was used for magnetic separation, and deionized water and ethanol were used to wash the reaction several times to remove the residual components and by-products, and the reaction was stored in deionized water to obtain superparamagnetic Fe 3 O 4 Magnetic particles.
[0044] (2) Preparation of magnetic agarose microspheres: Weigh 1 g of agarose and dissolve it in 20 mL of ultrapure water. Stir at 92° C. for 30 min to obtain a uniform and viscous agarose solution. Then weigh 1.5 g of the magnetic nanoparticles Fe prepared in step (1). 3 O 4 Continue stirring for 30 minutes until uniform; measure 150mL of liquid paraffin, 0.8g of magnesium stearate and 1mL of OP-10 in a container, mix until uniform to obtain an oil phase, and then heat to 92°C at room temperature; add the agarose solution to the oil phase, continue stirring until the droplets are evenly dispersed, react for 1 hour, and then gradually cool to room temperature, perform magnetic separation, and repeatedly wash with anhydrous ethanol and ultrapure water to obtain magnetic agarose microspheres. The scanning electron microscope is as follows Figure 1 , Figure 2 , transmission electron microscopy Figure 3 .
[0045] (3) Preparation of magnetic carboxyl modified agarose microspheres:
[0046] 60 mL of isopropanol and 20 mL of deionized water were weighed into a container, heated to 60° C. and stirred evenly. 1.5 g of the magnetic agarose microspheres obtained in step (2) and 4 g of NaOH were weighed in turn and placed into a container. The microspheres were swollen and alkalized at 60° C. for 2 h. 10 mL of an isopropanol solution of chloroacetic acid (0.3 g / mL) was added dropwise, and the reaction was continued at a constant temperature for 6 h. 20 mL of an ethanol solution (20% wt) was added to terminate the reaction. The microspheres were cooled to room temperature, subjected to magnetic separation, and repeatedly washed with an ethanol solution to obtain magnetic agarose microspheres functionalized with surface carboxyl groups.
[0047] Embodiment 2:
[0048] This embodiment 2 provides a magnetic carboxyl modified agarose microsphere, and the preparation method thereof comprises:
[0049] (1)Fe 3 O 4 Preparation of magnetic particles: weigh 2 g FeCl 3 6H 2 O and 3 g FeCl 2 ·4H 2O was dissolved in 50 mL of water, heated to 50 °C, 0.5 g of SDS was added, stirred at 330 rpm for 15 min, 30 mL of ammonia water was added, the temperature was raised to 60 °C, the reaction was continued for 30 min, stirring was stopped, and the mixture was allowed to stand for 1.5 h. After the reaction was completed, a magnet was used for magnetic separation, and the mixture was washed alternately with deionized water and ethanol for several times to remove the residual components and by-products of the reaction, and the mixture was stored in deionized water to obtain superparamagnetic Fe 3 O 4 Magnetic particles.
[0050] (2) Preparation of magnetic agarose microspheres: 0.6 g of agarose was weighed and dissolved in 20 mL of ultrapure water, and stirred at 95° C. for 30 min to obtain a uniform and viscous agarose solution. Then 1.5 g of the magnetic nanoparticles Fe prepared in step (1) was weighed. 3 O 4 Continue stirring for 30 minutes until uniform; measure 100 mL of liquid paraffin, 0.8 g of magnesium stearate and 1 mL of OP-7 in a container, mix until uniform to obtain an oil phase, and then heat to 95°C at room temperature; add the agarose solution to the oil phase, continue stirring until the droplets are evenly dispersed, react for 30 minutes, and then gradually cool to room temperature, perform magnetic separation, and repeatedly wash with anhydrous ethanol and ultrapure water to obtain magnetic agarose microspheres.
[0051] (3) Preparation of magnetic carboxyl modified agarose microspheres:
[0052] 100 mL of isopropanol and 20 mL of deionized water were weighed in a container, heated to 65° C. and stirred evenly. 1.5 g of the magnetic agarose microspheres obtained in step (2) and 3 g of NaOH were weighed in a container in turn, and swollen and alkalized at 60° C. for 4 h. 10 mL of an isopropanol solution of chloroacetic acid (1 g / mL) was added dropwise, and the reaction was continued at a constant temperature for 8 h. 20 mL of an ethanol solution (20% wt) was added to terminate the reaction. After cooling to room temperature, the reaction was subjected to magnetic separation, and the reaction was repeatedly washed with an ethanol solution to obtain magnetic agarose microspheres functionalized with surface carboxyl groups.
[0053] Embodiment 3:
[0054] This embodiment 3 provides a magnetic carboxyl modified agarose microsphere, and the preparation method thereof comprises:
[0055] (1)Fe 3 O 4 Preparation of magnetic particles: weigh 2 g FeCl 3 6H 2 O and 2.5 g FeCl 2 ·4H 2O was dissolved in 50 mL of water, heated to 60 ° C, 0.2 g of SDS was added, stirred at 330 rpm for 10 min, and then 30 mL of ammonia water was added, the temperature was raised to 80 ° C, the reaction was continued for 15 min, stirring was stopped, and the reaction was allowed to stand for 1.5 h. After the reaction was completed, a magnet was used for magnetic separation, and deionized water and ethanol were used to wash the reaction several times to remove the residual components and by-products, and the reaction was stored in deionized water to obtain superparamagnetic Fe 3 O 4 Magnetic particles.
[0056] (2) Preparation of magnetic agarose microspheres: 1.2 g of agarose was weighed and dissolved in 20 mL of ultrapure water, and stirred at 92° C. for 30 min to obtain a uniform and viscous agarose solution. Then 1.5 g of the magnetic nanoparticles Fe prepared in step (1) was weighed. 3 O 4 Continue stirring for 30 minutes until uniform; measure 400mL of liquid paraffin, 0.8g of magnesium stearate and 1mL of OP-4 in a container, mix until uniform to obtain an oil phase, and then heat to 92°C at room temperature; add the agarose solution to the oil phase, continue stirring until the droplets are evenly dispersed, react for 1 hour, then gradually cool to room temperature, perform magnetic separation, and repeatedly wash with anhydrous ethanol and ultrapure water to obtain magnetic agarose microspheres. The scanning electron microscope is as follows Figure 1 , Figure 2 , transmission electron microscopy Figure 3 .
[0057] (3) Preparation of magnetic carboxyl modified agarose microspheres:
[0058] 60 mL of isopropanol and 20 mL of deionized water were weighed in a container, heated to 60° C. and stirred evenly. 1.5 g of the magnetic agarose microspheres obtained in step (2) and 15 g of NaOH were weighed in a container in turn, and swollen and alkalized at 60° C. for 4 h. 10 mL of an isopropanol solution of chloroacetic acid (1 g / mL) was added dropwise, and the reaction was continued at a constant temperature for 6 h. 20 mL of an ethanol solution (20% wt) was added to terminate the reaction. After cooling to room temperature, the reaction was subjected to magnetic separation, and the reaction was repeatedly washed with an ethanol solution to obtain magnetic agarose microspheres functionalized with surface carboxyl groups.
[0059] The embodiments described above are only descriptions of the preferred implementation modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.
Claims
1. A method for preparing magnetic carboxyl modified agarose microspheres, characterized in that: The following steps are involved: (1) FeCl36H2O and FeCl24H2O are dissolved in water, stirred evenly, and then heated and sodium dodecyl sulfate is added to react by stirring, and then ammonia water is added to continue the reaction after heating, and stirring is stopped, and after cooling to room temperature, magnetic separation is performed, and repeated washing is performed to obtain Fe3O4 magnetic nanoparticles; (2) weighing the Fe3O4 magnetic nanoparticles prepared in step (1), continuing to stir evenly, adding the oil phase solution, heating, adding the agarose solution, continuing to stir until the droplets are evenly dispersed, reacting, cooling to room temperature, performing magnetic separation, and repeatedly washing with anhydrous ethanol and ultrapure water to obtain magnetic agarose microspheres; (3) isopropanol and water are weighed and mixed, heated and stirred evenly, the magnetic agarose microspheres obtained in the above step (2) and the alkaline compound are added in sequence, swelling and alkalization reaction is carried out at a constant temperature, an isopropanol solution of chloroacetic acid is added dropwise, the reaction is continued at a constant temperature, an ethanol solution is added to terminate the reaction, and after cooling to room temperature, magnetic separation is carried out, and the magnetic agarose microspheres functionalized with surface carboxyl groups are obtained by repeated washing with an ethanol solution.
2. The preparation method according to claim 1, characterized in that In step (1), the temperature is heated to 50-60° C.; The reaction time is 10 to 15 minutes; The temperature is raised to 60-80° C.; The time for the continued reaction is 15 to 30 minutes.
3. The preparation method according to claim 1, characterized in that: The mass ratio of FeCl3·6H2O, FeCl2·4H2O and sodium dodecyl sulfate in step (1) is 2:2.5:0.2 to 2:3:0.
5.
4. The preparation method according to claim 1, characterized in that: The heating in step (2) is to 92-95° C.; The reaction time is 1 to 1.5 hours; The mass volume ratio of the Fe3O4 magnetic nanoparticles, the oil phase solution and the agarose solution is 1.5g:150mL:20mL to 1.5g:200mL:30mL.
5. The preparation method according to claim 1, characterized in that: The mass fraction of agarose in the agarose solution in step (2) is 3-6%; The preparation process of the agarose solution is as follows: weigh agarose, dissolve it in water, and stir at high temperature to obtain the agarose solution; Preferably, the high temperature stirring conditions are: temperature 92-95° C., time 30-60 min.
6. The preparation method according to claim 1, characterized in that: The oil phase solution in step (2) comprises a long-chain alkane compound, a stearate, an alkylphenol and an ethylene oxide condensate; The mass volume ratio of the long-chain alkane compound, stearate, alkylphenol and ethylene oxide condensate is 150mL:0.8g:1mL to 180mL:1.2g:2mL; The volume ratio of the agarose solution to the long-chain alkane compound is 1:5-25; Preferably, the long-chain alkane compound is liquid paraffin or cyclohexane; the stearate is magnesium stearate or zinc stearate; and the condensate of alkylphenol and ethylene oxide is OP-10, OP-4, OP-7 or OP-9.
7. The preparation method according to claim 1, characterized in that: The heating in step (3) is raised to 60-65° C.; The swelling and alkalization reaction time is 1 to 4 hours; The constant temperature reaction time is 6-8h.
8. The preparation method according to claim 1, characterized in that: The alkaline compound in step (3) is aqueous ammonia, sodium hydroxide or sodium bicarbonate; The mass ratio of the magnetic agarose microspheres to the alkaline compound is 1:(2-10); The volume ratio of isopropanol to water is (1-10):1; The concentration of the isopropanol solution of chloroacetic acid is 0.5-1 g / mL.
9. A magnetic carboxyl modified agarose microsphere, characterized in that: The method is obtained by the preparation method according to any one of claims 1 to 8.
10. Use of the preparation method according to any one of claims 1 to 8 in preparing magnetic carboxyl modified agarose microspheres.