Agarose magnetic bead, preparation method of agarose magnetic bead, functionalized agarose magnetic bead and preparation method of functionalized agarose magnetic bead

Abstract

The invention relates to the technical field of microsphere modification, in particular to an agarose magnetic bead and a preparation method thereof, a functionalized agarose magnetic bead and a preparation method thereof, and the preparation method of the agarose magnetic bead comprises the following steps: dissolving agarose in water to obtain an agarose solution; and heating the agarose solution to boiling, adding the magnetic bead solution and the emulsion, stirring, cooling with cold water, and continuously stirring to obtain the agarose magnetic beads. According to the preparation method, the agarose solution and the magnetic bead solution are stirred together, so that the magnetic beads and the agarose are wrapped together, the agarose magnetic beads with the particle size ranging from 10 micrometers to 200 micrometers are synthesized under the action of the emulsifier, and the agarose magnetic beads prepared through the preparation method have the advantages of being high in magnetism, uniform in particle size distribution, simple in equipment and easy to produce on a large scale. Moreover, a suspension stirring method is utilized, the magnetic beads are coated in the agarose, so that the agarose has magnetism, and the agarose magnetic beads with relatively good uniformity can be prepared.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of protein purification chromatography medium, and particularly relates to an agarose magnetic bead and a preparation method thereof, and a functionalized agarose magnetic bead and a preparation method thereof. BACKGROUND

[0002] Protein separation and purification is one of important links in antibody preparation, vaccine preparation, protein structure analysis and drug development, and protein separation and purification has technologies such as affinity chromatography, hydrophobic chromatography and molecular sieve chromatography, and agarose gel and dextran gel are often used as chromatography medium, and affinity chromatography is one of important technologies for protein separation and purification. Affinity chromatography uses agarose gel as chromatography medium, and the agarose gel has characteristics such as porosity, hydrophilicity, electrical neutrality and easy extension, but the agarose gel as chromatography medium has a long experimental process time and needs expensive protein purification instruments.

[0003] Therefore, the present application proposes to use agarose magnetic beads as chromatography medium, but the existing agarose magnetic beads prepared by a hydrothermal method have small particle size and weak magnetism, which is not conducive to separation of samples and the agarose magnetic beads, and leads to serious loss of the agarose magnetic beads.

[0004] Therefore, the prior art still needs to be improved and developed. SUMMARY

[0005] In view of the above problems in the prior art, the present application aims to provide an agarose magnetic bead and a preparation method thereof, and a functionalized agarose magnetic bead and a preparation method thereof, and aims to solve the problems of small particle size and weak magnetism of the agarose magnetic beads prepared by the existing method.

[0006] The technical scheme of the present application is as follows:

[0007] A preparation method of an agarose magnetic bead comprises the following steps:

[0008] Dissolving agarose in water to obtain an agarose solution;

[0009] Heating the agarose solution to boiling, adding a magnetic bead solution and an emulsion and stirring, continuously stirring after cooling with cold water, and obtaining agarose magnetic beads.

[0010] The preparation method of the agarose magnetic bead, wherein the mass of the magnetic beads in the magnetic bead solution is 20%-70% of the mass of the agarose.

[0011] The preparation method of the agarose magnetic bead, wherein the emulsion comprises an oil phase solution and a surfactant; the oil phase solution comprises one or more of petroleum ether, cyclohexane, cycloheptane, liquid paraffin and cyclooctane; and the surfactant comprises one or more of Span 80, Span 85, Tween-20, Tween-85 and triton-X100.

[0012] The preparation method of the agarose magnetic beads, wherein the preparation of the magnetic bead solution comprises the steps of mixing iron salt, ethylene glycol, a constraint agent and sodium acetate, and then performing a solvothermal reaction to obtain the magnetic bead solution.

[0013] An agarose magnetic bead prepared by the preparation method of the agarose magnetic bead.

[0014] The agarose magnetic bead, wherein the particle size of the agarose magnetic bead is 10-200 mu m.

[0015] A preparation method of a functionalized agarose magnetic bead, comprising the steps of:

[0016] Activating the agarose magnetic bead of any one of claims 5-6 by using a crosslinking agent to obtain an activated agarose magnetic bead.

[0017] Mixing the activated agarose magnetic bead with a functional molecule to obtain a mixed solution.

[0018] Adjusting the pH value of the mixed solution, and obtaining a functionalized agarose magnetic bead after reaction.

[0019] The preparation method of the functionalized agarose magnetic bead, wherein the functional molecule comprises one of nitrilotriacetic acid, iminodiacetic acid, reduced glutathione, streptavidin, Staphylococcus aureus surface protein A (protein A), streptococcal protein G (protein G), Streptococcus pyogenes protein L (protein L) flag tag antibody.

[0020] The preparation method of the functionalized agarose magnetic bead, wherein the crosslinking agent comprises one or more of epichlorohydrin, 1,4-butanediol diglycidyl ether, 2,3-dibromopropanol.

[0021] A functionalized agarose magnetic bead prepared by the preparation method of the functionalized agarose magnetic bead.

[0022] Beneficial effects: the present application provides an agarose magnetic bead and a preparation method thereof, a functionalized agarose magnetic bead and a preparation method thereof, the preparation method of the agarose magnetic bead comprises the following steps: dissolving agarose in water to obtain an agarose solution; heating the agarose solution to boiling, adding a magnetic bead solution and an emulsion and stirring, continuously stirring after cooling with cold water, and obtaining the agarose magnetic bead. The present application utilizes the agarose solution and the magnetic bead solution to stir together, so that the magnetic beads are wrapped together with the agarose, so that the agarose magnetic beads with a particle size range of 10-200 mu m are synthesized under the action of the emulsifier, the agarose magnetic beads prepared by the preparation method have the advantages of strong magnetism, uniform particle size distribution, simple equipment and easy large-scale production. Moreover, the present application utilizes the suspension stirring method to coat the magnetic beads in the agarose, so that the agarose has magnetism and the agarose magnetic beads with good uniformity can be prepared. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 It is a flowchart of the preparation method of the agarose magnetic bead of the present application;

[0024] Figure 2 It is a flowchart of the preparation method of the functionalized agarose magnetic bead of the present application;

[0025] Figure 3 It is a transmission electron microscope image of the magnetic bead prepared in Example 1;

[0026] Figure 4 It is a microscope image of the agarose magnetic bead prepared in Example 2;

[0027] Figure 5 It is a characterization image of the Ni-IDA agarose magnetic bead of Example 3 applied to the purification of recombinant protein containing 6X His tag;

[0028] Figure 6 It is a characterization image of the GSH agarose magnetic bead of Example 4 applied to the purification of recombinant protein containing GST tag;

[0029] Figure 7 It is an SDS-PAGE electrophoresis image of the protein A protein agarose magnetic bead of Example 5 purifying rabbit antibody;

[0030] Figure 8 It is a magnetization intensity characterization image of the magnetic bead prepared in the comparative example and the agarose magnetic bead prepared in Example 2;

[0031] Figure 9 It is a magnetic attraction effect image of the magnetic bead prepared in the comparative example and the agarose magnetic bead prepared in Example 2. DETAILED DESCRIPTION

[0032] The application provides an agarose magnetic bead and a preparation method thereof, and a functionalized agarose magnetic bead and a preparation method thereof. To make the purpose, technical scheme and effects of the application clearer and more explicit, the application is further described in detail below. It should be understood that the specific embodiments described herein are only used to explain the application and not to limit the application.

[0033] Those skilled in the art can understand that, unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. It should also be understood that terms such as those defined in general dictionaries should be understood to have meanings consistent with those in the context of the prior art, and should not be interpreted in an idealized or overly formal sense unless specifically defined as such.

[0034] As shown in Figure 1 The application provides a preparation method of an agarose magnetic bead, comprising the steps of:

[0035] Step S10: dissolving agarose in water to obtain an agarose solution;

[0036] Step S20: heating the agarose solution to boiling, adding a magnetic bead solution and an emulsion and stirring, and continuously stirring after cooling with cold water to obtain an agarose magnetic bead.

[0037] In this embodiment, by heating the agarose solution to boiling, the hydrogen bonds between agarose molecules can be broken, so that the agarose is dissolved in the solution to form a uniform agarose solution. Then, the agarose solution and the magnetic bead solution are stirred together to wrap the magnetic beads with agarose, so that the agarose magnetic beads with a particle size range of 10-200 μm are synthesized under the action of the emulsifier. The agarose magnetic beads prepared by this preparation method have the advantages of strong magnetism, uniform particle size distribution, simple equipment and easy large-scale production. Moreover, the magnetic beads are coated inside the agarose by using the suspension stirring method, so that the agarose has magnetism and the agarose magnetic beads with good uniformity can be prepared.

[0038] Specifically, ferric chloride, sodium acetate and polyethylene glycol are used as raw materials to synthesize magnetic beads with uniform particle size, superparamagnetism and fast magnetic response speed under high temperature conditions. The advantages of the magnetic beads and the agarose are combined to prepare agarose magnetic beads with uniform particle size, strong magnetism and few non-specificities by using the suspension stirring method. The agarose magnetic beads can be surface-modified, and different proteins can be separated and purified by modifying different labels.

[0039] It should be noted that, in addition to the suspension stirring method of the application for preparing the agarose magnetic beads, the agarose magnetic beads can also be prepared by a membrane emulsification method, a jet method and a microfluidic method.

[0040] In the present embodiment, the steps of preparing the agarose magnetic beads by the suspension stirring method include:

[0041] 1) Prepare an organic phase solution (emulsion), configure petroleum ether, liquid paraffin and surfactant in proportion to a volume of 100 ml, prepare the organic phase solution in a 200 ml container, heat it to 70-80℃, and reserve it.

[0042] 2) Prepare a water phase solution, take 1 g of agarose, pour it into a 250 ml three-necked flask, disperse it with 25 ml of ultrapure water, fix a mechanical stirrer in the three-necked flask, fix it on a heating jacket and a mechanical stirrer, heat the heating jacket to 100℃ to completely dissolve the agarose to boiling. Stop heating and remove the heating jacket, start the mechanical stirrer, and let the agarose solution stir quickly, and add 10 ml of ferroferric oxide magnetic bead solution, uniformly stir for 2-3 minutes, pour the organic phase solution into the three-necked flask, uniformly stir for 2-3 minutes, quickly cool with cold water, and continue stirring for 30-60 minutes. After the reaction is completed, clean the agarose magnetic beads with water / ethanol solution repeatedly to obtain the agarose magnetic beads.

[0043] In some embodiments, in the step S20, the magnetic bead solution and the emulsion are added and stirred at a speed of 300-1500 rpm / min, to ensure that the agarose solution, the magnetic bead solution and the emulsion are uniformly mixed, and to promote the emulsification; then after cooling with cold water, continue stirring at a speed of 300-1500 rpm / min, to maintain the uniform state of the solution during the whole cooling process, and to remove the bubbles.

[0044] In some embodiments, the mass of the magnetic beads in the magnetic bead solution is 20%-70% of the mass of the agarose; the volume ratio of the emulsion to the magnetic bead solution is 1:(1-20). Controlling the amount of the magnetic beads added to be 20%-70% of the mass of the agarose can make the agarose wrap the magnetic beads, provide the agarose with magnetism, and facilitate the separation of the agarose magnetic beads from the sample.

[0045] In some embodiments, the emulsion includes but is not limited to an oil phase solution and a surfactant, and the amount of the surfactant accounts for 10%-20% of the volume of the oil phase solution; the oil phase solution includes but is not limited to one or more of petroleum ether, cyclohexane, cycloheptane, liquid paraffin and cyclooctane; the surfactant includes but is not limited to one or more of Span 80, Span 85, Tween-20, Tween-85 and triton-X100. The hot agarose solution forms water-in-oil in the emulsion, and under the action of stirring, the magnetic bead solution can generate agarose magnetic beads with uniform size.

[0046] In a specific embodiment, the emulsion is prepared by mixing petroleum ether, liquid paraffin, Span 80 and Tween-20 in a volume ratio of 5:4:0.8:0.2, and heating to 70-80℃ for standby.

[0047] In some embodiments, the preparation of the magnetic bead solution comprises the step of mixing iron salt, ethylene glycol, constraint agent and sodium acetate, and then performing a solvothermal reaction to obtain the magnetic bead solution. The magnetic beads prepared by the solvothermal reaction have uniform particle size and fast magnetic response speed.

[0048] Specifically, the iron salt provides iron element for the synthesis of the magnetic beads, the ethylene glycol is the reaction environment for the synthesis of the magnetic beads, and the sodium acetate prevents the formation of magnetic bead clusters. The iron element in the reaction system is derived from the iron salt, which can be selected from, but is not limited to, one or more of ferric chloride, ferrous chloride, ferric sulfate and ferrous sulfate. The constraint agent can be selected from, but is not limited to, one or more of polyethylene glycol, polyethyleneimine, citrate and chitosan with different molecular weights. The magnetic beads with uniform particle size are prepared by mixing the iron salt, ethylene glycol, constraint agent and sodium acetate, and then performing a solvothermal reaction. Compared with the magnetic beads prepared by the hydrothermal method, the magnetic beads prepared by the solvothermal method have larger particle size and stronger magnetism, and do not need to be treated with oleic acid to prepare agarose magnetic beads. The strong magnetism of the magnetic beads is conducive to the separation of the sample and the agarose magnetic beads, and reduces the loss of the agarose magnetic beads during use.

[0049] In some embodiments, the temperature of the solvothermal reaction is 150-180℃, and the time of the solvothermal reaction is 4-12h.

[0050] In a preferred embodiment, the temperature of the solvothermal reaction is 180℃, and the time of the solvothermal reaction is 4.5h.

[0051] In some embodiments, after the solvothermal reaction, the magnetic beads are washed, dried and weighed, and then dispersed into a magnetic bead solution with a certain concentration by using water.

[0052] In a preferred embodiment, the concentration of the magnetic bead solution is 100mg / ml.

[0053] In some embodiments, the concentration of the agarose solution is 0.02-0.1g / ml.

[0054] In addition, the application also provides an agarose magnetic bead prepared by the preparation method of the agarose magnetic bead.

[0055] In the embodiment, the two-step method for preparing the agarose magnetic beads is to first synthesize the magnetic beads, then stir the magnetic beads and the agarose hot solution together, so that the magnetic beads are wrapped together with the agarose, and under the action of the emulsifier, the agarose magnetic beads with a particle size range of 10-200 μm are synthesized. This method synthesizes magnetic beads with strong magnetism, uniform particle size distribution, simple equipment, and easy large-scale production. Moreover, the agarose magnetic beads can be surface-modified, and different proteins can be separated and purified by modifying different labels.

[0056] In some embodiments, the particle size of the agarose magnetic beads is 10 μm-200 μm.

[0057] In addition, as Figure 2 shown, the application also provides a preparation method of functionalized agarose magnetic beads, comprising the steps of:

[0058] Step S100: activating the agarose magnetic beads by using a crosslinking agent to obtain activated agarose magnetic beads;

[0059] Step S200: mixing the activated agarose magnetic beads with functional molecules to obtain a mixed solution;

[0060] Step S300: adjusting the pH value of the mixed solution, and obtaining functionalized agarose magnetic beads after reaction.

[0061] In the embodiment, the agarose magnetic beads are first activated by a crosslinking agent, so that the agarose molecules are crosslinked with each other, the agarose molecules become microspheres with rigidity and mechanical pressure resistance, and non-specific adsorption is reduced. Finally, different functional molecules are connected, so that the agarose magnetic beads have the advantages of agarose and magnetic beads, and can more efficiently separate and purify proteins.

[0062] In some embodiments, the functional molecules include one of nitrilotriacetic acid (NTA), iminodiacetic acid (IDA), reduced glutathione (GSH), streptavidin, Staphylococcus aureus surface protein A (protein A), streptococcal protein G (protein G), Streptococcus pyogenes protein L (protein L), and flag tag antibody. By connecting different functional molecules on the surface of the agarose magnetic beads, the agarose magnetic beads can be functionalized and different proteins can be purified. For example, iminodiacetic acid is connected on the surface of the agarose magnetic beads, which can be applied to the purification of recombinant proteins with 6X His tags; reduced glutathione is connected on the surface of the agarose magnetic beads, which can be applied to the purification of recombinant proteins containing GST tags; protein A protein is connected on the surface of the agarose magnetic beads, which can be applied to the purification of immunoglobulin G in rabbit, mouse, human, etc. serum.

[0063] In some embodiments, the cross-linking agent comprises one or more of epichlorohydrin, 1,4-butanediol diglycidyl ether, 2,3-dibromopropanol. The agarose magnetic beads are activated by using the above cross-linking agent, so that the agarose molecules are cross-linked intramolecularly and intermolecularly, so that they become microspheres with rigidity and mechanical pressure resistance, and non-specific adsorption is reduced.

[0064] In some embodiments, the cross-linking agent is added in an amount of 20%-30% of the mass of the agarose magnetic beads.

[0065] Specifically, the step S100, taking epichlorohydrin as an example of the cross-linking agent, comprises: taking 10 ml of agarose magnetic beads, dispersing a small amount of sodium borohydride with 1M sodium hydroxide solution, mixing uniformly, placing on a shaker, and mixing uniformly for 30 minutes; adding 20-30% of epichlorohydrin, and mixing uniformly for 6-7 hours. After the reaction is completed, the activated agarose magnetic beads are obtained by washing with ethanol three times, washing with 10% sodium carbonate solution three times, and washing with water three times.

[0066] In this embodiment, taking IDA (iminodiacetic acid) as an example of the modified functional molecule, 10 ml of activated agarose magnetic beads are taken, 0.5 g of IDA (iminodiacetic acid) is dissolved in 30 ml of water, the pH value is adjusted to 10, and the final volume of the solution is adjusted to 40 ml. The activated agarose magnetic beads are mixed uniformly with the IDA solution, and the reaction is carried out at room temperature for 24 hours. After the reaction is completed, the functional agarose magnetic beads are prepared by repeatedly washing with pure water and uniformly mixing 20 ml of 0.1M nickel sulfate solution with the agarose magnetic beads connected with IDA for 30 minutes. After the reaction is completed, the functional agarose magnetic beads are prepared by repeatedly washing with pure water until the solution is colorless. At this time, the prepared functional agarose magnetic beads are Ni-IDA agarose magnetic beads, which can be applied to the purification of recombinant proteins containing 6X His tags.

[0067] In addition, the application also provides a functional agarose magnetic bead prepared by the preparation method of the functional agarose magnetic bead.

[0068] The following examples are further used to illustrate the application. It should also be understood that the following examples are only used to further illustrate the application, and cannot be understood as limiting the protection scope of the application. Some non-essential improvements and adjustments made by those skilled in the art according to the above content of the application all belong to the protection scope of the application.

[0069] Example 1

[0070] In this embodiment, the magnetic beads are prepared by a solvothermal method, which comprises the following steps:

[0071] 1) 2.75 g of FeCl3·6H2O is weighed and dissolved in 50 mL of ethylene glycol to obtain a ferric chloride solution;

[0072] 2) Take 6.75 g of sodium acetate and add to the ferric chloride solution, stir and ultrasonic until dissolved;

[0073] 3) Take 1.85 g of PEG2000 and add to the sodium acetate + ferric chloride solution, stir and ultrasonic until dissolved;

[0074] 4) Pour the liquid obtained in step 3) into a reaction kettle, tighten the reaction kettle and place it in an oven at 200°C for 4.5 h, and let it cool naturally to room temperature;

[0075] 5) Take out the reaction kettle, pour out the supernatant, wash with ultrapure water three times, and then wash with 95% alcohol three times;

[0076] 6) Characterize the magnetic beads by transmission electron microscopy, as shown in Figure 3 , the size of the magnetic beads is 500 nm;

[0077] 7) Dry the magnetic beads, weigh them, and prepare a 100 mg / ml magnetic bead solution.

[0078] Example 2

[0079] This example uses a suspension stirring method to prepare agarose magnetic beads, including the following steps:

[0080] 1) Prepare an oil phase solution by taking 24 ml of Span 85, 6 ml of Tween-20, 120 ml of liquid paraffin, and 80 ml of petroleum ether, and heating them to 70°C for standby.

[0081] 2) Take 2 g of agarose and pour it into a three-necked flask, add 50 ml of ultrapure water, and fix the three-necked flask on a mechanical stirrer with a heating jacket;

[0082] 3) Heat the jacket to completely dissolve the agarose to boiling at 100°C, remove the heating jacket, start the stirrer, and add 5 ml of the magnetic bead solution prepared in Example 1, mix and stir for 2 minutes, when the liquid temperature decreases to 70°C, add the oil phase solution, stir for 3-5 minutes, quickly cool with cold water, and continue to stir for 60 minutes, after stirring is completed, wash repeatedly with water and ethanol until the liquid is clear and transparent, to obtain agarose magnetic beads.

[0083] Characterize the agarose magnetic beads prepared in this example, measure the particle size of the agarose magnetic beads under a microscope, and the results are shown in Figure 4 (the size of a small box is 20 μm), indicating that the particle size of the agarose magnetic beads is in the range of 20-80 μm.

[0084] Example 3

[0085] This example functionally modifies the agarose magnetic beads prepared in Example 2, including the following steps:

[0086] 1) Activation of agarose magnetic beads

[0087] Take 10 ml dry agarose magnetic beads, disperse with 1M sodium hydroxide solution, a small amount of sodium borohydride, mix evenly, put on a shaker, mix evenly for 30 minutes; add 20-30% epichlorohydrin, mix evenly for 6-7h, after the reaction is completed, wash with ethanol three times, wash with 10% sodium carbonate solution three times, wash with water three times, to obtain activated agarose magnetic beads.

[0088] 2) Modification of agarose magnetic beads

[0089] Take 0.5g IDA (iminodiacetic acid) and dissolve in 30ml water, adjust the pH value to 10, and the final volume of the solution is 40ml. Mix 10ml activated agarose magnetic beads with IDA solution evenly, and react at room temperature for 24h; after the reaction is completed, wash repeatedly with pure water, mix 20ml 0.1M nickel sulfate solution with the agarose magnetic beads with IDA evenly for 30 minutes, and wash repeatedly with pure water until colorless after the reaction is completed.

[0090] At this time, the prepared functional agarose magnetic beads are Ni-IDA agarose magnetic beads, which can be applied to the purification of recombinant proteins containing 6X His tag. Use the recombinant protein (molecular weight 65kDa) with 6X His tag to express the protein, use 0.5ml Ni-IDA agarose magnetic beads to purify the protein, a total of 3 tubes, each tube volume is 3ml, the calculation result is that 0.5ml Ni-IDA agarose magnetic beads can purify 22mg protein, the loading capacity of the agarose magnetic beads is 44mg / ml; through SDS-PAGE electrophoresis, the result shows that the molecular weight of the target protein is 65kDa, the protein purified by the agarose magnetic beads has very few impurities, and the purified protein has high purity. The specific data is shown in Table 1, and the results are shown in Figure 5

[0091] Table 1

[0092]

[0093]

[0094] Example 4

[0095] In this embodiment, the agarose magnetic beads prepared in Example 2 are functionally modified, including the following steps:

[0096] Take 0.5g GSH (reduced glutathione) and dissolve in 30ml water, adjust the pH value to 10, and the final volume of the solution is 40ml. Mix 10ml activated agarose magnetic beads with GSH solution evenly, and react at room temperature for 24h. After the reaction is completed, wash repeatedly with pure water. ​

[0097] At this time, the prepared functional agarose magnetic beads are GSH agarose magnetic beads, which can be applied to the purification of recombinant proteins containing GST tags. The recombinant protein (25 kDa in molecular weight) with a GST tag is expressed by a bacterial expression strain, and the protein is purified using 0.5 ml of GSH agarose magnetic beads. A total of 3 tubes are eluted, each with a volume of 3 ml. The results show that 5.4 mg of protein can be purified by 0.5 ml of GSH agarose magnetic beads, and the loading capacity of the agarose magnetic beads is 10.8 mg / ml. The results of SDS-PAGE electrophoresis show that the molecular weight of the target protein is 25 kDa, and the protein purified by the agarose magnetic beads has very few impurities, and the purified protein has high purity. The specific data are shown in Table 2, and the results are shown in Figure 6 .

[0098] Table 2

[0099]

[0100] Example 5

[0101] In this embodiment, the agarose magnetic beads prepared in Example 2 are functionally modified, including the following steps:

[0102] Add 20 ml of protein A protein to 10 ml of activated agarose magnetic beads, and the protein concentration is 2.5 mg / ml. Adjust the pH to 9-10. Mix the activated agarose magnetic beads and the protein A protein uniformly, and react at room temperature for 24 hours. After the reaction is completed, wash repeatedly with phosphate buffer.

[0103] At this time, the prepared functional agarose magnetic beads are protein A agarose magnetic beads, which can be applied to the purification of immunoglobulin G in rabbit, mouse, human, and the like. The rabbit serum containing polyclonal immunoglobulin G (IgG) is purified using 0.5 ml of protein A agarose magnetic beads. A total of 3 tubes are eluted, each with a volume of 3 ml. The results show that 24 mg of protein can be purified by 0.5 ml of protein A agarose magnetic beads, and the loading capacity of the agarose magnetic beads is 48 mg / ml. The results of SDS-PAGE electrophoresis show that the large subunit of the rabbit antibody has a molecular weight of 50 kDa, and the small subunit has a molecular weight of 25 kDa. The protein purified by the agarose magnetic beads has very few impurities, and the purified protein has high purity. The specific data are shown in Table 3, and the results are shown in Figure 7 .

[0104] Table 3

[0105]

[0106] Comparative Example 1

[0107] The comparative example uses a hydrothermal co-precipitation method to prepare magnetic beads, including the following steps:

[0108] 1) Take a 500ml clean three-necked flask, weigh 2g of iron trichloride hexahydrate and 0.9g of ferrous dichloride tetrahydrate, and fully dissolve them in 100ml of water.

[0109] 2) Place the three-necked flask on a mechanical stirrer, fill the flask with nitrogen, and heat to 100℃ using a heating jacket.

[0110] 3) The solution containing iron ions and ferrous ions is stirred for 30 minutes at 100℃, and 10ml of concentrated ammonia is added. At this time, the solution changes from a yellow clear liquid to a black particle, and the generated product is a magnetic bead. Continue to heat and stir for 1 hour.

[0111] 4) Stop heating, and after cooling the synthesized magnetic beads, wash them repeatedly with ethanol and water for three to five times.

[0112] 5) Dry the magnetic beads, weigh them, and prepare a 100mg / ml magnetic bead solution.

[0113] The magnetic beads prepared in the comparative example and the agarose magnetic beads prepared in Example 2 are tested for their magnetic field strength using a magnetometer, and the magnetic response time of the magnetic beads prepared by the two methods is compared. The magnetization strength results are shown in Figure 8 , and the magnetic attraction effect diagram is shown in Figure 9 .

[0114] In summary, the agarose magnetic beads and the preparation method thereof, and the functionalized agarose magnetic beads and the preparation method thereof provided by the present application, the preparation method of the agarose magnetic beads comprises the steps of: dissolving agarose in water to obtain an agarose solution; heating the agarose solution to boiling, adding a magnetic bead solution and an emulsion, and stirring, and continuously stirring after cooling with cold water to obtain agarose magnetic beads. The present application uses the agarose solution and the magnetic bead solution to co-stir, so that the magnetic beads are wrapped together with the agarose, and the agarose magnetic beads with a particle size range of 10-200μm are synthesized under the action of the emulsifier. The agarose magnetic beads prepared by the preparation method have the advantages of strong magnetism, uniform particle size distribution, simple equipment and easy large-scale production. Moreover, the present application uses a suspension stirring method to coat the magnetic beads inside the agarose, so that the agarose has magnetism, and the agarose magnetic beads with good uniformity can be prepared.

[0115] It should be understood that the application of the present application is not limited to the above examples, and those skilled in the art can make improvements or changes according to the above description, and all these improvements and changes shall belong to the protection scope of the appended claims of the present application.

Claims

1. A method for preparing an agarose magnetic bead, characterized by, The method comprises the steps of: dissolving agarose in water to obtain an agarose solution; heating the agarose solution to boiling, adding a magnetic bead solution and an emulsion, stirring, continuously stirring after cooling with cold water, and obtaining agarose magnetic beads.

2. The method for preparing agarose magnetic beads according to claim 1, characterized in that, The concentration of the agarose solution is 0.02 g / ml-0.1 g / ml; the mass of the magnetic beads in the magnetic bead solution is 20%-70% of the mass of the agarose; and the volume ratio of the emulsion to the magnetic bead solution is 1:(1-20).

3. The method for preparing agarose magnetic beads according to claim 1, characterized in that, The emulsion comprises an oil phase solution and a surfactant; the amount of the surfactant accounts for 10%-20% of the volume of the oil phase solution; the oil phase solution comprises one or more of petroleum ether, cyclohexane, cycloheptane, liquid paraffin, and cyclooctane; and the surfactant comprises one or more of Span 80, Span 85, Tween-20, Tween-85, and triton-X100.

4. The method for preparing agarose magnetic beads according to claim 1, characterized in that, The preparation of the magnetic bead solution comprises the steps of mixing a ferric salt, ethylene glycol, a constraint agent, and sodium acetate, and then performing a solvothermal reaction to obtain the magnetic bead solution.

5. An agarose magnetic bead, characterized in that, The agarose magnetic beads are prepared by using the preparation method of the agarose magnetic beads according to any one of claims 1-4.

6. The agarose magnetic bead of claim 5, wherein, The particle size of the agarose magnetic beads is 10 μm-200 μm.

7. A method for preparing functionalized agarose magnetic beads, characterized by, The method comprises the steps of: activating the agarose magnetic beads according to any one of claims 5-6 by using a crosslinking agent to obtain activated agarose magnetic beads; mixing the activated agarose magnetic beads with functional molecules to obtain a mixed solution; adjusting the pH value of the mixed solution, and obtaining functionalized agarose magnetic beads after reaction.

8. The method for preparing functionalized agarose magnetic beads according to claim 7, characterized in that, The functional molecules comprise one of nitrilotriacetic acid, iminodiacetic acid, reduced glutathione, streptavidin, staphylococcus aureus surface protein A, streptococcal protein G, peptostreptococcus protein L, and flag-tagged antibody.

9. The method for preparing functionalized agarose magnetic beads according to claim 7, characterized in that, The crosslinking agent comprises one or more of epichlorohydrin, 1,4-butanediol diglycidyl ether, and 2,3-dibromopropanol.

10. A functionalized agarose magnetic bead, characterized in that, The functionalized agarose magnetic beads are prepared by using the preparation method of the functionalized agarose magnetic beads according to any one of claims 7-9.

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