CN111135804A - Preparation method of multi-carboxyl magnetic microspheres for affinity purification - Google Patents
Preparation method of multi-carboxyl magnetic microspheres for affinity purification Download PDFInfo
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- CN111135804A CN111135804A CN201911354672.1A CN201911354672A CN111135804A CN 111135804 A CN111135804 A CN 111135804A CN 201911354672 A CN201911354672 A CN 201911354672A CN 111135804 A CN111135804 A CN 111135804A
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Abstract
The invention relates to a preparation method of a multi-carboxyl magnetic microsphere for affinity purification, which comprises the steps of coating an epoxy monomer, a comonomer, a cross-linking agent and an initiator on the surface of a seed microsphere by a water-phase in-situ polymerization method to form a uniform epoxy shell layer, then opening and connecting epoxy to a connecting carboxyl monomer by ring-opening reaction of an epoxy group, and finally connecting a third carboxyl to the surface of the microsphere by substitution reaction to form tridentate carboxyl. The double carboxyl on the surface of the microsphere is changed into tricarboxy by a post-modification method to form a tridentate carboxyl structure; the carboxyl on the surface of the magnetic microsphere is in a tridentate shape, and the carboxyl with a tridentate structure can better and more firmly grab and couple biomacromolecules, so that the high-efficiency and high-recovery of subsequent affinity purification is ensured.
Description
Technical Field
The invention relates to a preparation method of a polycarboxyl magnetic microsphere for affinity purification.
Background
In recent years, nanotechnology and biotechnology are changing day by day, the cross fusion of the two fields is deepened continuously, and the functionalized superparamagnetic composite microsphere is widely concerned by people due to excellent performance, and has a very good application prospect in the directions of bioseparation, immunochromatography and the like. In the field of affinity purification, natural polysaccharides, such as agarose microspheres, are mostly used as carriers. However, the single agarose microspheres have the problems of complex operation, complex washing and separation and the like in the using process. The functionalized superparamagnetic composite microsphere can meet the surface characteristics of natural polysaccharide after surface modification, can solve the problems of complex operation, washing, separation and the like, is simple and convenient, can firmly grab and couple biomacromolecules by the modified tridentate carboxyl on the surface of the microsphere, and can be used in the field of affinity purification.
The immunoaffinity purification technology is to extract a target compound from a complex sample according to the high selectivity of an antigen antibody by utilizing the characteristic of specific reversible binding of the antigen antibody. Taking purified antigen as an example, after an antibody is covalently bonded to a microsphere, the antigen is mixed with a solution containing the antigen, the antigen in the solution is captured by the antibody on the surface of the microsphere, and then irrelevant antigen or impurities are removed by washing. Finally, the microspheres are treated by elution buffer solution, so that the antigen bound on the surfaces of the microspheres is released, and the purpose of obtaining the purified antigen is achieved. The technology can extract target protein from a mixture with complex components and high impurity content, can separate active target protein from inactive target protein according to the biological performance of the target protein, and has the advantages of high efficiency, high recovery rate and the like.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provides a preparation method of a polycarboxyl magnetic microsphere for affinity purification.
The purpose of the invention is realized by the following technical scheme:
a preparation method of multi-carboxyl magnetic microspheres for affinity purification is characterized in that: the method comprises the steps of coating an epoxy monomer, a comonomer, a cross-linking agent and an initiator on the surface of a seed microsphere to form a uniform epoxy shell layer by a water-phase in-situ polymerization method, then connecting epoxy with a carboxyl monomer by opening an epoxy ring-opening reaction of an epoxy group, and finally connecting a third carboxyl to the surface of the microsphere to form a tridentate carboxyl by a substitution reaction.
Further, the preparation method of the polycarboxy magnetic microsphere for affinity purification comprises the steps of dispersing the seed microsphere in an organic solvent, adding a silane coupling agent under the stirring condition, adjusting the pH value to make the solution alkaline, and reacting to obtain the double-bond modified magnetic microsphere;
dispersing the double-bond modified magnetic microspheres in a water phase, adding an epoxy monomer, a comonomer, an initiator and a crosslinking agent, heating in a water bath to 40-90 ℃, and reacting for 1-24 hours to obtain epoxy shell layer coated magnetic microspheres;
dispersing the magnetic microspheres coated by the epoxy shell layer in deionized water under an alkaline condition, adding a carboxyl compound with amino and water, heating in a water bath to 40-90 ℃, and reacting for 1-24 hours to obtain carboxyl modified magnetic microspheres;
dispersing the carboxyl magnetic microspheres in water under an alkaline condition, adding a substitution reagent, adjusting the pH value to 9-11, stirring at room temperature for 8-24 hours, taking out, and carrying out magnetic separation and washing to obtain the tridentate carboxyl magnetic microspheres.
Further, the preparation method of the polycarboxyl magnetic microsphere for affinity purification is described, wherein the seed microsphere is a magnetic microsphere with the particle size of 1-20 μm; the thickness of the epoxy shell layer is 20 nm-200 nm.
Further, in the preparation method of the polycarboxy magnetic microsphere for affinity purification, the initiator is a peroxide initiator or a redox initiator.
Further, in the preparation method of the polycarboxyl magnetic microsphere for affinity purification, the initiator is one or more of benzoyl peroxide, potassium persulfate, ammonium persulfate and sodium bisulfite.
Further, in the preparation method of the polycarboxy magnetic microsphere for affinity purification, the cross-linking agent is one or more of a diene monomer, a triene monomer and a polyene monomer.
Further, the preparation method of the polycarboxyl magnetic microsphere for affinity purification is described, wherein the cross-linking agent is one or more of divinylbenzene, N' N methylene bisacrylamide and pentaerythritol triacrylate.
Further, the preparation method of the polycarboxyl magnetic microsphere for affinity purification is described, wherein the epoxy monomer is glycidyl methacrylate.
Further, in the above method for preparing the polycarboxyl magnetic microsphere for affinity purification, the carboxyl monomer is aspartic acid.
Further, the preparation method of the polycarboxyl magnetic microsphere for affinity purification is described above, wherein the magnetic microsphere is Fe3O4Magnetic microspheres, CoFe2O4Magnetic microspheres, NiFe2O4One or more of magnetic microspheres.
Compared with the prior art, the invention has obvious advantages and beneficial effects, and is embodied in the following aspects:
the invention changes the dicarboxyl on the surface of the microsphere into tricarboxy by a post-modification method to form a tridentate carboxyl structure; the carboxyl on the surface of the magnetic microsphere is in a tridentate shape, the carboxyl with a tridentate structure can better and more firmly grab and couple biomacromolecules, the high-efficiency and high-recovery of subsequent affinity purification is ensured, and the magnetic microsphere can be better applied to the field of affinity purification.
Additional features and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by the practice of the invention. The objectives and other advantages of the invention will be realized and attained by the structure particularly pointed out in the written description and claims thereof.
Drawings
FIG. 1: synthesizing a route map by using tridentate carboxyl magnetic microspheres;
FIG. 2: the carboxyl magnetic microsphere prepared by the invention is a scanning electron microscope photo.
Detailed Description
In order to more clearly understand the technical features, objects, and effects of the present invention, specific embodiments will now be described in detail.
The invention provides a preparation method of magnetic microspheres, which has high combination efficiency and simple process flow and is suitable for automation. Modifying the surface of the seed microsphere with silane with double bonds, so that subsequent shell coating can occur on the surface of the microsphere; opening double bonds on the surfaces of the microspheres in an in-situ polymerization mode, and coating epoxy shell layers on the surfaces of the seed microspheres; attaching a dicarboxyl group to the surface of the microsphere by a ring-opening reaction; and finally, connecting a third carboxyl to the surface of the microsphere through a substitution reaction to form a tridentate carboxyl.
The double bonds can be modified on the surface of the magnetic microsphere through hydrolysis of silane, the seed microsphere used by the silane-modified magnetic microsphere is a superparamagnetic microsphere, and the content of the double bonds on the surface of the magnetic microsphere can be adjusted by changing the using amount of a silane coupling agent; the shell thickness, the crosslinking degree and the epoxy group content of the magnetic microsphere coated by the epoxy shell can be adjusted; the crosslinking degree of the magnetic microspheres coated by the epoxy shell layer can be adjusted by the dosage of the crosslinking agent; the content of epoxy groups in the magnetic microspheres coated by the epoxy shell can be adjusted by the using amount of the epoxy monomer.
Firstly, dispersing seed microspheres in an organic solvent, wherein the seed microspheres are magnetic microspheres with the particle size of 1-20 mu m, and the magnetic microspheres are Fe3O4Magnetic microspheres, CoFe2O4Magnetic microspheres or NiFe2O4The magnetic microspheres are prepared by mixing ethanol, acetonitrile and water as organic solvents; adding a silane coupling agent under the condition of mechanical stirring, wherein the silane coupling agent can be gamma-methacryloxypropyltrimethoxysilane, adjusting the pH value to make the solution alkaline, and obtaining the double-bond modified magnetic microspheres after reaction;
then, dispersing the double-bond modified magnetic microspheres in a water phase, adding an epoxy monomer, a comonomer, an initiator and a crosslinking agent, heating in a water bath to 40-90 ℃, and reacting for 1-24 hours to obtain magnetic microspheres coated by an epoxy shell layer, wherein the thickness of the epoxy shell layer is 20-200 nm; wherein the initiator is a peroxide initiator or a redox initiator, and specifically can adopt one or more of benzoyl peroxide, potassium persulfate, ammonium persulfate and sodium bisulfite; the cross-linking agent is one or more of diene monomer, triene monomer and polyene monomer, and specifically can adopt one or more of divinylbenzene, N' N methylene bisacrylamide and pentaerythritol triacrylate; the epoxy monomer is glycidyl methacrylate, and the organic matter with amino and carboxyl is aspartic acid; the comonomer is styrene or methyl methacrylate;
dispersing the magnetic microspheres coated by the epoxy shell layer in deionized water under an alkaline condition, adding a carboxyl compound with amino and water, heating in a water bath to 40-90 ℃, and reacting for 1-24 hours to obtain carboxyl modified magnetic microspheres;
finally, dispersing the carboxyl magnetic microspheres in water under an alkaline condition, adding a substitution reagent, and adjusting the pH value to 9-11, wherein the substitution reagent is chloroacetic acid or bromoacetic acid; and stirring and reacting for 8-24 hours at room temperature, taking out, and performing magnetic separation and washing to obtain the tridentate carboxyl magnetic microspheres.
Example 1
Double bond modification: weighing 5g of ferroferric oxide magnetic microspheres (with the average particle size of 1 mu m) and dispersing in 500ml of ethanol solution, adding 3.3g of gamma-methacryloxypropyltrimethoxysilane while stirring, then adding 32.4g of sodium hydroxide solution (0.5g/ml), reacting for 15 hours at 40 ℃ to obtain the magnetic microspheres with surface double bond modification, and storing for later use.
Example 2
Double bond modification: weighing 5g of ferroferric oxide magnetic microspheres (the average particle size is 3 mu m) and dispersing the ferroferric oxide magnetic microspheres in 500ml of ethanol solution, adding 0.41g of gamma-methacryloxypropyltrimethoxysilane while stirring, then adding 32.4g of sodium hydroxide solution (0.5g/ml), reacting for 3 hours at 70 ℃ to obtain the magnetic microspheres with surface double bond modification, and storing for later use.
Example 3
Coating with an epoxy shell layer: 5g of double bond-modified ferroferric oxide magnetic microspheres (average particle size of 1 μm) are weighed and dispersed in 300ml of water, and 10g of glycidyl methacrylate, 0.4g of divinylbenzene and 0.2g of potassium persulfate are added. And (3) pouring the solution into a three-neck flask after ultrasonic treatment, uniformly stirring, heating to 70 ℃, and reacting for 5 hours to obtain the magnetic microsphere with the epoxy modified surface.
Example 4
Double bond modification: weighing 5g of ferroferric oxide magnetic microspheres (with the average particle size of 1 micron) and dispersing the ferroferric oxide magnetic microspheres in 500ml of ethanol solution, adding 3.3g of gamma-methacryloxypropyltrimethoxysilane while stirring, then adding 32.4g of ammonia water, reacting for 3 hours at 70 ℃ to obtain magnetic microspheres with surface double bond modification, and storing for later use.
Example 5
Double bond modification: weighing 5g of ferroferric oxide magnetic microspheres (the average particle size is 3 mu m) and dispersing in 500ml of ethanol solution, adding 0.41g of gamma-methacryloxypropyltrimethoxysilane while stirring, then adding 32.4g of ammonia water, reacting for 3 hours at 70 ℃ to obtain the magnetic microspheres with surface double bond modification, and storing for later use.
Example 6
Epoxy modification: 5g of double-bond modified ferroferric oxide magnetic microspheres (the average particle size is 3 microns) are weighed and dispersed in 300ml of deionized water, and 9g of glycidyl methacrylate, 0.4g of divinylbenzene, 1g of styrene and 0.2g of ammonium persulfate are added. And (3) pouring the solution into a three-neck flask after ultrasonic treatment, uniformly stirring, heating to 70 ℃, and reacting for 5 hours to obtain the magnetic microsphere with the epoxy modified surface.
Example 7
Carboxyl modification: weighing 5g of ferroferric oxide magnetic microspheres (with the average particle size of 1 mu m) with epoxy modified surfaces, dispersing the ferroferric oxide magnetic microspheres in 200ml of deionized water, and then adding 2.85g of NaOH, 6.8g of aspartic acid and a proper amount of Na2CO3Adjusting the pH value of the solution to 11, carrying out water bath reaction at 70 ℃ for 8 hours, carrying out magnetic separation and washing for 6 times, and then re-dispersing in 200ml of deionized water to obtain the magnetic microspheres with surface carboxyl modified.
Example 8
Carboxyl modification: weighing 5g of ferroferric oxide magnetic microspheres with epoxy-modified surfaces(average particle size 3 μm) was dispersed in 200ml of deionized water, and 0.36g of NaOH, 0.85g of aspartic acid and an appropriate amount of Na were added2CO3Adjusting the pH value of the solution to 11, carrying out water bath reaction at 70 ℃ for 8 hours, carrying out magnetic separation and washing for 6 times, and then re-dispersing in 200ml of deionized water to obtain the magnetic microspheres with surface carboxyl modified.
Example 9
Three-tooth-shaped carboxyl modification: 5g of ferroferric oxide magnetic microspheres (average particle size is 1 mu m) with surface carboxyl modified are weighed and dispersed in 400ml of deionized water, and then 1.13g of NaOH, 3.75g of bromoacetic acid and a proper amount of Na are added2CO3And (3) adjusting the pH value of the solution to 11, carrying out water bath reaction at 70 ℃ for 8 hours, carrying out magnetic separation and washing for 6 times, and dispersing in 200ml of deionized water again to obtain the magnetic microsphere with the surface modified by the tridentate carboxyl. The synthetic route is shown in figure 1, the carboxyl on the surface of the magnetic microsphere is tridentate, and biological macromolecules can be captured and coupled more firmly as shown in figure 2.
Example 10
Three-tooth-shaped carboxyl modification: 5g of ferroferric oxide magnetic microspheres (average particle size is 3 mu m) with surface carboxyl modified are weighed and dispersed in 400ml of deionized water, and then 0.19g of NaOH, 0.63g of bromoacetic acid and a proper amount of Na are added2CO3And (3) adjusting the pH value of the solution to 10, carrying out water bath reaction at 70 ℃ for 8 hours, carrying out magnetic separation and washing for 6 times, and dispersing in 200ml of deionized water again to obtain the magnetic microsphere with the surface modified by the tridentate carboxyl.
In conclusion, the invention changes the dicarboxyl on the surface of the microsphere into the tricarboxy by a post-modification method to form a tridentate carboxyl structure; the carboxyl on the surface of the magnetic microsphere is in a tridentate shape, the carboxyl with a tridentate structure can better and more firmly grab and couple biomacromolecules, the high-efficiency and high-recovery of subsequent affinity purification is ensured, and the magnetic microsphere can be better applied to the field of affinity purification.
It should be noted that: the above description is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention; while the foregoing is directed to embodiments of the present invention, other and further embodiments of the invention may be devised without departing from the basic scope thereof, and the scope thereof is determined by the claims that follow.
Claims (10)
1. A preparation method of a polycarboxyl magnetic microsphere for affinity purification is characterized by comprising the following steps: the method comprises the steps of coating an epoxy monomer, a comonomer, a cross-linking agent and an initiator on the surface of a seed microsphere to form a uniform epoxy shell layer by a water-phase in-situ polymerization method, then connecting epoxy with a carboxyl monomer by opening an epoxy ring-opening reaction of an epoxy group, and finally connecting a third carboxyl to the surface of the microsphere to form a tridentate carboxyl by a substitution reaction.
2. The method for preparing polycarboxy magnetic microsphere for affinity purification according to claim 1, wherein the method comprises the following steps: dispersing the seed microspheres in an organic solvent, adding a silane coupling agent under the stirring condition, adjusting the pH value to make the solution alkaline, and reacting to obtain double-bond modified magnetic microspheres;
dispersing the double-bond modified magnetic microspheres in a water phase, adding an epoxy monomer, a comonomer, an initiator and a crosslinking agent, heating in a water bath to 40-90 ℃, and reacting for 1-24 hours to obtain epoxy shell layer coated magnetic microspheres;
dispersing the magnetic microspheres coated by the epoxy shell layer in deionized water under an alkaline condition, adding a carboxyl compound with amino and water, heating in a water bath to 40-90 ℃, and reacting for 1-24 hours to obtain carboxyl modified magnetic microspheres;
dispersing the carboxyl magnetic microspheres in water under an alkaline condition, adding a substitution reagent, adjusting the pH value to 9-11, stirring at room temperature for 8-24 hours, taking out, and carrying out magnetic separation and washing to obtain the tridentate carboxyl magnetic microspheres.
3. The method for preparing polycarboxy magnetic microsphere for affinity purification according to claim 1 or 2, wherein: the seed microspheres are magnetic microspheres, and the particle size is 1-20 microns; the thickness of the epoxy shell layer is 20 nm-200 nm.
4. The method for preparing polycarboxy magnetic microsphere for affinity purification according to claim 1 or 2, wherein: the initiator is one or more of a peroxide initiator and a redox initiator.
5. The method for preparing polycarboxy magnetic microsphere for affinity purification according to claim 1 or 2, wherein: the initiator is one or more of benzoyl peroxide, potassium persulfate, ammonium persulfate and sodium bisulfite.
6. The method for preparing polycarboxy magnetic microsphere for affinity purification according to claim 1 or 2, wherein: the cross-linking agent is one or more of diene monomer, triene monomer and polyene monomer.
7. The method for preparing polycarboxy magnetic microsphere for affinity purification according to claim 1 or 2, wherein: the cross-linking agent is one or more of divinylbenzene, N' N methylene bisacrylamide and pentaerythritol triacrylate.
8. The method for preparing polycarboxy magnetic microsphere for affinity purification according to claim 1 or 2, wherein: the epoxy monomer is glycidyl methacrylate.
9. The method for preparing polycarboxy magnetic microsphere for affinity purification according to claim 1, wherein the method comprises the following steps: the carboxyl monomer is aspartic acid.
10. The method for preparing polycarboxy magnetic microsphere for affinity purification according to claim 1 or 2, wherein: the magnetic microspheres are Fe3O4Magnetic microspheres, CoFe2O4Magnetic microspheres, NiFe2O4One or more of magnetic microspheres.
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