CN112915977A - Magnetic microsphere for cell sorting and preparation method thereof - Google Patents
Magnetic microsphere for cell sorting and preparation method thereof Download PDFInfo
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- 239000004005 microsphere Substances 0.000 title claims abstract description 45
- 238000002360 preparation method Methods 0.000 title claims abstract description 14
- 239000002122 magnetic nanoparticle Substances 0.000 claims abstract description 39
- 239000004372 Polyvinyl alcohol Substances 0.000 claims abstract description 22
- 229920002451 polyvinyl alcohol Polymers 0.000 claims abstract description 22
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 20
- 238000000034 method Methods 0.000 claims abstract description 15
- 229910052710 silicon Inorganic materials 0.000 claims abstract description 9
- 239000010703 silicon Substances 0.000 claims abstract description 9
- 239000002245 particle Substances 0.000 claims abstract description 8
- -1 silicon ester Chemical class 0.000 claims abstract description 6
- 238000003980 solgel method Methods 0.000 claims abstract description 5
- 150000002505 iron Chemical class 0.000 claims abstract description 3
- 238000006243 chemical reaction Methods 0.000 claims description 17
- 239000007864 aqueous solution Substances 0.000 claims description 14
- 239000000377 silicon dioxide Substances 0.000 claims description 9
- 235000012239 silicon dioxide Nutrition 0.000 claims description 9
- 238000000151 deposition Methods 0.000 claims description 5
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 claims description 4
- 150000003839 salts Chemical class 0.000 claims description 3
- 229910021578 Iron(III) chloride Inorganic materials 0.000 claims description 2
- 239000003153 chemical reaction reagent Substances 0.000 claims description 2
- 229960002089 ferrous chloride Drugs 0.000 claims description 2
- 235000003891 ferrous sulphate Nutrition 0.000 claims description 2
- 239000011790 ferrous sulphate Substances 0.000 claims description 2
- 229910052742 iron Inorganic materials 0.000 claims description 2
- NMCUIPGRVMDVDB-UHFFFAOYSA-L iron dichloride Chemical compound Cl[Fe]Cl NMCUIPGRVMDVDB-UHFFFAOYSA-L 0.000 claims description 2
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 claims description 2
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 claims description 2
- RUTXIHLAWFEWGM-UHFFFAOYSA-H iron(3+) sulfate Chemical compound [Fe+3].[Fe+3].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O RUTXIHLAWFEWGM-UHFFFAOYSA-H 0.000 claims description 2
- 229910000359 iron(II) sulfate Inorganic materials 0.000 claims description 2
- 229910000360 iron(III) sulfate Inorganic materials 0.000 claims description 2
- 230000008569 process Effects 0.000 abstract description 7
- 230000001476 alcoholic effect Effects 0.000 abstract description 3
- 125000002887 hydroxy group Chemical group [H]O* 0.000 abstract description 3
- 239000006249 magnetic particle Substances 0.000 abstract description 3
- 229910052814 silicon oxide Inorganic materials 0.000 abstract description 2
- 230000006978 adaptation Effects 0.000 abstract 1
- 239000006185 dispersion Substances 0.000 abstract 1
- 239000010410 layer Substances 0.000 abstract 1
- 239000002344 surface layer Substances 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 17
- 239000000243 solution Substances 0.000 description 12
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 9
- 239000000203 mixture Substances 0.000 description 6
- BOTDANWDWHJENH-UHFFFAOYSA-N Tetraethyl orthosilicate Chemical compound CCO[Si](OCC)(OCC)OCC BOTDANWDWHJENH-UHFFFAOYSA-N 0.000 description 5
- 239000011324 bead Substances 0.000 description 5
- 238000001878 scanning electron micrograph Methods 0.000 description 4
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 2
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 2
- 239000003513 alkali Substances 0.000 description 2
- 235000011114 ammonium hydroxide Nutrition 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 229920001296 polysiloxane Polymers 0.000 description 2
- 108090001008 Avidin Proteins 0.000 description 1
- 102000018697 Membrane Proteins Human genes 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 241000238367 Mya arenaria Species 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000013399 early diagnosis Methods 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 229920001002 functional polymer Polymers 0.000 description 1
- 238000001879 gelation Methods 0.000 description 1
- 239000004519 grease Substances 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 238000007885 magnetic separation Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 230000004043 responsiveness Effects 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- LFQCEHFDDXELDD-UHFFFAOYSA-N tetramethyl orthosilicate Chemical compound CO[Si](OC)(OC)OC LFQCEHFDDXELDD-UHFFFAOYSA-N 0.000 description 1
- ZQZCOBSUOFHDEE-UHFFFAOYSA-N tetrapropyl silicate Chemical compound CCCO[Si](OCCC)(OCCC)OCCC ZQZCOBSUOFHDEE-UHFFFAOYSA-N 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
- B01J20/26—Synthetic macromolecular compounds
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- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
- B01J20/28002—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their physical properties
- B01J20/28009—Magnetic properties
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- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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Abstract
The invention relates to a magnetic microsphere for cell sorting and a preparation method thereof, wherein the preparation method of the magnetic microsphere comprises the following steps: under the condition of the rotating speed of 2000 plus 18000rpm, iron salt is utilized to form magnetic nano particles, then silicon ester is added for high-speed dispersion, silicon oxide is deposited on the surfaces of the magnetic nano particles through a sol-gel method to form magnetic microspheres, a small amount of polyvinyl alcohol is added in the middle of the magnetic nano particles, and finally a certain shell layer is formed on the surface layers of the magnetic nano particles, so that the surfaces of the magnetic particles are more hydrophilic. The magnetic microsphere prepared by the invention has good hydrophilicity and biocompatibility, wide pH adaptation range and abundant alcoholic hydroxyl and silicon hydroxyl on the surface; the particle size of the microspheres is uniform and ranges from tens of nanometers to tens of nanometers; the process route is very simple.
Description
Technical Field
The invention relates to the field of preparation of functional polymer microspheres, in particular to a magnetic microsphere for cell sorting and a preparation method thereof.
Background
Immunomagnetic bead cell sorting is a technique for separating cells of interest from a cell population by magnetic beads. First, the magnetic beads are bound to a specific cell surface protein on the target cells by an antibody, biotin or avidin. Then the sample is placed in a magnetic field, and the cells marked by the magnetic beads are adsorbed under the action of the magnetic field. The unlabeled cells are retained in the supernatant, thereby physically separating the cells of interest from the non-cells of interest in the sample.
The recent development trend of the biological magnetic separation technology is miniaturization, parallelization and automation, and the key problems are the preparation of superparamagnetic magnetic microspheres with uniform particle size, good dispersibility in water and strong magnetic responsiveness so as to ensure the high efficiency, reproducibility and controllability of the biological separation process. Magnetic nanoparticles have potential in early diagnosis and treatment of diseases, and development in the biomedical field is also receiving considerable attention. Because of its rapidity and simplicity of operation, immunomagnetic bead cell sorting is one of the most common methods used by researchers to isolate specific cell subsets of high purity. It has the advantages of high purity, rapidness, simple operation, low cost, capability of separating various cells at one time, automation, high cell survival rate and the like.
The existing methods have some defects, such as the defects that the particle size of the seed microsphere cannot be too small, the adsorbed magnetic particles are easy to fall off, the treatment process is complicated, the process route is long, and the like.
Disclosure of Invention
In order to solve the technical problems, the invention aims to provide a magnetic microsphere for cell sorting and a preparation method thereof.
The first object of the present invention is to provide a magnetic microsphere, which comprises magnetic nanoparticles and a shell layer coated on the surface of the magnetic nanoparticles, wherein the shell layer comprises silicon dioxide and polyvinyl alcohol (PVA).
Further, the magnetic nanoparticles are iron-based magnetic nanoparticles; the particle size of the magnetic nanoparticles is 10-100 nm.
Furthermore, the thickness of the shell layer is 10-100 nm.
Furthermore, the particle size of the magnetic microsphere is 50nm-200 nm.
Further, the mass of the silicon dioxide is 0.5 wt% to 20 wt% of the magnetic nanoparticles. The silicon dioxide is rich in a large number of silicon hydroxyl groups, can adsorb partial polyvinyl alcohol in the process of gelation, is deposited on the surfaces of the magnetic particles, has certain hydrophilicity on one hand, and can improve the dispersibility of the subsequent magnetic microspheres in a buffer solution in specific application.
Further, the mass of the polyvinyl alcohol is 0.5 wt% -10 wt% of the magnetic nano particles; the molecular weight of the polyvinyl alcohol may be 48000 to 220000. The polyvinyl alcohol is rich in a large amount of alcoholic hydroxyl groups, can improve the hydrophilicity of the surface of the magnetic microsphere, and meanwhile, the polyvinyl alcohol has good biocompatibility, and can be modified on the surface of the magnetic microsphere to further improve the biocompatibility of the magnetic microsphere.
The second purpose of the invention is to provide a preparation method of the magnetic microsphere, which comprises the following steps:
uniformly mixing magnetic nanoparticles and silicon ester in an aqueous solution with the pH value of 11-12, depositing silicon dioxide on the surfaces of the magnetic nanoparticles by a sol-gel method at the rotating speed of 2000-18000rpm, then adding polyvinyl alcohol for reaction, and obtaining magnetic microspheres after the reaction is completed; or
Uniformly mixing magnetic nanoparticles, silicon ester and polyvinyl alcohol in an aqueous solution with the pH value of 11-12, depositing silicon dioxide on the surfaces of the magnetic nanoparticles by a sol-gel method at the rotating speed of 2000-18000rpm, and simultaneously wrapping the surfaces of the magnetic nanoparticles with the polyvinyl alcohol to obtain the magnetic microspheres.
Further, the preparation method of the magnetic nanoparticles comprises the following steps:
adjusting the pH value of the ferric salt aqueous solution to 11-12 at the rotating speed of 2000-18000rpm, and reacting at 25-90 ℃ to obtain the magnetic nanoparticles after the reaction is completed. The reaction time is preferably 0.5-5 h.
Further, the iron salt is selected from one or more of ferric chloride, ferrous chloride, ferric sulfate and ferrous sulfate.
When the pH value of the ferric salt aqueous solution is adjusted, alkali liquor can be adopted for adjustment, the alkali liquor comprises sodium hydroxide solution or concentrated ammonia water, and the concentration of the sodium hydroxide solution can be 0.0001-1 mol/L.
Further, in the preparation process of the magnetic microspheres, the reaction is carried out for 0.5-5 hours at 50 ℃.
Further, the silicon ester comprises one or more of methyl orthosilicate, ethyl orthosilicate and propyl orthosilicate.
Further, the above-mentioned reactions can be carried out in a wall breaking machine. Through adjusting the gear of broken wall machine, and then adjust the rotational speed.
In the preparation method, the reaction is carried out at a high rotating speed, so that the reaction raw materials are uniformly dispersed, and the silicone ester is prevented from being agglomerated in the gelling process. The particle size, shell thickness and surface hydrophilicity and hydrophobicity of the reaction product magnetic microsphere can be adjusted. The shell layer of the magnetic microsphere can be a soft shell layer or a hard shell layer, and can be realized by adjusting the proportion of polyvinyl alcohol and silicone grease.
The invention also claims the application of the magnetic microspheres prepared by the invention in preparing a reagent for cell sorting.
By the scheme, the invention at least has the following advantages:
the magnetic microsphere prepared by the invention has very good hydrophilicity and biocompatibility, can be used for cell sorting, and has a large amount of alcoholic hydroxyl and silicon hydroxyl on the surface; the particle size of the microspheres is uniform and is 50nm-200 nm; the synthetic process route is very simple.
The preparation method of the magnetic microsphere comprises the steps of synthesizing magnetic nano particles, depositing silicon oxide on the surfaces of the magnetic nano particles and modifying the surfaces of the magnetic nano particles by polyvinyl alcohol macromolecules, and the process is simple and convenient.
The foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical solutions of the present invention more clearly understood and to implement them in accordance with the contents of the description, the following description is made with reference to the preferred embodiments of the present invention and the accompanying detailed drawings.
Drawings
FIG. 1 is a scanning electron micrograph of magnetic microspheres prepared in example 3;
FIG. 2 is a scanning electron micrograph of magnetic microspheres prepared in example 4;
FIG. 3 is a scanning electron micrograph of magnetic microspheres prepared according to example 5;
FIG. 4 is a scanning electron micrograph of the magnetic microspheres prepared in example 6.
Detailed Description
The following examples are given to further illustrate the embodiments of the present invention. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
Example 1
Adding 0.5mol/L Fe2+An aqueous solution and 0.5mol/L Fe3+Aqueous solution, according to 1: 2, pouring the mixture into a wall breaking machine, adjusting the gear to 4 (the rotating speed is 8000rpm), quickly adjusting the pH of the solution to 11 by using 0.1M NaOH, heating the solution to 80 ℃, and reacting for 30min to obtain the magnetic nanoparticles.
Example 2
Adding 0.5mol/L Fe2+An aqueous solution and 0.5mol/L Fe3+Aqueous solution, according to 1: mixing the components according to the volume ratio of 1.8, pouring the mixture into a wall breaking machine, adjusting the gear to 4 (the rotating speed is 8000rpm), quickly adjusting the pH value of the solution to 12 by using ammonia water, heating the solution to 90 ℃, and reacting for 30min to obtain the magnetic nanoparticles.
Example 3
96ml of ethyl orthosilicate is added into the reaction solution of the example 1, the gear is adjusted to 7, the mixture is heated to 50 ℃, and the reaction is carried out for 30min, so as to obtain the magnetic microspheres, as shown in figure 1.
Example 4
48ml of ethyl orthosilicate and 10ml of 10 wt% polyvinyl alcohol (molecular weight of 48000) aqueous solution are added to the reaction solution in example 1, the gear is adjusted to 7 (14000rpm), the mixture is heated to 50 ℃ and reacted for 30min, and magnetic microspheres are obtained, as shown in FIG. 2.
Example 5
24ml of ethyl orthosilicate and 30ml of 10 wt% polyvinyl alcohol (molecular weight of 48000) aqueous solution are added into the reaction solution in example 1, the gear is adjusted to 7 (rotation speed of 14000rpm), the mixture is heated to 50 ℃ and reacted for 30min, and magnetic microspheres are obtained, and the figure is 3.
Example 6
72ml of ethyl orthosilicate and 5ml of 4 wt% polyvinyl alcohol (molecular weight 220000) aqueous solution are added into the reaction solution in the example 1, the gear is adjusted to 7 (the rotating speed is 14000rpm), the mixture is heated to 50 ℃, and the reaction is carried out for 30min, so that the magnetic microspheres are obtained, and the figure 4 shows.
The above is only a preferred embodiment of the present invention, and is not intended to limit the present invention, it should be noted that, for those skilled in the art, many modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the protection scope of the present invention.
Claims (10)
1. A magnetic microsphere, characterized in that: the magnetic nano particle comprises a magnetic nano particle and a shell layer wrapped on the surface of the magnetic nano particle, wherein the shell layer comprises silicon dioxide and polyvinyl alcohol.
2. The magnetic microsphere of claim 1, wherein: the magnetic nanoparticles are iron-based magnetic nanoparticles; the particle size of the magnetic nanoparticles is 10-100 nm.
3. The magnetic microsphere of claim 1, wherein: the thickness of the shell layer is 10-100 nm.
4. The magnetic microsphere of claim 1, wherein: the mass of the silicon dioxide is 0.5 wt% -20 wt% of the magnetic nano particles.
5. The magnetic microsphere of claim 1, wherein: the mass of the polyvinyl alcohol is 0.5-10 wt% of that of the magnetic nano particles; the molecular weight of the polyvinyl alcohol is 48000-220000.
6. A method for preparing magnetic microspheres according to any one of claims 1 to 5, comprising the steps of:
uniformly mixing magnetic nanoparticles with silicon ester in an aqueous solution with the pH value of 11-12, depositing silicon dioxide on the surfaces of the magnetic nanoparticles by a sol-gel method at the rotating speed of 2000-18000rpm, then adding polyvinyl alcohol for reaction, and obtaining the magnetic microspheres after the reaction is completed; or
Uniformly mixing magnetic nanoparticles, silicon ester and polyvinyl alcohol in an aqueous solution with the pH value of 11-12, depositing silicon dioxide on the surfaces of the magnetic nanoparticles by a sol-gel method at the rotating speed of 2000-18000rpm, and simultaneously wrapping the surfaces of the magnetic nanoparticles with the polyvinyl alcohol to obtain the magnetic microspheres.
7. The method of claim 6, wherein the method of preparing the magnetic nanoparticles comprises the steps of:
adjusting the pH value of the ferric salt aqueous solution to 11-12 at the rotating speed of 2000-18000rpm, reacting at 25-90 ℃, and obtaining the magnetic nanoparticles after the reaction is completed.
8. The method of claim 7, wherein: the iron salt is selected from one or more of ferric chloride, ferrous chloride, ferric sulfate and ferrous sulfate.
9. The method of claim 6, wherein: the reaction is carried out at 50 ℃ for 0.5-5 h.
10. Use of magnetic microspheres according to any one of claims 1 to 5 for the preparation of a reagent for cell sorting.
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