CN115127972A - Magnetic beads and methods of making the same - Google Patents

Abstract

The application relates to the technical field of magnetic bead modification, and particularly discloses a magnetic bead and a manufacturing method thereof, wherein the magnetic bead comprises the following components: the magnetic microsphere comprises a core microsphere and first magnetic nanoparticles distributed in the core microsphere; a second magnetic nanoparticle coupled to an outer surface of the core microsphere; and the third magnetic nano-particles are at least dispersed and distributed in the core microsphere. By the mode, the magnetic response of the magnetic beads is improved, meanwhile, interference signals caused by impurities are reduced, autofluorescence of the magnetic microspheres can be reduced, and detection sensitivity is improved.

Description

Magnetic beads and methods of making the same

technical field

The present application relates to the technical field of magnetic bead modification, and in particular, to a magnetic bead and a manufacturing method thereof.

Background technique

Magnetic microspheres are a kind of spherical composite materials with a diameter of nanometer or micrometer. Magnetic microspheres are composed of microspheres and magnetic nanoparticles adsorbed on the surface of the microspheres.

In the long-term research and development process, the inventors of the present application found that if the surface of the microspheres adsorbs magnetic nanoparticles excessively, as shown in Figure 26, the impurities on the magnetic microspheres will increase, and the impurity signal will seriously interfere with the main group on the flow cytometer. signal, and heating is required in the process of coating the polymer protective layer, which easily causes the microspheres to generate strong autofluorescence, resulting in a decrease in detection sensitivity.

SUMMARY OF THE INVENTION

The present application aims to solve at least one of the technical problems existing in the prior art. Therefore, one purpose of the present application is to propose a magnetic bead and a method for making the same, which can reduce the interference signal caused by impurities while improving the magnetic response of the magnetic bead, and can reduce the autofluorescence of magnetic microspheres, improve detection sensitivity.

A first aspect of the present application provides a magnetic bead, the magnetic bead comprising: a magnetic microsphere, including a core microsphere and a first magnetic nanoparticle distributed inside the core microsphere; a second magnetic nanoparticle coupled to the core microsphere the outer surface of the sphere; the third magnetic nanoparticles, at least dispersed in the inner part of the core microsphere.

A second aspect of the present application provides a method for manufacturing a magnetic bead, the method comprising: providing a magnetic microsphere, the magnetic microsphere including a core microsphere and first magnetic nanoparticles distributed inside the core microsphere; adopting an adsorption technology The magnetic microspheres and the second magnetic nanoparticles are combined, so that the second magnetic nanoparticles are coupled to the outer surface of the core microsphere; wherein, the magnetic microspheres and the second magnetic nanoparticles are combined by using an adsorption technology, so that the second magnetic Before the step of coupling the nanoparticle to the outer surface of the core microsphere, or, using an adsorption technique to combine the magnetic microsphere and the second magnetic nanoparticle, so that the second magnetic nanoparticle is coupled to the outer surface of the core microsphere. Afterwards, the method further includes: combining the magnetic microspheres and the third magnetic nanoparticles by using a swelling technique, so that the third magnetic nanoparticles are dispersed and distributed inside the core microspheres.

Different from the situation in the prior art, the magnetic beads of the present application include: second magnetic nanoparticles coupled to the outer surface of the magnetic microspheres, and at least dispersed in the interior of the core microspheres and/or the interior of the gel layer The third magnetic nanoparticle, as shown in Figure 27, can enhance the magnetic response signal of the magnetic bead and reduce the interference signal caused by impurities on the premise of avoiding excessive adsorption of magnetic nanoparticles on the surface of the magnetic microsphere With the autofluorescence of magnetic microspheres, the detection sensitivity is improved.

Additional aspects and advantages of the present application will be set forth, in part, from the following description, and in part will be apparent from the following description, or may be learned by practice of the present application.

Description of drawings

The above and/or additional aspects and advantages of the present application will become apparent and readily understood from the following description of embodiments in conjunction with the accompanying drawings, wherein:

1 is a schematic flowchart of a method for making magnetic beads according to the first embodiment of the present application;

2 is a schematic flowchart of a method for making magnetic beads according to a second embodiment of the present application;

3 is a schematic flowchart of a method for making magnetic beads according to a third embodiment of the present application;

Fig. 4 is a schematic flowchart of step S10 in Figs. 1-3;

5 is a schematic flowchart of a method for manufacturing a magnetic bead according to a fourth embodiment of the present application;

6 is a schematic flowchart of a method for making magnetic beads according to the fifth embodiment of the present application;

FIG. 7 is a schematic flowchart of a method for making magnetic beads according to the sixth embodiment of the present application;

8 is a schematic flowchart of a method for making magnetic beads according to the seventh embodiment of the present application;

9 is a schematic flowchart of a method for manufacturing a magnetic bead according to an eighth embodiment of the present application;

10 is a schematic flowchart of a method for making magnetic beads according to the ninth embodiment of the present application;

11 is a schematic flowchart of a method for producing magnetic beads according to the tenth embodiment of the present application;

12 is a schematic flowchart of a method for manufacturing a magnetic bead according to the eleventh embodiment of the present application;

13 is a schematic flowchart of a method for making magnetic beads according to the twelfth embodiment of the present application;

14 is a schematic flowchart of a method for producing magnetic beads according to the thirteenth embodiment of the present application;

15 is a schematic flowchart of a method for manufacturing a magnetic bead according to the fourteenth embodiment of the present application;

16 is a schematic flowchart of a method for making magnetic beads according to the fifteenth embodiment of the present application;

17 is a schematic flowchart of a method for making magnetic beads according to the sixteenth embodiment of the present application;

18 is a schematic flowchart of a method for manufacturing a magnetic bead according to the seventeenth embodiment of the present application;

19 is a schematic flowchart of a method for making magnetic beads according to the eighteenth embodiment of the present application;

Fig. 20 is a schematic flowchart of step S30 in Fig. 1-Fig. 3 and Fig. 5-Fig. 19;

21 is a schematic diagram of the first structure of the magnetic bead proposed in the present application;

Fig. 22 is the second structural schematic diagram of the magnetic bead proposed by the present application;

23 is a schematic diagram of a method for making magnetic microspheres proposed by the present application;

Fig. 24 is the third structural schematic diagram of the magnetic bead proposed by the present application;

FIG. 25 is a schematic diagram of the fourth structure of the magnetic bead proposed by the present application;

Fig. 26 is the magnetic response signal diagram when the magnetic nanoparticles are excessively adsorbed on the surface of the microsphere in the prior art;

Fig. 27 is the magnetic response signal diagram of the magnetic beads of the present application;

Fig. 28 is the fifth structural schematic diagram of the magnetic bead proposed by the present application;

FIG. 29 is a schematic diagram of the sixth structure of the magnetic bead proposed in the present application.

Detailed ways

The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in this application, all other embodiments obtained by those of ordinary skill in the art without creative work, all belong to the scope of protection of this application.

Throughout the specification, unless specifically stated otherwise, terms used herein should be understood as commonly used in the art. Therefore, unless otherwise defined, all 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. In case of conflict, this manual takes precedence.

It should be noted that, in the embodiments of the present application, the terms "comprising", "comprising" or any other variations thereof are intended to cover non-exclusive inclusion, so that a method or device including a series of elements not only includes the explicitly stated elements, but also other elements not expressly listed or inherent to the implementation of the method or apparatus. Without further limitation, an element defined by the phrase "comprising a..." does not preclude the presence of additional related elements (eg, steps in a method or elements in an apparatus) in the method or apparatus that includes the element , where a unit may be part of a circuit, part of a processor, part of a program or software, etc.).

It should be noted that the term "first\second\third" involved in the embodiments of the present application is only to distinguish similar objects, and does not represent a specific ordering of objects. It is understandable that "first\second\third" "Three" may be interchanged in a particular order or sequence where permitted. It should be understood that the "first\second\third" distinctions may be interchanged under appropriate circumstances, so that the embodiments of the present application described herein can be implemented in sequences other than those illustrated or described herein.

Referring to Fig. 1, the first embodiment of the present application proposes a method for making a magnetic bead, the method comprising the following steps:

S10: Provide a magnetic microsphere, the magnetic microsphere includes a core microsphere and first magnetic nanoparticles distributed inside the core microsphere.

The above-mentioned first magnetic nanoparticles may be at least one of lipid-soluble magnetic nanoparticles or water-soluble magnetic nanoparticles.

S20: The magnetic microspheres and the second magnetic nanoparticles are combined by the adsorption technology, so that the second magnetic nanoparticles are coupled to the outer surface of the core microspheres.

Specifically, the second magnetic nanoparticles can be water-soluble magnetic nanoparticles, and the coupling between the core microspheres and the second magnetic nanoparticles can be realized by adsorption, van der Waals force and/or covalent bonding.

Optionally, the outer surface of the core microspheres can be chemically modified with required charged functional groups, wherein the charged functional groups include at least one of charged carboxyl groups, amino groups, sulfonic acid groups or sulfhydryl groups, which are core microspheres. Coupling a second magnetic nanoparticle offers the possibility.

Dissolve the second magnetic nanoparticles in deionized water, add magnetic microspheres, and cross-link the second magnetic nanoparticles with the magnetic microspheres, so that the outer surface of the core microspheres is coupled with the second magnetic nanoparticles .

S30: A gel layer is coated on the outer surface of the magnetic microspheres coupled with the second magnetic nanoparticles, and the gel layer coats at least part of the second magnetic nanoparticles.

Mix the magnetic microspheres freshly prepared in step S10 with water-soluble magnetic nanoparticles coupled on the outer surface with the gel material, add a cross-linking agent, stir the reaction, and after the obtained product is allowed to stand, magnetic separation is performed to remove the residual gel material , to obtain microspheres whose outer surface is covered with a gel layer. Wherein, the gel layer covers at least part of the second magnetic nanoparticles.

Wherein, the material of the gel layer may be at least one of chitosan, sodium alginate, polyacrylic acid, polymethacrylic acid, polyacrylamide, and polyN-polyacrylamide.

S40: The magnetic microspheres and the third magnetic nanoparticles are combined by the swelling technology, so that the third magnetic nanoparticles are dispersed and distributed in the interior of the core microspheres and/or the interior of the gel layer.

Specifically, the third magnetic nanoparticles can be lipid-soluble magnetic nanoparticles, the magnetic microspheres are dispersed in the first medium, a second medium containing the third magnetic nanoparticles is provided, mixed, and then subjected to a swelling reaction (vortex). Dispersion is uniform, and the rotation reaction time is preset), the third magnetic nanoparticles are embedded in the interior of the magnetic microspheres and/or the interior of the gel layer, the supernatant is removed by magnetic separation, and the third magnetic nanoparticles are distributed inside. of microspheres. Wherein, the first medium and the second medium are both swelling media, specifically a single or mixed solvent that can not only disperse and distribute the third magnetic nanoparticles, but also swell magnetic microspheres, for example, the swelling medium can be chloroform, two One or a combination of methyl chloride, ethanol, methanol, hexanediol, n-butanol, isobutanol, n-hexane, cyclohexane, and tetrahydrofuran, but not limited to the above-mentioned substances.

Referring to Fig. 2, the second embodiment of the present application proposes a method for making a magnetic bead, the method comprising the following steps:

S10: Provide a magnetic microsphere, the magnetic microsphere includes a core microsphere and first magnetic nanoparticles distributed inside the core microsphere.

The above-mentioned first magnetic nanoparticles may be at least one of lipid-soluble magnetic nanoparticles or water-soluble magnetic nanoparticles.

S40: the swelling technology is used to combine the magnetic microspheres and the third magnetic nanoparticles, so that the third magnetic nanoparticles are dispersed and distributed inside the core microspheres.

The above-mentioned third magnetic nanoparticles may be fat-soluble magnetic nanoparticles.

S20: The magnetic microspheres and the second magnetic nanoparticles are combined by the adsorption technology, so that the second magnetic nanoparticles are coupled to the outer surface of the core microspheres.

The above-mentioned second magnetic nanoparticles are water-soluble magnetic nanoparticles.

S30: A gel layer is coated on the outer surface of the magnetic microspheres coupled with the second magnetic nanoparticles, and the gel layer coats at least part of the second magnetic nanoparticles.

Different from the first embodiment, the second embodiment “combines magnetic microspheres and third magnetic nanoparticles using swelling technology” occurs before “combining magnetic microspheres and second magnetic nanoparticles using adsorption technology”, so , which can avoid the difficulty of the fat-soluble magnetic nanoparticles entering the interior of the magnetic microspheres due to the hydrophilicity (ie oleophobicity) of the water-soluble magnetic nanoparticles, and greatly improve the encapsulation of the lipid-soluble magnetic nanoparticles in the magnetic microspheres. Buried quantity.

Referring to Fig. 3, the third embodiment of the present application proposes a method for making a magnetic bead, the method comprising the following steps:

S10: Provide a magnetic microsphere, the magnetic microsphere includes a core microsphere and first magnetic nanoparticles distributed inside the core microsphere.

The above-mentioned first magnetic nanoparticles may be at least one of lipid-soluble magnetic nanoparticles or water-soluble magnetic nanoparticles.

S20: The magnetic microspheres and the second magnetic nanoparticles are combined by the adsorption technology, so that the second magnetic nanoparticles are coupled to the outer surface of the core microspheres.

The above-mentioned second magnetic nanoparticles may be water-soluble magnetic nanoparticles.

S40: the swelling technology is used to combine the magnetic microspheres and the third magnetic nanoparticles, so that the third magnetic nanoparticles are dispersed and distributed inside the core microspheres.

The above-mentioned third magnetic nanoparticles may be fat-soluble magnetic nanoparticles.

S30: A gel layer is coated on the outer surface of the magnetic microspheres coupled with the second magnetic nanoparticles, and the gel layer coats at least part of the second magnetic nanoparticles.

Different from the first embodiment, the “combination of magnetic microspheres and third magnetic nanoparticles using swelling technology” in the third embodiment occurs before the coating of the gel layer, so the third magnetic nanoparticles are more likely to enter the core microparticles. Inside the sphere, the embedding quantity of the third magnetic nanoparticles in the magnetic microsphere is greatly increased.

Optionally, the gel layer in step S30 of the first embodiment to the third embodiment can be replaced with a polymer coating layer, and the material of the polymer coating layer is polystyrene, polymethyl methacrylate, poly At least one of ethylene-methyl methacrylate, polyacrylonitrile, polyethylene, polypropylene, and polyethyl acrylate.

Therefore, the structure of the magnetic bead 10 prepared by the above-mentioned first to third embodiments is shown in FIG. 21 or FIG. 22 . The magnetic bead 10 includes: magnetic microspheres 11 , second magnetic nanoparticles 12 , third magnetic Magnetic nanoparticles 13 and coating layer 14 . The magnetic microspheres 11 include core microspheres 111 and first magnetic nanoparticles 112 distributed inside the core microspheres 111 . The second magnetic nanoparticles 12 can be coupled to the outer surface of the magnetic microspheres 11. The coating layer 14 coats the outer surface of the magnetic microspheres 11 , and the coating layer 14 coats at least part of the second magnetic nanoparticles 12 . Optionally, the coating layer 14 covers all the second magnetic nanoparticles 12, and the outer surface of the magnetic beads 10 is a smooth surface. The third magnetic nanoparticles 13 are dispersed and distributed at least inside the core microspheres 111. It can be understood that after the third magnetic nanoparticles 13 enter the interior of the core microspheres 111 through swelling, the third magnetic nanoparticles 13 are embedded in the core microspheres. Inside of ball 111. Wherein, the above-mentioned coating layer 14 can be a gel layer or a polymer coating layer. It can be understood that the third magnetic nanoparticles 13 can be dispersed in the gel layer, and limited by the material of the polymer coating layer, the third magnetic nanoparticles 13 cannot be dispersed in the polymer coating layer. .

Further, step S30 in the above-mentioned first embodiment to third embodiment can be deleted, and the structure of the magnetic bead 10 thus obtained is shown in FIG. 28 . The magnetic bead 10 includes: a magnetic microsphere 11 , a second Magnetic nanoparticles 12 and third magnetic nanoparticles 13 . The magnetic microspheres 11 include core microspheres 111 and first magnetic nanoparticles 112 distributed inside the core microspheres 111. The second magnetic nanoparticles 12 can be coupled to the outer surface of the magnetic microspheres 11. The third magnetic nanoparticles 13 are dispersed and distributed at least inside the core microspheres 111. It can be understood that after the third magnetic nanoparticles 13 enter the interior of the core microspheres 111 through swelling, the third magnetic nanoparticles 13 are embedded in the core microspheres. Inside of ball 111.

Different from the situation in the prior art, the magnetic beads prepared in the embodiments of the present application include: second magnetic nanoparticles coupled to the outer surface of the magnetic microspheres, and at least third magnetic nanoparticles dispersed inside the core microspheres. Particles, as shown in Figure 27, under the premise of avoiding the excessive adsorption of magnetic nanoparticles on the surface of the magnetic microspheres, the magnetic beads of the present application can be enhanced by swelling the third magnetic nanoparticles inside and coupling the second magnetic nanoparticles outside. The magnetic response signal (ie the main group signal) of the magnetic beads can reduce the interference signal caused by impurities, and can reduce the autofluorescence of the magnetic microspheres and improve the detection sensitivity.

In some embodiments, the core microspheres 111 are polymer core microspheres 111 .

Specifically, the material of the core microspheres 111 is a polymer of organic monomer molecules. More specifically, the material of the core microspheres 111 may include polystyrene, polymethyl methacrylate, polyethylene-methyl methacrylate At least one of ester, polyacrylonitrile, polyethylene, polypropylene, and polyethyl acrylate.

In some embodiments, the outer surface of the above-mentioned core microspheres 111 contains charged functional groups, and the charged functional groups include at least one of charged carboxyl groups, amino groups, sulfonic acid groups or thiol groups. The core microspheres 111 are coupled to the second magnetic nanoparticles 12 through charged functional groups.

In some embodiments, the above-mentioned water-soluble magnetic nanoparticles are paramagnetic nanoparticles, wherein the paramagnetic nanoparticles can be selected from ferric tetroxide, ferric oxide, and alloy-type paramagnetism containing nickel or cobalt at least one of the magnetic particles.

In some embodiments, the above-mentioned fat-soluble magnetic nanoparticles are nanoparticles with paramagnetic properties, wherein the nanoparticles with paramagnetic properties can be selected from at least one of ferric oxide or ferric oxide. In addition, the fat-soluble magnetic nanoparticles contain a fat-soluble ligand of at least one of unsaturated fatty acids, saturated fatty acids, unsaturated fatty amines or saturated fatty amines. The above-mentioned lipid-soluble ligands can bind to the surface of the lipid-soluble magnetic nanoparticles, thereby stabilizing the lipid-soluble magnetic nanoparticles. Wherein, the fat-soluble ligand may include at least one of oleic acid, oleylamine or stearic acid.

In some embodiments, the particle size of the magnetic microspheres 11 is 1 μm˜50 μm (eg 1 μm, 5 μm, 10 μm, 20 μm, 50 μm), and the particle size of the third magnetic nanoparticles 13 is 1 nm˜200 nm (eg 1 nm, 5 nm, 50 nm, 100 nm, 200 nm), the particle size of the second magnetic nanoparticles 12 is 1 nm˜200 nm (for example, 1 nm, 5 nm, 50 nm, 100 nm, 200 nm).

In some embodiments, by mass percentage, the magnetic beads 10 include: 50%-99.5% of the magnetic microspheres 11, 0.1%-49.9% of the second magnetic nanoparticles 12, 0.1%-49.9% of the third magnetic Nanoparticles 13. The applicant found that within the above numerical range, the magnetic response of the magnetic beads can be improved, the interference signal caused by impurities can be reduced, and the magnetic microspheres can be reduced under the premise of avoiding excessive adsorption of magnetic nanoparticles on the surface of the magnetic microspheres. Autofluorescence improves detection sensitivity.

Referring to FIG. 4, in some embodiments, the above step S10 includes the following steps:

S11: Mix the first magnetic nanoparticles with organic monomer molecules to form a mixed fluid.

S12: Prepare the mixed fluid into magnetic microspheres.

Specifically, as shown in FIG. 23, the organic monomer molecules can be styrene monomer, methyl methacrylate monomer, ethylene-methyl methacrylate monomer, acrylonitrile monomer, ethylene monomer, propylene monomer At least one of monomer and ethyl acrylate monomer. The first magnetic nanoparticles are added to the solution of organic monomer molecules to form a mixed fluid. The mixed fluid is polymerized to produce magnetic microspheres, wherein the first magnetic nanoparticles are distributed inside the core microspheres. More specifically, the superparamagnetic magnetic nanoparticles, styrene monomer, and octane are mixed and ultrasonically dispersed to obtain a mixed fluid; the self-crosslinkable nonionic water-soluble surfactant and the mixed fluid are added to water, and the mixture is fully mixed. Mix and react at 0-65°C (eg, 0°C, 25°C, 30°C, 50°C, 65°C) to obtain a magnetic nanoparticle-polystyrene core microsphere mixed emulsion. The catalyst solution is added to the magnetic nanoparticle-polystyrene core microsphere mixed emulsion, stirred, left to stand for precipitation, and washed with water to obtain magnetically separated magnetic microspheres.

Referring to Figure 5, the fourth embodiment of the present application proposes a method for making a magnetic bead, the method comprising the following steps:

S10: Provide a magnetic microsphere, the magnetic microsphere includes a core microsphere and first magnetic nanoparticles distributed inside the core microsphere.

S50: Swelling technology is used to combine magnetic microspheres and lipid-soluble fluorescent dyes, so that the lipid-soluble fluorescent dyes are dispersed and distributed inside the core microspheres.

Specifically, the magnetic microspheres are dispersed in the first medium, a third medium containing a fat-soluble fluorescent dye is provided, mixed, and then a swelling reaction is performed (the vortex is dispersed uniformly, and the rotation reaction time is preset), and the fat-soluble fluorescent dye is The dye is embedded in the core microspheres, and the supernatant is magnetically separated to obtain fluorescent microspheres with lipid-soluble fluorescent dyes distributed inside. Wherein, the first medium and the third medium are both swelling media, specifically a single or mixed solvent that can not only disperse and distribute fat-soluble magnetic nanoparticles, but also swell magnetic microspheres. One or a combination of methyl chloride, ethanol, methanol, hexanediol, n-butanol, isobutanol, n-hexane, cyclohexane, and tetrahydrofuran, but not limited to the above-mentioned substances.

Further, step S50 may be performed multiple times, respectively combining lipid-soluble fluorescent dyes with different fluorescence characteristics and magnetic microspheres, or, respectively combining lipid-soluble fluorescent dyes and magnetic microspheres with different concentrations, to obtain a plurality of different fluorescent dyes. Intensity of the microspheres, that is, fluorescently encoded microspheres. Wherein, different liposoluble fluorescent dyes can be mixed in different proportions to prepare different encoded microspheres. It is precisely due to the different ratios of different lipid-soluble fluorescent dyes that endow the fluorescently encoded microspheres with different fluorescent characteristics.

S20: The magnetic microspheres and the second magnetic nanoparticles are combined by the adsorption technology, so that the second magnetic nanoparticles are coupled to the outer surface of the core microspheres.

S30: A gel layer is coated on the outer surface of the magnetic microspheres coupled with the second magnetic nanoparticles, and the gel layer coats at least part of the second magnetic nanoparticles.

S40: The magnetic microspheres and the third magnetic nanoparticles are combined by the swelling technology, so that the third magnetic nanoparticles are dispersed and distributed in the interior of the core microspheres and/or the interior of the gel layer.

Referring to Fig. 6, the fifth embodiment of the present application proposes a method for making a magnetic bead, the method comprising the following steps:

S10: Provide a magnetic microsphere, the magnetic microsphere includes a core microsphere and first magnetic nanoparticles distributed inside the core microsphere.

S20: The magnetic microspheres and the second magnetic nanoparticles are combined by the adsorption technology, so that the second magnetic nanoparticles are coupled to the outer surface of the core microspheres.

S50: Swelling technology is used to combine magnetic microspheres and lipid-soluble fluorescent dyes, so that the lipid-soluble fluorescent dyes are dispersed and distributed inside the core microspheres.

S30: A gel layer is coated on the outer surface of the magnetic microspheres coupled with the second magnetic nanoparticles, and the gel layer coats at least part of the second magnetic nanoparticles.

S40: The magnetic microspheres and the third magnetic nanoparticles are combined by the swelling technology, so that the third magnetic nanoparticles are dispersed and distributed in the interior of the core microspheres and/or the interior of the gel layer.

Referring to Fig. 7, the sixth embodiment of the present application proposes a method for making a magnetic bead, the method comprising the following steps:

S10: Provide a magnetic microsphere, the magnetic microsphere includes a core microsphere and first magnetic nanoparticles distributed inside the core microsphere.

S20: The magnetic microspheres and the second magnetic nanoparticles are combined by the adsorption technology, so that the second magnetic nanoparticles are coupled to the outer surface of the core microspheres.

S30: A gel layer is coated on the outer surface of the magnetic microspheres coupled with the second magnetic nanoparticles, and the gel layer coats at least part of the second magnetic nanoparticles.

S50: Magnetic microspheres and liposoluble fluorescent dyes are combined using swelling technology, so that the liposoluble fluorescent dyes are dispersed and distributed in the interior of the core microspheres and/or the interior of the gel layer.

Further, in the process of embedding the lipid-soluble fluorescent dye into the interior of the core microspheres, the lipid-soluble fluorescent dye may be embedded into the interior of the gel layer.

S40: The magnetic microspheres and the third magnetic nanoparticles are combined by the swelling technology, so that the third magnetic nanoparticles are dispersed and distributed in the interior of the core microspheres and/or the interior of the gel layer.

Referring to Figure 8, the seventh embodiment of the present application proposes a method for making a magnetic bead, the method comprising the following steps:

S10: Provide a magnetic microsphere, the magnetic microsphere includes a core microsphere and first magnetic nanoparticles distributed inside the core microsphere.

S20: The magnetic microspheres and the second magnetic nanoparticles are combined by the adsorption technology, so that the second magnetic nanoparticles are coupled to the outer surface of the core microspheres.

S30: A gel layer is coated on the outer surface of the magnetic microspheres coupled with the second magnetic nanoparticles, and the gel layer coats at least part of the second magnetic nanoparticles.

S40: The magnetic microspheres and the third magnetic nanoparticles are combined by the swelling technology, so that the third magnetic nanoparticles are dispersed and distributed in the interior of the core microspheres and/or the interior of the gel layer.

S50: Magnetic microspheres and liposoluble fluorescent dyes are combined using swelling technology, so that the liposoluble fluorescent dyes are dispersed and distributed in the interior of the core microspheres and/or the interior of the gel layer.

Referring to Fig. 9, the eighth embodiment of the present application proposes a method for making a magnetic bead, the method comprising the following steps:

S10: Provide a magnetic microsphere, the magnetic microsphere includes a core microsphere and first magnetic nanoparticles distributed inside the core microsphere.

S20: The magnetic microspheres and the second magnetic nanoparticles are combined by the adsorption technology, so that the second magnetic nanoparticles are coupled to the outer surface of the core microspheres.

S30: A gel layer is coated on the outer surface of the magnetic microspheres coupled with the second magnetic nanoparticles, and the gel layer coats at least part of the second magnetic nanoparticles.

S60: Using swelling technology to combine magnetic microspheres and lipid-soluble fluorescent dyes, magnetic microspheres and third magnetic nanoparticles simultaneously, so that the lipid-soluble fluorescent dyes and third magnetic nanoparticles are dispersed and distributed inside the core microspheres and/or inside the gel layer.

Specifically, magnetic microspheres are dispersed in a first medium, a second medium containing lipid-soluble magnetic nanoparticles is provided, a third medium containing lipid-soluble fluorescent dyes is provided, mixed, and then a swelling reaction (vortex) is performed. Disperse uniformly, rotate for a preset time), liposoluble magnetic nanoparticles and liposoluble fluorescent dyes are embedded in the interior of the core microspheres, magnetically separate the supernatant, and obtain the core microspheres with lipid-soluble magnetic nanoparticles and lipids distributed inside the core microspheres. Fluorescent magnetic beads for soluble fluorescent dyes.

Further, in the process of embedding the lipid-soluble magnetic nanoparticles and lipid-soluble fluorescent dyes into the interior of the core microspheres, the lipid-soluble magnetic nanoparticles and lipid-soluble fluorescent dyes can be embedded into the interior of the gel layer.

Further, step S60 can be performed multiple times, respectively combining lipid-soluble fluorescent dyes with different fluorescent characteristics and magnetic microspheres, or, respectively combining lipid-soluble fluorescent dyes with different concentrations and magnetic microspheres, and combining lipid-soluble magnetic nanospheres. particles and magnetic microspheres to obtain multiple magnetic beads with different fluorescence intensities, that is, fluorescently encoded magnetic beads. Among them, different liposoluble fluorescent dyes can be mixed in different proportions to prepare different encoded magnetic beads. It is precisely because of the different ratios of different lipid-soluble fluorescent dyes that the prepared fluorescently encoded magnetic beads have different fluorescent characteristics.

Referring to Figure 10, the ninth embodiment of the present application proposes a method for making a magnetic bead, the method comprising the following steps:

S10: Provide a magnetic microsphere, the magnetic microsphere includes a core microsphere and first magnetic nanoparticles distributed inside the core microsphere.

S40: the swelling technology is used to combine the magnetic microspheres and the third magnetic nanoparticles, so that the third magnetic nanoparticles are dispersed and distributed inside the core microspheres.

S50: Swelling technology is used to combine magnetic microspheres and lipid-soluble fluorescent dyes, so that the lipid-soluble fluorescent dyes are dispersed and distributed inside the core microspheres.

S20: The magnetic microspheres and the second magnetic nanoparticles are combined by the adsorption technology, so that the second magnetic nanoparticles are coupled to the outer surface of the core microspheres.

S30: A gel layer is coated on the outer surface of the magnetic microspheres coupled with the second magnetic nanoparticles, and the gel layer coats at least part of the second magnetic nanoparticles.

Referring to Figure 11, the tenth embodiment of the present application proposes a method for making magnetic beads, the method comprising the following steps:

S10: Provide a magnetic microsphere, the magnetic microsphere includes a core microsphere and first magnetic nanoparticles distributed inside the core microsphere.

S40: the swelling technology is used to combine the magnetic microspheres and the third magnetic nanoparticles, so that the third magnetic nanoparticles are dispersed and distributed inside the core microspheres.

S20: The magnetic microspheres and the second magnetic nanoparticles are combined by the adsorption technology, so that the second magnetic nanoparticles are coupled to the outer surface of the core microspheres.

S50: Swelling technology is used to combine magnetic microspheres and lipid-soluble fluorescent dyes, so that the lipid-soluble fluorescent dyes are dispersed and distributed inside the core microspheres.

S30: A gel layer is coated on the outer surface of the magnetic microspheres coupled with the second magnetic nanoparticles, and the gel layer coats at least part of the second magnetic nanoparticles.

Referring to Figure 12, the eleventh embodiment of the present application proposes a method for making a magnetic bead, the method comprising the following steps:

S10: Provide a magnetic microsphere, the magnetic microsphere includes a core microsphere and first magnetic nanoparticles distributed inside the core microsphere.

S50: Swelling technology is used to combine magnetic microspheres and lipid-soluble fluorescent dyes, so that the lipid-soluble fluorescent dyes are dispersed and distributed inside the core microspheres.

S40: the swelling technology is used to combine the magnetic microspheres and the third magnetic nanoparticles, so that the third magnetic nanoparticles are dispersed and distributed inside the core microspheres.

S20: The magnetic microspheres and the second magnetic nanoparticles are combined by the adsorption technology, so that the second magnetic nanoparticles are coupled to the outer surface of the core microspheres.

S30: A gel layer is coated on the outer surface of the magnetic microspheres coupled with the second magnetic nanoparticles, and the gel layer coats at least part of the second magnetic nanoparticles.

Referring to Figure 13, the twelfth embodiment of the present application proposes a method for making a magnetic bead, the method comprising the following steps:

S10: Provide a magnetic microsphere, the magnetic microsphere includes a core microsphere and first magnetic nanoparticles distributed inside the core microsphere.

S40: the swelling technology is used to combine the magnetic microspheres and the third magnetic nanoparticles, so that the third magnetic nanoparticles are dispersed and distributed inside the core microspheres.

S20: The magnetic microspheres and the second magnetic nanoparticles are combined by the adsorption technology, so that the second magnetic nanoparticles are coupled to the outer surface of the core microspheres.

S30: A gel layer is coated on the outer surface of the magnetic microspheres coupled with the second magnetic nanoparticles, and the gel layer coats at least part of the second magnetic nanoparticles.

S50: Magnetic microspheres and liposoluble fluorescent dyes are combined using swelling technology, so that the liposoluble fluorescent dyes are dispersed and distributed in the interior of the core microspheres and/or the interior of the gel layer.

Referring to Figure 14, the thirteenth embodiment of the present application proposes a method for making magnetic beads, the method comprising the following steps:

S10: Provide a magnetic microsphere, the magnetic microsphere includes a core microsphere and first magnetic nanoparticles distributed inside the core microsphere.

S60: The swelling technology is used to combine magnetic microspheres and lipid-soluble fluorescent dyes, magnetic microspheres and third magnetic nanoparticles, so that the lipid-soluble fluorescent dyes and the third magnetic nanoparticles are dispersed and distributed inside the core microspheres.

S20: The magnetic microspheres and the second magnetic nanoparticles are combined by the adsorption technology, so that the second magnetic nanoparticles are coupled to the outer surface of the core microspheres.

S30: A gel layer is coated on the outer surface of the magnetic microspheres coupled with the second magnetic nanoparticles, and the gel layer coats at least part of the second magnetic nanoparticles.

Referring to Figure 15, the fourteenth embodiment of the present application proposes a method for making magnetic beads, the method comprising the following steps:

S10: Provide a magnetic microsphere, the magnetic microsphere includes a core microsphere and first magnetic nanoparticles distributed inside the core microsphere.

S50: Swelling technology is used to combine magnetic microspheres and lipid-soluble fluorescent dyes, so that the lipid-soluble fluorescent dyes are dispersed and distributed inside the core microspheres.

S20: The magnetic microspheres and the second magnetic nanoparticles are combined by the adsorption technology, so that the second magnetic nanoparticles are coupled to the outer surface of the core microspheres.

S40: the swelling technology is used to combine the magnetic microspheres and the third magnetic nanoparticles, so that the third magnetic nanoparticles are dispersed and distributed inside the core microspheres.

S30: A gel layer is coated on the outer surface of the magnetic microspheres coupled with the second magnetic nanoparticles, and the gel layer coats at least part of the second magnetic nanoparticles.

Referring to Figure 16, a fifteenth embodiment of the present application proposes a method for making magnetic beads, the method comprising the following steps:

S10: Provide a magnetic microsphere, the magnetic microsphere includes a core microsphere and first magnetic nanoparticles distributed inside the core microsphere.

S20: The magnetic microspheres and the second magnetic nanoparticles are combined by the adsorption technology, so that the second magnetic nanoparticles are coupled to the outer surface of the core microspheres.

S40: the swelling technology is used to combine the magnetic microspheres and the third magnetic nanoparticles, so that the third magnetic nanoparticles are dispersed and distributed inside the core microspheres.

S50: Swelling technology is used to combine magnetic microspheres and lipid-soluble fluorescent dyes, so that the lipid-soluble fluorescent dyes are dispersed and distributed inside the core microspheres.

S30: A gel layer is coated on the outer surface of the magnetic microspheres coupled with the second magnetic nanoparticles, and the gel layer coats at least part of the second magnetic nanoparticles.

Referring to Figure 17, the sixteenth embodiment of the present application proposes a method for making magnetic beads, the method comprising the following steps:

S10: Provide a magnetic microsphere, the magnetic microsphere includes a core microsphere and first magnetic nanoparticles distributed inside the core microsphere.

S20: The magnetic microspheres and the second magnetic nanoparticles are combined by the adsorption technology, so that the second magnetic nanoparticles are coupled to the outer surface of the core microspheres.

S50: Swelling technology is used to combine magnetic microspheres and lipid-soluble fluorescent dyes, so that the lipid-soluble fluorescent dyes are dispersed and distributed inside the core microspheres.

S40: the swelling technology is used to combine the magnetic microspheres and the third magnetic nanoparticles, so that the third magnetic nanoparticles are dispersed and distributed inside the core microspheres.

S30: A gel layer is coated on the outer surface of the magnetic microspheres coupled with the second magnetic nanoparticles, and the gel layer coats at least part of the second magnetic nanoparticles.

Referring to Figure 18, the seventeenth embodiment of the present application proposes a method for making a magnetic bead, the method comprising the following steps:

S10: Provide a magnetic microsphere, the magnetic microsphere includes a core microsphere and first magnetic nanoparticles distributed inside the core microsphere.

S20: The magnetic microspheres and the second magnetic nanoparticles are combined by the adsorption technology, so that the second magnetic nanoparticles are coupled to the outer surface of the core microspheres.

S40: the swelling technology is used to combine the magnetic microspheres and the third magnetic nanoparticles, so that the third magnetic nanoparticles are dispersed and distributed inside the core microspheres.

S30: A gel layer is coated on the outer surface of the magnetic microspheres coupled with the second magnetic nanoparticles, and the gel layer coats at least part of the second magnetic nanoparticles.

S50: Magnetic microspheres and liposoluble fluorescent dyes are combined using swelling technology, so that the liposoluble fluorescent dyes are dispersed and distributed in the interior of the core microspheres and/or the interior of the gel layer.

Referring to Figure 19, the eighteenth embodiment of the present application proposes a method for making magnetic beads, the method comprising the following steps:

S10: Provide a magnetic microsphere, the magnetic microsphere includes a core microsphere and first magnetic nanoparticles distributed inside the core microsphere.

S20: The magnetic microspheres and the second magnetic nanoparticles are combined by the adsorption technology, so that the second magnetic nanoparticles are coupled to the outer surface of the core microspheres.

S60: The swelling technology is used to combine magnetic microspheres and lipid-soluble fluorescent dyes, magnetic microspheres and third magnetic nanoparticles, so that the lipid-soluble fluorescent dyes and the third magnetic nanoparticles are dispersed and distributed inside the core microspheres.

S30: A gel layer is coated on the outer surface of the magnetic microspheres coupled with the second magnetic nanoparticles, and the gel layer coats at least part of the second magnetic nanoparticles.

Optionally, the gel layer in step S30 of the fourth embodiment to the eighteenth embodiment can be replaced with a polymer coating layer, and the material of the polymer coating layer is polystyrene, polymethyl methacrylate, Polyethylene - at least one of methyl methacrylate, polyacrylonitrile, polyethylene, polypropylene, polyethyl acrylate.

Thus, the magnetic bead structures prepared by the fourth embodiment to the eighteenth embodiment or their alternatives are shown in FIG. 24 or 25. The magnetic bead 10 includes: magnetic microspheres 11, liposoluble fluorescent dyes 15, The second magnetic nanoparticles 12 , the third magnetic nanoparticles 13 and the coating layer 14 . The magnetic microspheres 11 include core microspheres 111 and first magnetic nanoparticles 112 distributed inside the core microspheres 111 . Among them, the lipid-soluble fluorescent dye 15 and the third magnetic nanoparticles 13 are dispersed and distributed inside the core microsphere 111. It can be understood that, after entering the interior of the magnetic microsphere 11 through swelling, the lipid-soluble fluorescent dye 15, the third magnetic The nanoparticles 13 are embedded inside the core microspheres 111 . The second magnetic nanoparticles 12 may be coupled to the outer surface of the core microspheres 111. The coating layer 14 is coated on the outer surface of the magnetic microspheres 11, and the coating layer 14 is coated with at least part of the second magnetic nanoparticles 12. Optionally, the coating layer 14 covers all the second magnetic nanoparticles 12, and the outer surface of the magnetic beads 10 is a smooth surface. Wherein, the above-mentioned coating layer 14 may be a gel layer or a polymer coating layer. It can be understood that the third magnetic nanoparticles 13 and the liposoluble fluorescent dyes 15 can be dispersed in the gel layer, but limited by the material of the polymer coating layer, the third magnetic nanoparticles 13 and the liposoluble fluorescent dyes 15 is not dispersible in the interior of the polymer coating.

Further, step S30 in the fourth embodiment to the tenth embodiment above can be deleted, and the structure of the magnetic bead 10 obtained therefrom is shown in FIG. 28 . The magnetic bead 10 includes: Fluorescent dye 15 , second magnetic nanoparticles 12 and third magnetic nanoparticles 13 . The magnetic microspheres 11 include core microspheres 111 and first magnetic nanoparticles 112 distributed inside the core microspheres 111 . Among them, the lipid-soluble fluorescent dye 15 and the third magnetic nanoparticles 13 are dispersed and distributed inside the core microsphere 111. It can be understood that, after entering the interior of the magnetic microsphere 11 through swelling, the lipid-soluble fluorescent dye 15, the third magnetic The nanoparticles 13 are embedded inside the core microspheres 111 . The second magnetic nanoparticles 12 may be coupled to the outer surface of the core microspheres 111.

Referring to Figure 20, further, in the above-mentioned first embodiment to the eighteenth embodiment, step S30 specifically includes the following steps:

S31: Mix the magnetic microspheres coupled with the second magnetic nanoparticles and the gel material, add a cross-linking agent, and perform a cross-linking reaction, so that the outer surfaces of the magnetic microspheres coupled with the second magnetic nanoparticles are Coat the gel layer.

When the gel material is chitosan, the cross-linking agent can be glutaraldehyde, wherein the mass ratio of chitosan to magnetic microspheres is 0.01-20%, and chitosan interacts with glutaraldehyde through electrostatic interaction. A chemical reaction occurs to produce cross-linking.

Further, the operating temperature of step S20 in the above embodiment is 0-100°C (for example, 0°C, 25°C, 30°C, 50°C, 65°C, 100°C), and the operating temperature of step S30 is 20-65°C ( For example, 20°C, 30°C, 50°C, 65°C), the operating temperature of step S40 is 0-65°C (for example, 0°C, 25°C, 30°C, 50°C, 65°C), and the operating temperature of step S50 is 0-65°C 65°C (eg 0°C, 25°C, 30°C, 50°C, 65°C). Within the above temperature range, the increase in autofluorescence of the magnetic microspheres caused by heating can be effectively avoided, thereby improving the detection sensitivity.

Preferably, the operating temperature of step S20 in the above embodiment is 0-30°C (eg 0°C, 25°C, 30°C), and the operating temperature of step S30 is 20-30°C (eg 20°C, 25°C, 30°C) ), the operating temperature of step S40 is 0-30°C (eg 0°C, 25°C, 30°C), and the operating temperature of step S50 is 0-30°C (eg 0°C, 25°C, 30°C). Within the above operating temperature range, the autofluorescence increase of the non-magnetic and magnetic microspheres caused by heating can be effectively avoided, thereby improving the detection sensitivity.

More preferably, the operating temperature of step S20 in the above-mentioned embodiment is room temperature (23 ℃ ± 2 ℃), the operating temperature of step S30 is room temperature (23 ℃ ± 2 ℃), and the operating temperature of step S40 is room temperature (23 ℃ ± 2 ℃). 2° C.), and the operating temperature of step S50 is room temperature (23° C.±2° C.). Within the above operating temperature range, the autofluorescence increase of the non-magnetic and magnetic microspheres caused by heating can be effectively avoided, thereby improving the detection sensitivity.

In the several embodiments provided in this application, it should be understood that the disclosed method and device may be implemented in other manners. For example, the device implementations described above are only illustrative. For example, the division of the modules or units is only a logical function division. In actual implementation, there may be other divisions. For example, multiple units or components may be Incorporation may either be integrated into another system, or some features may be omitted, or not implemented.

The units described as separate components may or may not be physically separated, and components shown as units may or may not be physical units, that is, may be located in one place, or may be distributed to multiple network units. Some or all of the units may be selected according to actual needs to achieve the purpose of the solution in this implementation manner.

In addition, each functional unit in each embodiment of the present application may be integrated into one processing unit, or each unit may exist physically alone, or two or more units may be integrated into one unit. The above-mentioned integrated units can be implemented in the form of hardware, and can also be implemented in the form of software functional units.

The above are only the embodiments of the present application, and are not intended to limit the scope of the patent of the present application. Any equivalent structure or equivalent process transformation made by using the contents of the description and drawings of the present application, or directly or indirectly applied to other related technologies Fields are similarly included within the scope of patent protection of this application.

Claims (19)

1. A magnetic bead, comprising:

the magnetic microsphere comprises a core microsphere and first magnetic nanoparticles distributed in the core microsphere;

a second magnetic nanoparticle coupled to the outer surface of the core microsphere

And the third magnetic nano-particles are at least dispersed and distributed in the core microsphere.

2. The magnetic bead of claim 1, further comprising:

and the coating layer is coated on the outer surface of the magnetic microsphere and at least partially coats the second magnetic nanoparticles.

3. The magnetic bead of claim 2,

the coating layer is a gel layer or a polymer coating layer;

and the third magnetic nanoparticles are dispersed in the gel layer.

4. A magnetic bead as defined in claim 3,

the gel layer is made of at least one of chitosan, sodium alginate, polyacrylic acid, polymethacrylic acid, polyacrylamide and poly-N-substituted acrylamide;

the polymer coating layer is made of at least one of polystyrene, polymethyl methacrylate, polyethylene-methyl methacrylate, polyacrylonitrile, polyethylene, polypropylene and polyethylacrylate.

5. The magnetic bead of claim 1, wherein the core microsphere is a polymeric core microsphere.

6. The magnetic bead of claim 1, wherein an outer surface of the core microsphere comprises a charged functional group, and wherein the core microsphere is coupled to the second magnetic nanoparticle via the charged functional group.

7. The magnetic bead of claim 1, wherein the first magnetic nanoparticle is at least one of a fat-soluble magnetic nanoparticle or a water-soluble magnetic nanoparticle.

8. The magnetic bead of claim 1, wherein the second magnetic nanoparticle is a water-soluble magnetic nanoparticle and the third magnetic nanoparticle is a fat-soluble magnetic nanoparticle.

9. The magnetic bead of claim 7 or 8,

the fat-soluble magnetic nanoparticles are paramagnetic nanoparticles, and contain at least one fat-soluble ligand of unsaturated fatty acid, saturated fatty acid, unsaturated fatty amine or saturated fatty amine.

10. The magnetic bead of claim 7 or 8,

the water-soluble magnetic nanoparticles are paramagnetic nanoparticles.

11. The magnetic bead of claim 1, further comprising:

and the fat-soluble fluorescent dye is at least dispersed and distributed in the core microsphere.

12. The magnetic bead of claim 3, further comprising:

and the fat-soluble fluorescent dye is at least dispersed and distributed in the gel layer.

13. The magnetic bead of claim 1, wherein the magnetic bead is a magnetic bead,

the magnetic bead comprises the following components in percentage by mass: 50% -99.5% of the magnetic microspheres, 0.1% -49.9% of the second magnetic nanoparticles and 0.1% -49.9% of the third magnetic nanoparticles.

14. A method of making a magnetic bead, the method comprising:

providing a magnetic microsphere, wherein the magnetic microsphere comprises a core microsphere and first magnetic nanoparticles distributed in the core microsphere;

combining the magnetic microsphere and a second magnetic nanoparticle using an adsorption technique such that the second magnetic nanoparticle is coupled to the outer surface of the core microsphere;

wherein prior to the step of combining the magnetic microsphere and the second magnetic nanoparticle using an adsorption technique such that the second magnetic nanoparticle is coupled to the outer surface of the core microsphere, or after the step of combining the magnetic microsphere and the second magnetic nanoparticle using an adsorption technique such that the second magnetic nanoparticle is coupled to the outer surface of the core microsphere, the method further comprises: and combining the magnetic microspheres and the third magnetic nanoparticles by adopting a swelling technology, so that the third magnetic nanoparticles are dispersed in the core microspheres.

15. The method of claim 14, wherein after the step of combining the magnetic microsphere and the second magnetic nanoparticle using an adsorption technique such that the second magnetic nanoparticle is coupled to the outer surface of the core microsphere, the method further comprises: and coating a polymer coating layer on the outer surface of the magnetic microsphere coupled with the second magnetic nanoparticle, wherein the gel layer coats at least part of the second magnetic nanoparticle.

16. The method of claim 14, wherein after the step of combining the magnetic microsphere and the second magnetic nanoparticle using an adsorption technique such that the second magnetic nanoparticle is coupled to the outer surface of the core microsphere, the method further comprises: coating a gel layer on the outer surface of the magnetic microsphere coupled with the second magnetic nanoparticle, wherein the gel layer coats at least part of the second magnetic nanoparticle.

17. The method of claim 14, wherein the step of providing a magnetic microsphere comprises:

mixing the first magnetic nanoparticles with organic monomer molecules to form a mixed fluid;

preparing the mixed fluid into the magnetic microspheres.

18. The method of claim 16,

the magnetic microspheres and the second magnetic nanoparticles are combined by adopting an adsorption technology, so that the operating temperature of coupling the second magnetic nanoparticles to the outer surfaces of the core microspheres is 0-100 ℃;

coating a gel layer on the outer surface of the magnetic microsphere coupled with the second magnetic nanoparticles, wherein the operation temperature of coating at least part of the second magnetic nanoparticles by the gel layer is 20-65 ℃;

and combining the magnetic microspheres and the third magnetic nanoparticles by adopting a swelling technology, so that the operating temperature of the third magnetic nanoparticles dispersed in the core microspheres and/or the gel layer is 0-65 ℃.

19. The method of claim 14, further comprising:

and combining the magnetic microspheres and the fat-soluble fluorescent dye by adopting a swelling technology, so that the fat-soluble fluorescent dye is dispersed and distributed in the core microspheres.

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