CN115127978A - Fluorescent magnetic beads and method of making the same - Google Patents

Abstract

The application relates to the technical field of magnetic bead modification, and particularly discloses a fluorescent magnetic bead and a manufacturing method thereof, wherein the fluorescent magnetic bead comprises the following components: a magnetic bead body; fat-soluble fluorescent dye molecules are dispersed in the magnetic bead body; and the gel layer is coated on the outer surface of the magnetic bead body, wherein water-soluble fluorescent dye molecules are combined on the outer surface and/or the inner part of the gel layer. By the method, the fluorescent signal of the fluorescent magnetic bead can be improved, and the detection sensitivity can be improved.

Description

Fluorescent magnetic beads and method of making the same

technical field

The present application relates to the technical field of magnetic bead modification, and in particular, to a fluorescent 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. The 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 38, the impurities on the magnetic microspheres will increase, and the signal of the impurities will seriously interfere with the target 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 provide a fluorescent magnetic bead and a manufacturing method thereof, which can improve the fluorescent signal of the fluorescent magnetic bead and improve the detection sensitivity.

A first aspect of the present application provides a fluorescent magnetic bead, which includes: a magnetic bead body; lipid-soluble fluorescent dye molecules dispersed in the interior of the magnetic bead body; a gel layer coated on the outer surface of the magnetic bead body, Wherein, water-soluble fluorescent dye molecules are combined on the outer surface and/or inside of the gel layer.

A second aspect of the present application provides a method for producing fluorescent magnetic beads, the method comprising: providing a method for producing fluorescent magnetic beads, using adsorption technology to combine microspheres and water-soluble magnetic nanoparticles to couple the water-soluble magnetic nanoparticles on the outer surface of the microsphere; after the step of combining the microsphere and the water-soluble magnetic nanoparticles by adsorption technology, so that the water-soluble magnetic nanoparticles are coupled to the outer surface of the microsphere, the method further includes: after the water-soluble magnetic nanoparticles are coupled with the water-soluble magnetic nanoparticles The outer surface of the microsphere of the nanoparticle is covered with a gel layer, and the gel layer covers at least part of the water-soluble magnetic nanoparticles, and the adsorption technology is used to combine the microsphere and the water-soluble fluorescent dye molecule, so that the water-soluble fluorescent dye molecule is combined with the water-soluble fluorescent dye molecule. The outer surface of the gel layer, or, combine water-soluble fluorescent dye molecules with gel molecules to obtain fluorescent dye-gel complexes, and coat the fluorescent dye-gel complexes on coupled water-soluble magnetic nanoparticles The outer surface of the microspheres is formed to form a gel layer with water-soluble fluorescent dye molecules incorporated therein, wherein the gel layer coats at least part of the water-soluble magnetic nanoparticles; particles, after the step of coupling the water-soluble magnetic nanoparticles to the outer surface of the microspheres, or, using an adsorption technique to combine the microspheres and the water-soluble magnetic nanoparticles, so that the water-soluble magnetic nanoparticles are coupled to the outer surface of the microspheres Before the step of the method, the method further includes: combining the microspheres and the lipid-soluble fluorescent dye molecules by a swelling technique, so that the lipid-soluble fluorescent dye molecules are dispersed and distributed in the interior of the microspheres; using the swelling technique to combine the microspheres and the lipid-soluble fluorescent dye molecules, so that Before the step of dispersing and distributing the lipid-soluble fluorescent dye molecules in the interior of the microspheres, or, after the step of combining the microspheres with the lipid-soluble fluorescent dye molecules using a swelling technique, so that the lipid-soluble fluorescent dye molecules are dispersed and distributed in the interior of the microspheres, or , while using the swelling technology to combine the microspheres and the lipid-soluble fluorescent dye molecules, so that the lipid-soluble fluorescent dye molecules are dispersed in the interior of the microspheres, the method also includes: using the swelling technology to combine the microspheres and the lipid-soluble magnetic nanoparticles, so that the lipids The soluble magnetic nanoparticles are dispersed in the interior of the microspheres and/or the interior of the gel layer.

Different from the situation in the prior art, the fluorescent magnetic beads of the present application include: a magnetic bead body, a lipid-soluble fluorescent dye molecule dispersed and distributed inside the magnetic bead body, a gel layer coated on the outer surface of the magnetic bead body, and a gel layer bound to the magnetic bead body. The water-soluble fluorescent dye molecules on the outer surface and/or the inner part of the adhesive layer can improve the fluorescent signal of the fluorescent magnetic beads and improve the detection sensitivity.

Additional aspects and advantages of the present application will be set forth, in part, from the following description, and in part will become 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 producing fluorescent magnetic beads according to the first embodiment of the present application;

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

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

4 is a schematic flowchart of a method for manufacturing fluorescent magnetic beads according to the fourth embodiment of the present application;

5 is a schematic flowchart of a method for manufacturing fluorescent magnetic beads according to the fifth embodiment of the present application;

6 is a schematic flowchart of a method for manufacturing fluorescent magnetic beads proposed in the sixth embodiment of the present application;

7 is a schematic flowchart of a method for manufacturing fluorescent magnetic beads according to the seventh embodiment of the present application;

8 is a schematic flowchart of a method for manufacturing fluorescent magnetic beads according to the eighth embodiment of the present application;

9 is a schematic flowchart of a method for producing fluorescent magnetic beads according to the ninth embodiment of the present application;

10 is a schematic flowchart of a method for manufacturing fluorescent magnetic beads according to the tenth embodiment of the present application;

11 is a schematic flowchart of a method for manufacturing fluorescent magnetic beads according to the eleventh embodiment of the present application;

12 is a schematic flowchart of a method for manufacturing fluorescent magnetic beads according to the twelfth embodiment of the present application;

13 is a schematic flowchart of a method for manufacturing fluorescent magnetic beads according to the thirteenth embodiment of the present application;

14 is a schematic flowchart of a method for manufacturing fluorescent magnetic beads according to the fourteenth embodiment of the present application;

15 is a schematic flowchart of a method for manufacturing fluorescent magnetic beads according to the fifteenth embodiment of the present application;

16 is a schematic flowchart of a method for manufacturing fluorescent magnetic beads according to the sixteenth embodiment of the present application;

17 is a schematic flowchart of a method for manufacturing fluorescent magnetic beads according to the seventeenth embodiment of the present application;

18 is a schematic flowchart of a method for manufacturing fluorescent magnetic beads according to the eighteenth embodiment of the present application;

19 is a schematic flowchart of a method for producing fluorescent magnetic beads according to the nineteenth embodiment of the present application;

20 is a schematic flowchart of a method for manufacturing fluorescent magnetic beads according to the twentieth embodiment of the present application;

21 is a schematic flowchart of a method for manufacturing fluorescent magnetic beads according to the twenty-first embodiment of the present application;

22 is a schematic flowchart of a method for manufacturing fluorescent magnetic beads according to the twenty-second embodiment of the present application;

23 is a schematic flowchart of a method for manufacturing fluorescent magnetic beads according to the twenty-third embodiment of the present application;

24 is a schematic flowchart of a method for manufacturing fluorescent magnetic beads according to the twenty-fourth embodiment of the present application;

25 is a schematic flowchart of a method for manufacturing fluorescent magnetic beads according to the twenty-fifth embodiment of the present application;

26 is a schematic flowchart of a method for manufacturing fluorescent magnetic beads according to the twenty-sixth embodiment of the present application;

27 is a schematic flowchart of a method for manufacturing fluorescent magnetic beads according to the twenty-seventh embodiment of the present application;

28 is a schematic flowchart of a method for manufacturing fluorescent magnetic beads according to the twenty-eighth embodiment of the present application;

29 is a schematic flowchart of a method for manufacturing fluorescent magnetic beads according to the twenty-ninth embodiment of the present application;

30 is a schematic flowchart of a method for manufacturing fluorescent magnetic beads according to the thirtieth embodiment of the present application;

31 is a schematic diagram of the first structure of the fluorescent magnetic beads proposed in this application;

32 is a schematic diagram of the first structure of the fluorescent magnetic beads proposed in the present application;

33 is a schematic diagram of the first structure of the fluorescent magnetic beads proposed in the present application;

34 is a schematic diagram of the first structure of the fluorescent magnetic beads proposed in the present application;

FIG. 35 is a schematic diagram of the first structure of the microsphere body 111 in FIG. 32 or 33;

FIG. 36 is a schematic diagram of the second structure of the microsphere body 111 in FIG. 32 or 33;

FIG. 37 is a third structural schematic diagram of the microsphere body 111 in FIG. 32 or 33;

Fig. 38 is the magnetic response signal graph when the magnetic nanoparticles are excessively adsorbed on the surface of the microsphere in the prior art;

FIG. 39 is a graph of the magnetic response signal of the fluorescent magnetic beads of 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 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 the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present application.

Throughout the specification, unless specifically stated otherwise, terms used herein are to 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, the present specification 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.

As shown in FIG. 1 , the first embodiment of the present application proposes a method for manufacturing fluorescent magnetic beads, and the method includes the following steps:

S10: Combining the microspheres and the water-soluble magnetic nanoparticles using adsorption technology, so that the water-soluble magnetic nanoparticles are coupled to the outer surface of the microspheres.

Specifically, the coupling between the microspheres and the water-soluble magnetic nanoparticles is achieved by adsorption, van der Waals forces and/or covalent bonding.

Optionally, the outer surface of the 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 thiol groups, which are used for coupling water-soluble. Magnetic nanoparticles offer the possibility.

Dissolving the water-soluble magnetic nanoparticles in deionized water, adding microspheres, and rotating the water-soluble magnetic nanoparticles and the microspheres to cross-link the water-soluble magnetic nanoparticles to obtain the microspheres with the outer surfaces coupled with the water-soluble magnetic nanoparticles.

S21: Coating a gel layer on the outer surface of the microspheres coupled with water-soluble magnetic nanoparticles, and the gel layer coating at least part of the water-soluble magnetic nanoparticles, and combining the microspheres and the water-soluble fluorescent dye molecules by adsorption technology , so that the water-soluble fluorescent dye molecules are bound to the outer surface of the gel layer.

Specifically, the microspheres with water-soluble magnetic nanoparticles coupled to the outer surface prepared in step S10 are mixed with gel molecules, a cross-linking agent is added, and the reaction is stirred. After the product is allowed to stand, magnetic separation is performed to remove the residual gel. molecules to obtain microspheres whose outer surfaces are coated with a gel layer.

Wherein, the material of the gel layer (ie, the gel molecules) may include at least one of chitosan, sodium alginate, polyacrylic acid, polymethacrylic acid, polyacrylamide, and polyN-polyacrylamide.

The coupling between the gel layer and the water-soluble fluorescent dye molecules is achieved by adsorption, van der Waals force and/or covalent bonding. Wherein, the outer surface of the gel layer can be chemically modified with required charged functional groups, wherein the charged functional groups can include at least one of charged carboxyl groups, amino groups, sulfonic acid groups or sulfhydryl groups, which are water-soluble for subsequent coupling. Sexual fluorescent dye molecules offer the possibility. The water-soluble fluorescent dye molecules may have active groups, and the water-soluble fluorescent dye molecules are combined with the gel molecules through the active groups. The active group includes at least one of N-hydroxysuccinimide group, carboxyl group, sulfhydryl group, epoxy group or tosyl group, which provides the possibility for water-soluble fluorescent dye molecules to combine with gel molecules.

S30: Combining the microspheres and the lipid-soluble fluorescent dye molecules by means of swelling technology, so that the lipid-soluble fluorescent dye molecules are dispersed and distributed in the interior of the microspheres and/or the interior of the gel layer.

Specifically, the microspheres are dispersed in the first medium, a second medium containing lipid-soluble fluorescent dye molecules is provided, mixed, and then a swelling reaction is performed (uniform vortex dispersion, rotation reaction preset time), the lipid-soluble fluorescent dye Molecules are embedded in the interior of the microspheres, and the supernatant is magnetically separated to obtain microspheres with lipid-soluble fluorescent dye molecules distributed inside. Wherein, the first medium and the second medium are both swelling media, specifically a single or mixed solvent that can not only disperse the lipid-soluble fluorescent dye molecules, but also swell the microspheres, for example, the swelling media can be chloroform, dichloromethane , ethanol, methanol, n-butanol, isobutanol, n-hexane, cyclohexane, tetrahydrofuran, but not limited to the above substances.

Wherein, the microspheres may be at least one of non-crosslinked microspheres, crosslinked porous microspheres or hollow mesoporous microspheres. Cross-linked porous microspheres and hollow mesoporous microspheres have the characteristics of high porosity and high specific surface area, therefore, the embedding capacity of lipid-soluble fluorescent dye molecules in the microspheres can be improved, thereby increasing the fluorescence signal value of the fluorescent magnetic beads.

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

Further, step S30 may be performed multiple times, respectively combining lipid-soluble fluorescent dye molecules and microspheres with different fluorescence characteristics, or combining lipid-soluble fluorescent dye molecules and microspheres with different concentrations, respectively, to obtain a plurality of different fluorescence intensities. Microspheres, namely fluorescently encoded microspheres. Wherein, different lipid-soluble fluorescent dye molecules can be mixed in different proportions to prepare different encoded microspheres. It is precisely due to the different ratios of different lipid-soluble fluorescent dye molecules that endow the prepared fluorescently encoded microspheres with different fluorescent characteristics.

S40: Combining the microspheres and the liposoluble magnetic nanoparticles using swelling technology, so that the liposoluble magnetic nanoparticles are dispersed and distributed in the interior of the microspheres and/or the interior of the gel layer.

Specifically, the microspheres are dispersed in the first medium, a third medium containing lipid-soluble magnetic nanoparticles is provided, mixed, and then a swelling reaction is performed (uniform vortex dispersion, rotation reaction preset time), the lipid-soluble magnetic nanoparticles The particles are embedded in the interior of the microspheres, and the supernatant is magnetically separated to obtain fluorescent microspheres with lipid-soluble magnetic nanoparticles 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 the fat-soluble magnetic nanoparticles, but also swell the microspheres, for example, the swelling medium can be chloroform, dichloromethane , ethanol, methanol, n-butanol, isobutanol, n-hexane, cyclohexane, tetrahydrofuran, but not limited to the above substances.

Further, in the process of embedding the lipid-soluble magnetic nanoparticles into the inside of the microspheres, the lipid-soluble magnetic nanoparticles can be embedded into the inside of the gel layer.

When the microspheres are cross-linked porous microspheres or hollow mesoporous microspheres, they have the characteristics of high porosity and high specific surface area. Therefore, the embedding capacity of the lipid-soluble magnetic nanoparticles in the microspheres can be improved, thereby improving the microspheres. Magnetic responsivity of the ball.

Different from the situation in the prior art, the fluorescent magnetic beads of the present application include: a magnetic bead body, a lipid-soluble fluorescent dye molecule dispersed and distributed inside the magnetic bead body, a gel layer coated on the outer surface of the magnetic bead body, and a gel layer bound to the magnetic bead body. The water-soluble fluorescent dye molecules on the outer surface and/or the inner part of the adhesive layer can improve the fluorescent signal of the fluorescent magnetic beads and improve the detection sensitivity. In addition, as shown in Figure 39, under the premise of avoiding excessive adsorption of magnetic nanoparticles on the surface of the microspheres, the magnetic properties of fluorescent magnetic beads can be enhanced by dispersing lipid-soluble magnetic nanoparticles inside and coupling water-soluble magnetic nanoparticles outside. The response signal (ie the main group signal), and the interference signal caused by impurities can be reduced, the autofluorescence of the microspheres can be reduced, and the detection sensitivity can be improved.

As shown in FIG. 2 , a second embodiment of the present application proposes a method for manufacturing fluorescent magnetic beads, which includes the following steps:

S10: Combining the microspheres and the water-soluble magnetic nanoparticles using adsorption technology, so that the water-soluble magnetic nanoparticles are coupled to the outer surface of the microspheres.

S21: Coating a gel layer on the outer surface of the microspheres coupled with water-soluble magnetic nanoparticles, and the gel layer coating at least part of the water-soluble magnetic nanoparticles, and combining the microspheres and the water-soluble fluorescent dye molecules by adsorption technology , so that the water-soluble fluorescent dye molecules are bound to the outer surface of the gel layer.

S40: Combining the microspheres and the liposoluble magnetic nanoparticles using swelling technology, so that the liposoluble magnetic nanoparticles are dispersed and distributed in the interior of the microspheres and/or the interior of the gel layer.

S30: Combining the microspheres and the lipid-soluble fluorescent dye molecules by means of swelling technology, so that the lipid-soluble fluorescent dye molecules are dispersed and distributed in the interior of the microspheres and/or the interior of the gel layer.

As shown in FIG. 3 , a third embodiment of the present application proposes a method for manufacturing fluorescent magnetic beads, and the method includes the following steps:

S10: Combining the microspheres and the water-soluble magnetic nanoparticles using adsorption technology, so that the water-soluble magnetic nanoparticles are coupled to the outer surface of the microspheres.

S40: Using swelling technology to combine microspheres and lipid-soluble magnetic nanoparticles, the lipid-soluble magnetic nanoparticles are dispersed in the interior of the microspheres.

S21: Coating a gel layer on the outer surface of the microspheres coupled with water-soluble magnetic nanoparticles, and the gel layer coating at least part of the water-soluble magnetic nanoparticles, and combining the microspheres and the water-soluble fluorescent dye molecules by adsorption technology , so that the water-soluble fluorescent dye molecules are bound to the outer surface of the gel layer.

S30: Combining the microspheres and the lipid-soluble fluorescent dye molecules by means of swelling technology, so that the lipid-soluble fluorescent dye molecules are dispersed and distributed in the interior of the microspheres and/or the interior of the gel layer.

Different from the first embodiment, in the third embodiment, "combining microspheres and lipid-soluble magnetic nanoparticles using swelling technology" occurs before "coating the gel layer", so the lipid-soluble magnetic nanoparticles are more likely to enter the microspheres. Internally, the embedding capacity of the lipid-soluble magnetic nanoparticles within the microspheres is greatly improved.

As shown in FIG. 4 , a fourth embodiment of the present application proposes a method for manufacturing fluorescent magnetic beads, which includes the following steps:

S40: Using swelling technology to combine microspheres and lipid-soluble magnetic nanoparticles, the lipid-soluble magnetic nanoparticles are dispersed in the interior of the microspheres.

S10: Combining the microspheres and the water-soluble magnetic nanoparticles using adsorption technology, so that the water-soluble magnetic nanoparticles are coupled to the outer surface of the microspheres.

S21: Coating a gel layer on the outer surface of the microspheres coupled with water-soluble magnetic nanoparticles, and the gel layer coating at least part of the water-soluble magnetic nanoparticles, and combining the microspheres and the water-soluble fluorescent dye molecules by adsorption technology , so that the water-soluble fluorescent dye molecules are bound to the outer surface of the gel layer.

S30: Combining the microspheres and the lipid-soluble fluorescent dye molecules by means of swelling technology, so that the lipid-soluble fluorescent dye molecules are dispersed and distributed in the interior of the microspheres and/or the interior of the gel layer.

Different from the first embodiment, in the fourth embodiment, "combining microspheres and water-soluble magnetic nanoparticles by adsorption technology" occurs before "coating the gel layer", so the water-soluble magnetic nanoparticles are more likely to enter the microspheres. Internally, the embedding capacity of the water-soluble magnetic nanoparticles in the microspheres is greatly improved.

As shown in FIG. 5 , a fifth embodiment of the present application proposes a method for manufacturing fluorescent magnetic beads, and the method includes the following steps:

S10: Combining the microspheres and the water-soluble magnetic nanoparticles using adsorption technology, so that the water-soluble magnetic nanoparticles are coupled to the outer surface of the microspheres.

S21: Coating a gel layer on the outer surface of the microspheres coupled with water-soluble magnetic nanoparticles, and the gel layer coating at least part of the water-soluble magnetic nanoparticles, and combining the microspheres and the water-soluble fluorescent dye molecules by adsorption technology , so that the water-soluble fluorescent dye molecules are bound to the outer surface of the gel layer.

S50: Using swelling technology to simultaneously combine microspheres with lipid-soluble fluorescent dye molecules, microspheres and lipid-soluble magnetic nanoparticles, so that lipid-soluble fluorescent dye molecules and lipid-soluble magnetic nanoparticles are dispersed in the interior of the microspheres and/or in the gel layer internal.

Specifically, microspheres are dispersed in a first medium, a second medium containing lipid-soluble fluorescent dye molecules is provided, a third medium containing lipid-soluble magnetic nanoparticles is provided, mixed, and then subjected to a swelling reaction (vortex dispersion Uniform, rotating reaction preset time), lipid-soluble magnetic nanoparticles and lipid-soluble fluorescent dye molecules are simultaneously embedded in the interior of the microspheres, and the supernatant is removed by magnetic separation to obtain the internal distribution of lipid-soluble magnetic nanoparticles and lipid-soluble fluorescent dyes Molecular fluorescent beads.

Further, step S50 can be performed multiple times, respectively combining lipid-soluble fluorescent dye molecules and microspheres with different fluorescence characteristics, or, respectively combining lipid-soluble fluorescent dye molecules and microspheres with different concentrations, and combining lipid-soluble magnetic nanoparticles and microspheres. microspheres to obtain multiple magnetic beads with different fluorescence intensities, that is, fluorescently encoded magnetic beads. Wherein, different lipid-soluble fluorescent dye molecules 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 dye molecules that the prepared fluorescently encoded magnetic beads have different fluorescent characteristics.

Different from the first embodiment, the fifth embodiment embeds lipid-soluble magnetic nanoparticles and lipid-soluble fluorescent dye molecules into microspheres simultaneously by swelling technology, which can simplify the operation steps and save the amount of reagents.

As shown in FIG. 6 , the sixth embodiment of the present application proposes a method for manufacturing fluorescent magnetic beads, and the method includes the following steps:

S10: Combining the microspheres and the water-soluble magnetic nanoparticles using adsorption technology, so that the water-soluble magnetic nanoparticles are coupled to the outer surface of the microspheres.

S40: Using swelling technology to combine microspheres and lipid-soluble magnetic nanoparticles, the lipid-soluble magnetic nanoparticles are dispersed in the interior of the microspheres.

S30: The swelling technology is used to combine the microspheres with the lipid-soluble fluorescent dye molecules, so that the lipid-soluble fluorescent dye molecules are dispersed in the interior of the microspheres.

S21: Coating a gel layer on the outer surface of the microspheres coupled with water-soluble magnetic nanoparticles, and the gel layer coating at least part of the water-soluble magnetic nanoparticles, and combining the microspheres and the water-soluble fluorescent dye molecules by adsorption technology , so that the water-soluble fluorescent dye molecules are bound to the outer surface of the gel layer.

Different from the first embodiment, the sixth embodiment “combines microspheres and lipid-soluble magnetic nanoparticles by swelling technology” and “combines microspheres and lipid-soluble fluorescent dye molecules by swelling technology” occurs in the “coated gel layer”. "Before, therefore, the lipid-soluble magnetic nanoparticles and lipid-soluble fluorescent dye molecules can more easily enter the interior of the microspheres, which greatly improves the embedding capacity of the lipid-soluble magnetic nanoparticles and lipid-soluble fluorescent dye molecules in the microspheres.

As shown in FIG. 7 , a seventh embodiment of the present application proposes a method for manufacturing fluorescent magnetic beads, which includes the following steps:

S40: Using swelling technology to combine microspheres and lipid-soluble magnetic nanoparticles, the lipid-soluble magnetic nanoparticles are dispersed in the interior of the microspheres.

S10: Combining the microspheres and the water-soluble magnetic nanoparticles using adsorption technology, so that the water-soluble magnetic nanoparticles are coupled to the outer surface of the microspheres.

S30: The swelling technology is used to combine the microspheres with the lipid-soluble fluorescent dye molecules, so that the lipid-soluble fluorescent dye molecules are dispersed in the interior of the microspheres.

S21: Coating a gel layer on the outer surface of the microspheres coupled with water-soluble magnetic nanoparticles, and the gel layer coating at least part of the water-soluble magnetic nanoparticles, and combining the microspheres and the water-soluble fluorescent dye molecules by adsorption technology , so that the water-soluble fluorescent dye molecules are bound to the outer surface of the gel layer.

Different from the first embodiment, the seventh embodiment “combines microspheres and lipid-soluble magnetic nanoparticles by swelling technology”, “combines microspheres and water-soluble magnetic nanoparticles by adsorption technology” and “combines microspheres by swelling technology”. "With lipid-soluble fluorescent dye molecules" occurs before the "coating gel layer", therefore, lipid-soluble magnetic nanoparticles, water-soluble magnetic nanoparticles and lipid-soluble fluorescent dye molecules are more likely to enter the interior of the microspheres, and enhance the microspheres. Embedding capacity of lipid-soluble magnetic nanoparticles, water-soluble magnetic nanoparticles and lipid-soluble fluorescent dye molecules.

As shown in FIG. 8 , an eighth embodiment of the present application proposes a method for manufacturing fluorescent magnetic beads, which includes the following steps:

S10: Combining the microspheres and the water-soluble magnetic nanoparticles using adsorption technology, so that the water-soluble magnetic nanoparticles are coupled to the outer surface of the microspheres.

S30: The swelling technology is used to combine the microspheres with the lipid-soluble fluorescent dye molecules, so that the lipid-soluble fluorescent dye molecules are dispersed in the interior of the microspheres.

S40: Using swelling technology to combine microspheres and lipid-soluble magnetic nanoparticles, the lipid-soluble magnetic nanoparticles are dispersed in the interior of the microspheres.

S21: Coating a gel layer on the outer surface of the microspheres coupled with water-soluble magnetic nanoparticles, and the gel layer coating at least part of the water-soluble magnetic nanoparticles, and combining the microspheres and the water-soluble fluorescent dye molecules by adsorption technology , so that the water-soluble fluorescent dye molecules are bound to the outer surface of the gel layer.

As shown in FIG. 9 , the ninth embodiment of the present application proposes a method for manufacturing fluorescent magnetic beads, and the method includes the following steps:

S10: Combining the microspheres and the water-soluble magnetic nanoparticles using adsorption technology, so that the water-soluble magnetic nanoparticles are coupled to the outer surface of the microspheres.

S30: The swelling technology is used to combine the microspheres with the lipid-soluble fluorescent dye molecules, so that the lipid-soluble fluorescent dye molecules are dispersed in the interior of the microspheres.

S21: Coating a gel layer on the outer surface of the microspheres coupled with water-soluble magnetic nanoparticles, and the gel layer coating at least part of the water-soluble magnetic nanoparticles, and combining the microspheres and the water-soluble fluorescent dye molecules by adsorption technology , so that the water-soluble fluorescent dye molecules are bound to the outer surface of the gel layer.

S40: Combining the microspheres and the liposoluble magnetic nanoparticles using swelling technology, so that the liposoluble magnetic nanoparticles are dispersed and distributed in the interior of the microspheres and/or the interior of the gel layer.

As shown in FIG. 10 , the tenth embodiment of the present application proposes a method for manufacturing fluorescent magnetic beads, and the method includes the following steps:

S10: Combining the microspheres and the water-soluble magnetic nanoparticles using adsorption technology, so that the water-soluble magnetic nanoparticles are coupled to the outer surface of the microspheres.

S50: Using swelling technology to combine microspheres and lipid-soluble fluorescent dye molecules, microspheres and lipid-soluble magnetic nanoparticles at the same time, so that lipid-soluble fluorescent dye molecules and lipid-soluble magnetic nanoparticles are dispersed in the interior of the microspheres.

S21: Coating a gel layer on the outer surface of the microspheres coupled with water-soluble magnetic nanoparticles, and the gel layer coating at least part of the water-soluble magnetic nanoparticles, and combining the microspheres and the water-soluble fluorescent dye molecules by adsorption technology , so that the water-soluble fluorescent dye molecules are bound to the outer surface of the gel layer.

As shown in FIG. 11 , the eleventh embodiment of the present application proposes a method for manufacturing fluorescent magnetic beads, and the method includes the following steps:

S40: Using swelling technology to combine microspheres and lipid-soluble magnetic nanoparticles, the lipid-soluble magnetic nanoparticles are dispersed in the interior of the microspheres.

S30: The swelling technology is used to combine the microspheres with the lipid-soluble fluorescent dye molecules, so that the lipid-soluble fluorescent dye molecules are dispersed in the interior of the microspheres.

S10: Combining the microspheres and the water-soluble magnetic nanoparticles using adsorption technology, so that the water-soluble magnetic nanoparticles are coupled to the outer surface of the microspheres.

S21: Coating a gel layer on the outer surface of the microspheres coupled with water-soluble magnetic nanoparticles, and the gel layer coating at least part of the water-soluble magnetic nanoparticles, and combining the microspheres and the water-soluble fluorescent dye molecules by adsorption technology , so that the water-soluble fluorescent dye molecules are bound to the outer surface of the gel layer.

Different from the first embodiment, in the eleventh embodiment, "using swelling technology to combine microspheres and lipid-soluble magnetic nanoparticles" and "using swelling technology to combine microspheres and lipid-soluble fluorescent dye molecules" occurred in "using adsorption technology to combine Therefore, it can avoid the difficulty of lipid-soluble magnetic nanoparticles and lipid-soluble fluorescent dye molecules entering the interior of the microspheres due to the hydrophilicity (ie oleophobicity) of the water-soluble magnetic nanoparticles. It can greatly increase the embedded quantity of lipid-soluble magnetic nanoparticles and lipid-soluble fluorescent dye molecules in the microspheres.

As shown in FIG. 12 , the twelfth embodiment of the present application proposes a method for manufacturing fluorescent magnetic beads. The method includes the following steps:

S30: The swelling technology is used to combine the microspheres with the lipid-soluble fluorescent dye molecules, so that the lipid-soluble fluorescent dye molecules are dispersed in the interior of the microspheres.

S40: Using swelling technology to combine microspheres and lipid-soluble magnetic nanoparticles, the lipid-soluble magnetic nanoparticles are dispersed in the interior of the microspheres.

S10: Combining the microspheres and the water-soluble magnetic nanoparticles using adsorption technology, so that the water-soluble magnetic nanoparticles are coupled to the outer surface of the microspheres.

S21: Coating a gel layer on the outer surface of the microspheres coupled with water-soluble magnetic nanoparticles, and the gel layer coating at least part of the water-soluble magnetic nanoparticles, and combining the microspheres and the water-soluble fluorescent dye molecules by adsorption technology , so that the water-soluble fluorescent dye molecules are bound to the outer surface of the gel layer.

As shown in FIG. 13 , the thirteenth embodiment of the present application proposes a method for manufacturing fluorescent magnetic beads. The method includes the following steps:

S30: The swelling technology is used to combine the microspheres with the lipid-soluble fluorescent dye molecules, so that the lipid-soluble fluorescent dye molecules are dispersed in the interior of the microspheres.

S10: Combining the microspheres and the water-soluble magnetic nanoparticles using adsorption technology, so that the water-soluble magnetic nanoparticles are coupled to the outer surface of the microspheres.

S40: Using swelling technology to combine microspheres and lipid-soluble magnetic nanoparticles, the lipid-soluble magnetic nanoparticles are dispersed in the interior of the microspheres.

S21: Coating a gel layer on the outer surface of the microspheres coupled with water-soluble magnetic nanoparticles, and the gel layer coating at least part of the water-soluble magnetic nanoparticles, and combining the microspheres and the water-soluble fluorescent dye molecules by adsorption technology , so that the water-soluble fluorescent dye molecules are bound to the outer surface of the gel layer.

As shown in FIG. 14 , the fourteenth embodiment of the present application proposes a method for manufacturing fluorescent magnetic beads, and the method includes the following steps:

S30: The swelling technology is used to combine the microspheres with the lipid-soluble fluorescent dye molecules, so that the lipid-soluble fluorescent dye molecules are dispersed in the interior of the microspheres.

S10: Combining the microspheres and the water-soluble magnetic nanoparticles using adsorption technology, so that the water-soluble magnetic nanoparticles are coupled to the outer surface of the microspheres.

S21: Coating a gel layer on the outer surface of the microspheres coupled with water-soluble magnetic nanoparticles, and the gel layer coating at least part of the water-soluble magnetic nanoparticles, and combining the microspheres and the water-soluble fluorescent dye molecules by adsorption technology , so that the water-soluble fluorescent dye molecules are bound to the outer surface of the gel layer.

S40: Combining the microspheres and the liposoluble magnetic nanoparticles using swelling technology, so that the liposoluble magnetic nanoparticles are dispersed and distributed in the interior of the microspheres and/or the interior of the gel layer.

As shown in FIG. 15 , a fifteenth embodiment of the present application proposes a method for manufacturing fluorescent magnetic beads, and the method includes the following steps:

S50: Using swelling technology to combine microspheres and lipid-soluble fluorescent dye molecules, microspheres and lipid-soluble magnetic nanoparticles at the same time, so that lipid-soluble fluorescent dye molecules and lipid-soluble magnetic nanoparticles are dispersed in the interior of the microspheres.

S10: Combining the microspheres and the water-soluble magnetic nanoparticles using adsorption technology, so that the water-soluble magnetic nanoparticles are coupled to the outer surface of the microspheres.

S21: Coating a gel layer on the outer surface of the microspheres coupled with water-soluble magnetic nanoparticles, and the gel layer coating at least part of the water-soluble magnetic nanoparticles, and combining the microspheres and the water-soluble fluorescent dye molecules by adsorption technology , so that the water-soluble fluorescent dye molecules are bound to the outer surface of the gel layer.

As shown in FIG. 16 , the sixteenth embodiment of the present application proposes a method for manufacturing fluorescent magnetic beads. The method includes the following steps:

S10: Combining the microspheres and the water-soluble magnetic nanoparticles using adsorption technology, so that the water-soluble magnetic nanoparticles are coupled to the outer surface of the microspheres.

S22: combine water-soluble fluorescent dye molecules with gel molecules to obtain fluorescent dye-gel complexes, and coat the fluorescent dye-gel complexes on the outer surface of microspheres coupled with water-soluble magnetic nanoparticles , to form a gel layer with water-soluble fluorescent dye molecules combined therein, wherein the gel layer covers at least part of the water-soluble magnetic nanoparticles.

Specifically, the gel molecules include at least one of chitosan, sodium alginate, polyacrylic acid, polymethacrylic acid, polyacrylamide, and polyN-polyacrylamide. The water-soluble fluorescent dye molecules have active groups, and the water-soluble fluorescent dye molecules are combined with the gel layer through the active groups. The active group includes N-hydroxysuccinimide group, at least one of carboxyl group, sulfhydryl group, epoxy group or tosyl group, which provides the possibility for water-soluble fluorescent dye molecules to combine with gel molecules.

S30: Combining the microspheres and the lipid-soluble fluorescent dye molecules by means of swelling technology, so that the lipid-soluble fluorescent dye molecules are dispersed and distributed in the interior of the microspheres and/or the interior of the gel layer.

S40: Combining the microspheres and the liposoluble magnetic nanoparticles using swelling technology, so that the liposoluble magnetic nanoparticles are dispersed and distributed in the interior of the microspheres and/or the interior of the gel layer.

Different from the situation in the prior art, the fluorescent magnetic beads of the present application include: water-soluble magnetic nanoparticles coupled to the outer surface of the microspheres, a gel layer with water-soluble fluorescent dye molecules bound inside, and dispersed and distributed on the microspheres. The lipid-soluble magnetic nanoparticles in the inner and/or gel layer, as shown in Figure 39, under the premise of avoiding excessive adsorption of magnetic nanoparticles on the surface of the microspheres, the lipid-soluble magnetic nanoparticles are dispersed and distributed in the inner and externally coupled with the magnetic nanoparticles. The water-soluble magnetic nanoparticles can enhance the magnetic response signal (ie the main group signal) of the fluorescent magnetic beads, reduce the interference signal caused by impurities, and can reduce the autofluorescence of the microspheres and improve the detection sensitivity.

Further, in the above embodiment, step S22 specifically includes the following steps:

S221: Mix the microspheres coupled with water-soluble magnetic nanoparticles and the fluorescent dye-gel composite, add a cross-linking agent, and carry out a cross-linking reaction, so that the outer surface of the microspheres coupled with water-soluble magnetic nanoparticles is Coat the gel layer.

When the fluorescent dye-gel complex is chitosan, the cross-linking agent can be glutaraldehyde, wherein the mass ratio of chitosan to microspheres is 0.01-20%, and chitosan interacts with pentane through electrostatic interaction. The dialdehyde undergoes a chemical reaction to generate a cross-linking effect, and within the above temperature range, the autofluorescence increase of the microspheres caused by heating can be effectively avoided, thereby improving the detection sensitivity.

As shown in FIG. 17 , the seventeenth embodiment of the present application proposes a method for manufacturing fluorescent magnetic beads, and the method includes the following steps:

S10: Combining the microspheres and the water-soluble magnetic nanoparticles using adsorption technology, so that the water-soluble magnetic nanoparticles are coupled to the outer surface of the microspheres.

S22: combine water-soluble fluorescent dye molecules with gel molecules to obtain fluorescent dye-gel complexes, and coat the fluorescent dye-gel complexes on the outer surface of microspheres coupled with water-soluble magnetic nanoparticles , to form a gel layer with water-soluble fluorescent dye molecules combined therein, wherein the gel layer covers at least part of the water-soluble magnetic nanoparticles.

S40: Combining the microspheres and the liposoluble magnetic nanoparticles using swelling technology, so that the liposoluble magnetic nanoparticles are dispersed and distributed in the interior of the microspheres and/or the interior of the gel layer.

S30: The swelling technology is used to combine the microspheres with the lipid-soluble fluorescent dye molecules, so that the lipid-soluble fluorescent dye molecules are dispersed in the interior of the microspheres.

As shown in FIG. 18 , the eighteenth embodiment of the present application proposes a method for manufacturing fluorescent magnetic beads, and the method includes the following steps:

S10: Combining the microspheres and the water-soluble magnetic nanoparticles using adsorption technology, so that the water-soluble magnetic nanoparticles are coupled to the outer surface of the microspheres.

S40: Combining the microspheres and the liposoluble magnetic nanoparticles using swelling technology, so that the liposoluble magnetic nanoparticles are dispersed and distributed in the interior of the microspheres and/or the interior of the gel layer.

S22: combine water-soluble fluorescent dye molecules with gel molecules to obtain fluorescent dye-gel complexes, and coat the fluorescent dye-gel complexes on the outer surface of microspheres coupled with water-soluble magnetic nanoparticles , to form a gel layer with water-soluble fluorescent dye molecules combined therein, wherein the gel layer covers at least part of the water-soluble magnetic nanoparticles.

S30: The swelling technology is used to combine the microspheres with the lipid-soluble fluorescent dye molecules, so that the lipid-soluble fluorescent dye molecules are dispersed in the interior of the microspheres.

As shown in FIG. 19 , the nineteenth embodiment of the present application proposes a method for manufacturing fluorescent magnetic beads. The method includes the following steps:

S40: Combining the microspheres and the liposoluble magnetic nanoparticles using swelling technology, so that the liposoluble magnetic nanoparticles are dispersed and distributed in the interior of the microspheres and/or the interior of the gel layer.

S10: Combining the microspheres and the water-soluble magnetic nanoparticles using adsorption technology, so that the water-soluble magnetic nanoparticles are coupled to the outer surface of the microspheres.

S22: combine water-soluble fluorescent dye molecules with gel molecules to obtain fluorescent dye-gel complexes, and coat the fluorescent dye-gel complexes on the outer surface of microspheres coupled with water-soluble magnetic nanoparticles , to form a gel layer with water-soluble fluorescent dye molecules combined therein, wherein the gel layer covers at least part of the water-soluble magnetic nanoparticles.

S30: The swelling technology is used to combine the microspheres with the lipid-soluble fluorescent dye molecules, so that the lipid-soluble fluorescent dye molecules are dispersed in the interior of the microspheres.

As shown in FIG. 20 , the twentieth embodiment of the present application proposes a method for manufacturing fluorescent magnetic beads. The method includes the following steps:

S10: Combining the microspheres and the water-soluble magnetic nanoparticles using adsorption technology, so that the water-soluble magnetic nanoparticles are coupled to the outer surface of the microspheres.

S22: combine water-soluble fluorescent dye molecules with gel molecules to obtain fluorescent dye-gel complexes, and coat the fluorescent dye-gel complexes on the outer surface of microspheres coupled with water-soluble magnetic nanoparticles , to form a gel layer with water-soluble fluorescent dye molecules combined therein, wherein the gel layer covers at least part of the water-soluble magnetic nanoparticles.

S50: Using swelling technology to simultaneously combine microspheres with lipid-soluble fluorescent dye molecules, microspheres and lipid-soluble magnetic nanoparticles, so that lipid-soluble fluorescent dye molecules and lipid-soluble magnetic nanoparticles are dispersed in the interior of the microspheres and/or in the gel layer internal.

As shown in FIG. 21 , the twenty-first embodiment of the present application proposes a method for manufacturing fluorescent magnetic beads. The method includes the following steps:

S10: Combining the microspheres and the water-soluble magnetic nanoparticles using adsorption technology, so that the water-soluble magnetic nanoparticles are coupled to the outer surface of the microspheres.

S40: Combining the microspheres and the liposoluble magnetic nanoparticles using swelling technology, so that the liposoluble magnetic nanoparticles are dispersed and distributed in the interior of the microspheres and/or the interior of the gel layer.

S30: The swelling technology is used to combine the microspheres with the lipid-soluble fluorescent dye molecules, so that the lipid-soluble fluorescent dye molecules are dispersed in the interior of the microspheres.

S22: combine water-soluble fluorescent dye molecules with gel molecules to obtain fluorescent dye-gel complexes, and coat the fluorescent dye-gel complexes on the outer surface of microspheres coupled with water-soluble magnetic nanoparticles , to form a gel layer with water-soluble fluorescent dye molecules combined therein, wherein the gel layer covers at least part of the water-soluble magnetic nanoparticles.

As shown in FIG. 22 , the twenty-second embodiment of the present application proposes a method for manufacturing fluorescent magnetic beads. The method includes the following steps:

S40: Combining the microspheres and the liposoluble magnetic nanoparticles using swelling technology, so that the liposoluble magnetic nanoparticles are dispersed and distributed in the interior of the microspheres and/or the interior of the gel layer.

S10: Combining the microspheres and the water-soluble magnetic nanoparticles using adsorption technology, so that the water-soluble magnetic nanoparticles are coupled to the outer surface of the microspheres.

S30: The swelling technology is used to combine the microspheres with the lipid-soluble fluorescent dye molecules, so that the lipid-soluble fluorescent dye molecules are dispersed in the interior of the microspheres.

S22: combine water-soluble fluorescent dye molecules with gel molecules to obtain fluorescent dye-gel complexes, and coat the fluorescent dye-gel complexes on the outer surface of microspheres coupled with water-soluble magnetic nanoparticles , to form a gel layer with water-soluble fluorescent dye molecules combined therein, wherein the gel layer covers at least part of the water-soluble magnetic nanoparticles.

As shown in FIG. 23 , the twenty-third embodiment of the present application proposes a method for manufacturing fluorescent magnetic beads. The method includes the following steps:

S10: Combining the microspheres and the water-soluble magnetic nanoparticles using adsorption technology, so that the water-soluble magnetic nanoparticles are coupled to the outer surface of the microspheres.

S30: The swelling technology is used to combine the microspheres with the lipid-soluble fluorescent dye molecules, so that the lipid-soluble fluorescent dye molecules are dispersed in the interior of the microspheres.

S40: Combining the microspheres and the liposoluble magnetic nanoparticles using swelling technology, so that the liposoluble magnetic nanoparticles are dispersed and distributed in the interior of the microspheres and/or the interior of the gel layer.

S22: combine water-soluble fluorescent dye molecules with gel molecules to obtain fluorescent dye-gel complexes, and coat the fluorescent dye-gel complexes on the outer surface of microspheres coupled with water-soluble magnetic nanoparticles , to form a gel layer with water-soluble fluorescent dye molecules combined therein, wherein the gel layer covers at least part of the water-soluble magnetic nanoparticles.

As shown in FIG. 24 , the twenty-fourth embodiment of the present application proposes a method for manufacturing fluorescent magnetic beads. The method includes the following steps:

S10: Combining the microspheres and the water-soluble magnetic nanoparticles using adsorption technology, so that the water-soluble magnetic nanoparticles are coupled to the outer surface of the microspheres.

S30: The swelling technology is used to combine the microspheres with the lipid-soluble fluorescent dye molecules, so that the lipid-soluble fluorescent dye molecules are dispersed in the interior of the microspheres.

S22: combine water-soluble fluorescent dye molecules with gel molecules to obtain fluorescent dye-gel complexes, and coat the fluorescent dye-gel complexes on the outer surface of microspheres coupled with water-soluble magnetic nanoparticles , to form a gel layer with water-soluble fluorescent dye molecules combined therein, wherein the gel layer covers at least part of the water-soluble magnetic nanoparticles.

S40: Combining the microspheres and the liposoluble magnetic nanoparticles using swelling technology, so that the liposoluble magnetic nanoparticles are dispersed and distributed in the interior of the microspheres and/or the interior of the gel layer.

As shown in FIG. 25 , the twenty-fifth embodiment of the present application proposes a method for manufacturing fluorescent magnetic beads. The method includes the following steps:

S10: Combining the microspheres and the water-soluble magnetic nanoparticles using adsorption technology, so that the water-soluble magnetic nanoparticles are coupled to the outer surface of the microspheres.

S50: Using swelling technology to combine microspheres and lipid-soluble fluorescent dye molecules, microspheres and lipid-soluble magnetic nanoparticles at the same time, so that lipid-soluble fluorescent dye molecules and lipid-soluble magnetic nanoparticles are dispersed in the interior of the microspheres.

S22: combine water-soluble fluorescent dye molecules with gel molecules to obtain fluorescent dye-gel complexes, and coat the fluorescent dye-gel complexes on the outer surface of microspheres coupled with water-soluble magnetic nanoparticles , to form a gel layer with water-soluble fluorescent dye molecules combined therein, wherein the gel layer covers at least part of the water-soluble magnetic nanoparticles.

As shown in FIG. 26 , the twenty-sixth embodiment of the present application proposes a method for manufacturing fluorescent magnetic beads. The method includes the following steps:

S40: Combining the microspheres and the liposoluble magnetic nanoparticles using swelling technology, so that the liposoluble magnetic nanoparticles are dispersed and distributed in the interior of the microspheres and/or the interior of the gel layer.

S30: The swelling technology is used to combine the microspheres with the lipid-soluble fluorescent dye molecules, so that the lipid-soluble fluorescent dye molecules are dispersed in the interior of the microspheres.

S10: Combining the microspheres and the water-soluble magnetic nanoparticles using adsorption technology, so that the water-soluble magnetic nanoparticles are coupled to the outer surface of the microspheres.

S22: combine water-soluble fluorescent dye molecules with gel molecules to obtain fluorescent dye-gel complexes, and coat the fluorescent dye-gel complexes on the outer surface of microspheres coupled with water-soluble magnetic nanoparticles , to form a gel layer with water-soluble fluorescent dye molecules combined therein, wherein the gel layer covers at least part of the water-soluble magnetic nanoparticles.

As shown in FIG. 27 , the twenty-seventh embodiment of the present application proposes a method for manufacturing fluorescent magnetic beads. The method includes the following steps:

S30: The swelling technology is used to combine the microspheres with the lipid-soluble fluorescent dye molecules, so that the lipid-soluble fluorescent dye molecules are dispersed in the interior of the microspheres.

S40: Combining the microspheres and the liposoluble magnetic nanoparticles using swelling technology, so that the liposoluble magnetic nanoparticles are dispersed and distributed in the interior of the microspheres and/or the interior of the gel layer.

S10: Combining the microspheres and the water-soluble magnetic nanoparticles using adsorption technology, so that the water-soluble magnetic nanoparticles are coupled to the outer surface of the microspheres.

S22: combine water-soluble fluorescent dye molecules with gel molecules to obtain fluorescent dye-gel complexes, and coat the fluorescent dye-gel complexes on the outer surface of microspheres coupled with water-soluble magnetic nanoparticles , to form a gel layer with water-soluble fluorescent dye molecules combined therein, wherein the gel layer covers at least part of the water-soluble magnetic nanoparticles.

As shown in FIG. 28 , the twenty-eighth embodiment of the present application proposes a method for manufacturing fluorescent magnetic beads. The method includes the following steps:

S30: The swelling technology is used to combine the microspheres with the lipid-soluble fluorescent dye molecules, so that the lipid-soluble fluorescent dye molecules are dispersed in the interior of the microspheres.

S10: Combining the microspheres and the water-soluble magnetic nanoparticles using adsorption technology, so that the water-soluble magnetic nanoparticles are coupled to the outer surface of the microspheres.

S40: Combining the microspheres and the liposoluble magnetic nanoparticles using swelling technology, so that the liposoluble magnetic nanoparticles are dispersed and distributed in the interior of the microspheres and/or the interior of the gel layer.

S22: combine water-soluble fluorescent dye molecules with gel molecules to obtain fluorescent dye-gel complexes, and coat the fluorescent dye-gel complexes on the outer surface of microspheres coupled with water-soluble magnetic nanoparticles , to form a gel layer with water-soluble fluorescent dye molecules combined therein, wherein the gel layer covers at least part of the water-soluble magnetic nanoparticles.

As shown in FIG. 29 , the twenty-ninth embodiment of the present application proposes a method for manufacturing fluorescent magnetic beads. The method includes the following steps:

S30: The swelling technology is used to combine the microspheres with the lipid-soluble fluorescent dye molecules, so that the lipid-soluble fluorescent dye molecules are dispersed in the interior of the microspheres.

S10: Combining the microspheres and the water-soluble magnetic nanoparticles using adsorption technology, so that the water-soluble magnetic nanoparticles are coupled to the outer surface of the microspheres.

S22: combine water-soluble fluorescent dye molecules with gel molecules to obtain fluorescent dye-gel complexes, and coat the fluorescent dye-gel complexes on the outer surface of microspheres coupled with water-soluble magnetic nanoparticles , to form a gel layer with water-soluble fluorescent dye molecules combined therein, wherein the gel layer covers at least part of the water-soluble magnetic nanoparticles.

S40: Combining the microspheres and the liposoluble magnetic nanoparticles using swelling technology, so that the liposoluble magnetic nanoparticles are dispersed and distributed in the interior of the microspheres and/or the interior of the gel layer.

As shown in FIG. 30 , the thirtieth embodiment of the present application proposes a method for manufacturing fluorescent magnetic beads. The method includes the following steps:

S50: Using swelling technology to combine microspheres and lipid-soluble fluorescent dye molecules, microspheres and lipid-soluble magnetic nanoparticles at the same time, so that lipid-soluble fluorescent dye molecules and lipid-soluble magnetic nanoparticles are dispersed in the interior of the microspheres.

S10: Combining the microspheres and the water-soluble magnetic nanoparticles using adsorption technology, so that the water-soluble magnetic nanoparticles are coupled to the outer surface of the microspheres.

S22: combine water-soluble fluorescent dye molecules with gel molecules to obtain fluorescent dye-gel complexes, and coat the fluorescent dye-gel complexes on the outer surface of microspheres coupled with water-soluble magnetic nanoparticles , to form a gel layer with water-soluble fluorescent dye molecules combined therein, wherein the gel layer covers at least part of the water-soluble magnetic nanoparticles.

Fluorescent magnetic beads with different structures can be prepared by omitting some of the steps in the above embodiments.

As shown in FIG. 31 or 32 , the fluorescent magnetic bead 10 includes a magnetic bead body 11 , a lipid-soluble fluorescent dye molecule 12 and a gel layer 13 . The lipid-soluble fluorescent dye molecules 12 are dispersed and distributed in the interior of the magnetic bead body 11, and the gel layer 13 is coated on the outer surface of the magnetic bead body 11, wherein the outer surface and/or the interior of the gel layer 13 is combined with a water-soluble fluorescent dye. Molecule 14.

As shown in FIG. 33 , in some embodiments, the magnetic bead body 11 includes: microspheres 111 and water-soluble magnetic nanoparticles 112 , and the water-soluble magnetic nanoparticles 112 are coupled to the outer surface of the microspheres 111 . The gel layer 13 covers at least part of the water-soluble magnetic nanoparticles 112 . Optionally, the gel layer 13 covers all the water-soluble magnetic nanoparticles 112, and the outer surface of the fluorescent magnetic beads 10 is a smooth surface.

Wherein, in terms of mass percentage, the fluorescent magnetic beads 10 include: 50%-99.5% of microspheres 111, 0.1%-10.0% of water-soluble fluorescent dye molecules 14, 0.1%-10.0% of lipid-soluble fluorescent dye molecules 12, 0.1% %~49.5% of water-soluble magnetic nanoparticles 112.

In some embodiments, the magnetic bead body 11 includes: microspheres 111 and liposoluble magnetic nanoparticles 113 , and the liposoluble magnetic nanoparticles 113 are dispersed in the interior of the microspheres 111 and/or the interior of the gel layer 13 .

Wherein, in terms of mass percentage, the fluorescent magnetic beads 10 include: 50%-99.5% of microspheres 111, 0.1%-10.0% of water-soluble fluorescent dye molecules 14, 0.1%-10.0% of lipid-soluble fluorescent dye molecules 12, 0.1% % ~ 49.5% of liposoluble magnetic nanoparticles 113.

As shown in FIG. 34 , in some embodiments, the magnetic bead body 11 includes: microspheres 111 , water-soluble magnetic nanoparticles 112 , and fat-soluble magnetic nanoparticles 113 . The water-soluble magnetic nanoparticles 112 are coupled to the outer surface of the microspheres 111 , and the fat-soluble magnetic nanoparticles 113 are dispersed in the interior of the microspheres 111 and/or the interior of the gel layer 13 . The gel layer 13 covers at least part of the water-soluble magnetic nanoparticles 112 .

Wherein, in terms of mass percentage, the fluorescent magnetic beads 10 include: 50%-99.5% of microspheres 111, 0.1%-10.0% of water-soluble fluorescent dye molecules 14, 0.1%-10.0% of lipid-soluble fluorescent dye molecules 12, 0.1% ~49.5% of fat-soluble magnetic nanoparticles 113, 0.1% to 49.5% of water-soluble magnetic nanoparticles 112.

In some embodiments, the water-soluble fluorescent dye molecules 14 have reactive groups, and the water-soluble fluorescent dye molecules 14 are combined with the gel layer 13 through the reactive groups. The active group includes at least one of N-hydroxysuccinimide group, carboxyl group, sulfhydryl group, epoxy group or tosyl group, which provides the possibility for the water-soluble fluorescent dye molecule 14 to combine with the gel molecule.

In some embodiments, the material of the gel layer 13 is at least one of chitosan, sodium alginate, polyacrylic acid, polymethacrylic acid, polyacrylamide, and polyN-polyacrylamide.

In some embodiments, the material of the water-soluble magnetic nanoparticles 112 is paramagnetic nanoparticles, wherein the paramagnetic nanoparticles can be selected from ferric oxide, ferric oxide, alloys containing nickel or cobalt At least one of the paramagnetic magnetic particles.

In some embodiments, the material of the fat-soluble magnetic nanoparticles 113 is paramagnetic nanoparticles, wherein the paramagnetic nanoparticles can be selected from at least one of ferric oxide or ferric oxide. In addition, the outer surface of the fat-soluble magnetic nanoparticles 113 contains a fat-soluble ligand having at least one of unsaturated fatty acid, saturated fatty acid, unsaturated fatty amine or saturated fatty amine. The above-mentioned lipid-soluble ligands can bind to the surface of the lipid-soluble magnetic nanoparticles 113 , thereby stabilizing the lipid-soluble magnetic nanoparticles 113 . The fat-soluble ligand may include at least one of oleic acid, oleylamine, or stearic acid.

In some embodiments, the particle size of the microspheres 111 is 1 μm˜50 μm, the particle size of the fat-soluble magnetic nanoparticles 113 is 1 nm˜200 nm, and the particle size of the water-soluble magnetic nanoparticles 112 is 1 nm˜200 nm.

In some embodiments, the microspheres 111 may be at least one of magnetic microspheres 111 or non-magnetic microspheres 111 .

In some embodiments, as shown in FIG. 35 , the magnetic microspheres 111 at least include: silica core microspheres 101 , a water-soluble magnetic nanoparticle layer 102 coupled to the outer surface of the silica core microspheres 101 , and a polymer coating layer 103 coated on the outer surface of the water-soluble magnetic nanoparticle layer 102 .

Specifically, the manufacturing method of the magnetic microspheres 111 shown in FIG. 35 is as follows: using adsorption technology to combine the silica core microspheres 101 and water-soluble magnetic nanoparticles, so that the water-soluble magnetic nanoparticles are coupled to the silica core The outer surface of the microspheres 101 . A polymer coating layer 103 is coated on the outer surface of the silica core microspheres 101 coupled with water-soluble magnetic nanoparticles, and the polymer coating layer 103 coats at least part of the water-soluble magnetic nanoparticles.

Further, the silica core microspheres 101 can be hollow mesoporous silica core microspheres 101, and optionally, the outer surface of the silica core microspheres 101 can be modified with required charged functional groups by chemical means, wherein , the charged functional group includes at least one of charged carboxyl group, amino group, sulfonic acid group or sulfhydryl group, which provides the possibility for the silica core microsphere 101 to couple with water-soluble magnetic nanoparticles.

In other embodiments, soluble divalent and ferric ion salts can be used as raw materials, dissolved in ammonia water or sodium hydroxide solution, and react; or ferric ion salts can be used as raw materials, dissolved in ethylene glycol solution , solvothermal reaction is carried out to prepare ferric oxide magnetic nanoparticles, and then the prepared ferric oxide magnetic nanoparticles are synthesized and coupled with ferric oxide magnetic nanoparticles using tetraethyl orthosilicate with the participation of ammonia water Silica core microspheres 101.

In some embodiments, as shown in FIG. 36 , the magnetic microspheres 111 at least include: polymer core microspheres 104 and magnetic nanoparticles 105 distributed inside the polymer core microspheres 104 .

Specifically, the manufacturing method of the magnetic microspheres 111 shown in FIG. 36 is as follows: the magnetic nanoparticles 105 are mixed with organic monomer molecules to form a mixed fluid, and the mixed fluid is prepared into the magnetic microspheres 111 .

Specifically, the organic monomer molecule can be a polymer monomer, such as 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 water-soluble magnetic nanoparticles 105 or the lipid-soluble magnetic nanoparticles 105 are added to the organic monomer molecule solution to form a mixed fluid. The mixed fluid is polymerized to produce magnetic microspheres 111 , wherein the magnetic nanoparticles 105 are distributed inside the polymer core microspheres 104 . More specifically, the superparamagnetic magnetic nanoparticles 105, 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, Mix well and react at 0-65°C (for example, 0°C, 25°C, 30°C, 50°C, 65°C) to obtain magnetic nanoparticle 105-polystyrene core microsphere mixed emulsion. The catalyst solution is added into the magnetic nanoparticle 105-polystyrene core microsphere mixed emulsion, stirred, left to stand for precipitation, and then washed with water to obtain magnetically separable magnetic microspheres 111 .

In some embodiments, as shown in FIG. 37 , the magnetic microspheres 111 at least include: a polymer core microsphere 106 , a water-soluble magnetic nanoparticle layer 107 coupled to the outer surface of the polymer core microsphere 106 , a coating The polymer coating layer 108 on the outer surface of the water-soluble magnetic nanoparticle layer 107 , and the lipid-soluble magnetic nanoparticles 109 distributed in the polymer core microspheres 106 and/or the polymer coating layer 108 .

Specifically, the manufacturing method of the magnetic microspheres 111 shown in FIG. 37 is as follows: using adsorption technology to combine the polymer core microspheres 106 and water-soluble magnetic nanoparticles, so that the water-soluble magnetic nanoparticles are coupled to the polymer core microspheres 106 on the outer surface. A polymer coating layer 108 is coated on the outer surface of the polymer core microspheres 106 coupled with water-soluble magnetic nanoparticles, and the polymer coating layer 108 coats the outer surface of the core microspheres and the water-soluble magnetic nanoparticles The outer surface of the particle layer 107 . The polymer core microspheres 106 and the lipid-soluble magnetic nanoparticles 109 are combined using the swelling technique, so that the lipid-soluble magnetic nanoparticles 109 are dispersed and distributed in the polymer core microspheres 106 and/or the polymer coating layer 108 .

Specifically, the material of the polymer core microspheres 106 may include polystyrene, polymethyl methacrylate, polyethylene-methyl methacrylate, polyacrylonitrile, polyethylene, polypropylene, and polyethyl acrylate. at least one of. The outer surface of the core microsphere 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 material of the polymer coating layer 108 may include at least one of polystyrene, polymethyl methacrylate, polyethylene-methyl methacrylate, polyacrylonitrile, polyethylene, polypropylene, and polyethyl acrylate.

In some embodiments, the non-magnetic microspheres are at least one of cross-linked non-magnetic microspheres, non-cross-linked non-magnetic microspheres, or hollow mesoporous non-magnetic microspheres.

When the non-magnetic microspheres are cross-linked porous non-magnetic microspheres or hollow mesoporous non-magnetic microspheres, they have the characteristics of high porosity and high specific surface area. Therefore, the encapsulation of lipid-soluble magnetic nanoparticles in the microspheres can be improved. Buried capacity, thereby improving the fluorescence intensity and magnetic responsivity of the microspheres.

Further, depending on the sequence of the preparation steps, when the microspheres are coated with a gel layer, the lipid-soluble magnetic nanoparticles can be embedded in the gel layer during the process of embedding the lipid-soluble magnetic nanoparticles into the interior of the microspheres. internal.

The microspheres and the water-soluble magnetic nanoparticles are combined by adsorption technology, so that the operating temperature of the water-soluble magnetic nanoparticles coupled to the outer surface of the microspheres is 0 to 100°C (for example, 0°C, 25°C, 30°C, 50°C, 65°C). °C, 100 °C), preferably 0 to 65 °C (eg 0 °C, 25 °C, 30 °C, 50 °C, 65 °C), more preferably room temperature (23 °C ± 2 °C).

The operating temperature for coating the gel layer on the outer surface of the microspheres coupled with water-soluble magnetic nanoparticles is 20-65°C (eg 20°C, 30°C, 50°C, 65°C), preferably, 20-65°C 30°C (eg 20°C, 25°C, 30°C), more preferably room temperature (23°C ± 2°C).

The fluorescent dye-gel complex is coated on the outer surface of the microspheres coupled with water-soluble magnetic nanoparticles to form a gel layer with water-soluble fluorescent dye molecules bound inside. The operating temperature is 20～65 ℃ ( For example, 20°C, 30°C, 50°C, 65°C), preferably 20-30°C (eg 20°C, 25°C, 30°C), more preferably room temperature (23°C±2°C).

The adsorption technology is used to combine the microspheres and the water-soluble fluorescent dye molecules, so that the operating temperature for the water-soluble fluorescent dye molecules to bind to the outer surface of the gel layer is 20 to 65 °C (for example, 20 °C, 30 °C, 50 °C, 65 °C), Preferably, it is 20 to 30°C (eg, 20°C, 25°C, 30°C), and more preferably, it is room temperature (23°C±2°C).

The swelling technology is used to combine the microspheres and the lipid-soluble fluorescent dye molecules, so that the operating temperature at which the lipid-soluble fluorescent dye molecules are dispersed and distributed inside the microspheres is 20 to 65°C (for example, 20°C, 30°C, 50°C, 65°C), preferably Ground, it is 20 to 30°C (eg, 20°C, 25°C, 30°C), and more preferably, it is room temperature (23°C±2°C).

The microspheres and the liposoluble magnetic nanoparticles are combined using swelling technology, so that the liposoluble magnetic nanoparticles are dispersed in the interior of the microspheres and/or the operating temperature of the gel layer is 0 to 65°C (for example, 0°C, 25°C, 30°C, 50°C, 65°C), preferably 0-30°C (eg 0°C, 25°C, 30°C), more preferably room temperature (23°C±2°C).

Within the above-mentioned preferred operating temperature range or more preferred operating temperature range, the increase of autofluorescence of the 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 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 combined or Can be integrated into another system, or some features can be ignored, or not implemented.

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 may be implemented in the form of hardware, or may 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 in other related technical fields, All are similarly included in the scope of patent protection of the present application.

Claims (18)

1. a fluorescent magnetic bead, is characterized in that, described fluorescent magnetic bead comprises: Magnetic bead body; The lipid-soluble fluorescent dye molecules are dispersed and distributed inside the magnetic bead body; The gel layer is coated on the outer surface of the magnetic bead body, wherein, water-soluble fluorescent dye molecules are bound to the outer surface and/or the interior of the gel layer. 2. The fluorescent magnetic bead according to claim 1, wherein the magnetic bead body comprises: Microspheres; water-soluble magnetic nanoparticles coupled to the outer surface of the microspheres; Wherein, the gel layer covers at least part of the water-soluble magnetic nanoparticles. 3. The fluorescent magnetic bead according to claim 1, wherein the magnetic bead body comprises: Microspheres; The fat-soluble magnetic nanoparticles are dispersed and distributed in the interior of the microspheres and/or the interior of the gel layer. 4. The fluorescent magnetic bead according to claim 1, wherein the magnetic bead body comprises: Microspheres; water-soluble magnetic nanoparticles coupled to the outer surface of the microspheres; Fat-soluble magnetic nanoparticles, dispersed in the interior of the microspheres and/or the interior of the gel layer; Wherein, the gel layer covers at least part of the water-soluble magnetic nanoparticles. 5. The magnetic bead according to claim 1, wherein, The water-soluble fluorescent dye molecules have active groups, and the water-soluble fluorescent dye molecules are combined with the gel layer through the active groups. 6. The magnetic bead according to claim 1, characterized in that, The material of the gel layer is at least one of chitosan, sodium alginate, polyacrylic acid, polymethacrylic acid, polyacrylamide, and polyN-polyacrylamide. 7 . The fluorescent magnetic beads according to claim 2 , wherein the microspheres are at least one of magnetic microspheres or non-magnetic microspheres. 8 . 8. The fluorescent magnetic beads according to claim 7, wherein the magnetic microspheres at least comprise: A magnetic nanoparticle layer, and a polystyrene coating layer covering the outer surface of the water-soluble magnetic nanoparticle layer. 9 . The fluorescent magnetic beads according to claim 7 , wherein the magnetic microspheres at least comprise: polymer core microspheres and magnetic nanoparticles distributed inside the polymer core microspheres. 10 . 10 . The fluorescent magnetic beads according to claim 7 , wherein the magnetic microspheres at least comprise: polymer core microspheres, water-soluble magnetic nanoparticles coupled to the outer surface of the polymer core microspheres. 11 . A particle layer, a polymer coating layer coated on the outer surface of the water-soluble magnetic nanoparticle layer, and lipid-soluble magnetic nanoparticles distributed inside the polymer core microspheres and/or inside the polymer coating layer. 11. The fluorescent magnetic beads according to claim 7, wherein the non-magnetic microspheres are at least one of cross-linked non-magnetic microspheres, non-cross-linked non-magnetic microspheres or hollow mesoporous non-magnetic microspheres. kind. 12. The fluorescent magnetic bead according to any one of claims 3 or 4, characterized in that, The fat-soluble magnetic nanoparticles are nanoparticles with paramagnetic properties, and the fat-soluble magnetic nanoparticles contain at least one fat-soluble ligand of unsaturated fatty acid, saturated fatty acid, unsaturated fatty amine or saturated fatty amine. 13. The fluorescent magnetic bead according to any one of claims 2 or 4, characterized in that, The water-soluble magnetic nanoparticles are paramagnetic nanoparticles. 14. The fluorescent magnetic bead according to claim 2, wherein, By mass percentage, the fluorescent magnetic beads include: 50%-99.5% of the microspheres, 0.1%-10.0% of the water-soluble fluorescent dye molecules: 0.1%-10.0% of the fat-soluble fluorescent dye molecules : 0.1% to 49.5% of the water-soluble magnetic nanoparticles. 15. The fluorescent magnetic bead according to claim 3, characterized in that, By mass percentage, the fluorescent magnetic beads include: 50%-99.5% of the microspheres, 0.1%-10.0% of the water-soluble fluorescent dye molecules, and 0.1%-10.0% of the fat-soluble fluorescent dye molecules , 0.1% to 49.5% of the fat-soluble magnetic nanoparticles. 16. The fluorescent magnetic bead according to claim 4, characterized in that, By mass percentage, the fluorescent magnetic beads include: 50%-99.5% of the microspheres, 0.1%-10.0% of the water-soluble fluorescent dye molecules, and 0.1%-10.0% of the fat-soluble fluorescent dye molecules , 0.1%-49.5% of the fat-soluble magnetic nanoparticles, and 0.1%-49.5% of the water-soluble magnetic nanoparticles. 17. A method for making fluorescent magnetic beads, wherein the method comprises: Using adsorption technology to combine microspheres and water-soluble magnetic nanoparticles, so that the water-soluble magnetic nanoparticles are coupled to the outer surface of the microspheres; After the step of combining the microspheres and the water-soluble magnetic nanoparticles using adsorption technology, so that the water-soluble magnetic nanoparticles are coupled to the outer surface of the microspheres, the method further includes: after the coupling with the water-soluble magnetic nanoparticles The outer surface of the microspheres of the water-soluble magnetic nanoparticles is coated with a gel layer, and the gel layer coats at least part of the water-soluble magnetic nanoparticles, and the adsorption technology is used to combine the microspheres and the water-soluble fluorescent dye molecules , so that the water-soluble fluorescent dye molecules are combined with the outer surface of the gel layer, or, the water-soluble fluorescent dye molecules are combined with the gel molecules to obtain a fluorescent dye-gel complex, and the fluorescent dye- The gel complex is coated on the outer surface of the microspheres coupled with the water-soluble magnetic nanoparticles to form a gel layer with the water-soluble fluorescent dye molecules bound inside, wherein the gel a layer covering at least a portion of the water-soluble magnetic nanoparticles; After the step of combining the microspheres and the water-soluble magnetic nanoparticles by using the adsorption technology, so that the water-soluble magnetic nanoparticles are coupled to the outer surface of the microspheres, or, after the combining the microspheres and the water-soluble magnetic nanoparticles by using the adsorption technology Water-soluble magnetic nanoparticles, before the step of coupling the water-soluble magnetic nanoparticles to the outer surface of the microspheres, the method further includes: combining the microspheres and the lipid-soluble fluorescent dye molecules by a swelling technique, so that the The lipid-soluble fluorescent dye molecules are dispersed in the interior of the microsphere; Before the step of combining the microspheres with lipid-soluble fluorescent dye molecules by using swelling technology, so that the lipid-soluble fluorescent dye molecules are dispersed and distributed in the interior of the microspheres, or, before combining the microspheres by using swelling technology Microspheres and lipid-soluble fluorescent dye molecules, after the step of dispersing the lipid-soluble fluorescent dye molecules in the interior of the microspheres, or, after the combination of the microspheres and the lipid-soluble fluorescent dye molecules using the swelling technology, While making the lipid-soluble fluorescent dye molecules disperse and distribute inside the microspheres, the method further includes: combining the microspheres and the lipid-soluble magnetic nanoparticles by a swelling technique, so that the lipid-soluble magnetic nanoparticles are dispersed Distributed inside the microspheres and/or inside the gel layer. 18. The method of claim 17, wherein: The adsorption technology is used to combine the microspheres and the water-soluble magnetic nanoparticles, so that the operating temperature of the water-soluble magnetic nanoparticles coupled to the outer surface of the microspheres is 0-100°C; The operating temperature of coating the gel layer on the outer surface of the microspheres coupled with the water-soluble magnetic nanoparticles is 20-65°C; The fluorescent dye-gel complex is coated on the outer surface of the microspheres coupled with the water-soluble magnetic nanoparticles to form a gel with the water-soluble fluorescent dye molecules combined inside The operating temperature of the layer is 20 to 65°C; The adsorption technology is used to combine the microspheres and the water-soluble fluorescent dye molecules, so that the operating temperature of the water-soluble fluorescent dye molecules combined with the outer surface of the gel layer is 20-65°C; The swelling technology is used to combine the microspheres and the lipid-soluble fluorescent dye molecules, so that the operating temperature of the lipid-soluble fluorescent dye molecules dispersed in the microspheres is 20-65°C; The swelling technology is used to combine the microspheres and the fat-soluble magnetic nanoparticles, so that the fat-soluble magnetic nanoparticles are dispersed and distributed in the interior of the microspheres and/or the operating temperature of the gel layer is 0～ 65°C.

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