CN115127975A - Fluorescent magnetic beads and method of making the same - Google Patents

Abstract

The present application relates to the technical field of fluorescent magnetic bead modification, and specifically discloses a fluorescent magnetic bead and a manufacturing method thereof. The fluorescent magnetic bead comprises: microspheres; water-soluble magnetic nanoparticles coupled to the outer surface of the microspheres; Dye, at least disperse in the interior of the microspheres; lipid-soluble magnetic nanoparticles, at least disperse and distribute in the interior of the microspheres. Through the above method, while improving the magnetic response of the fluorescent magnetic beads, the interference signal caused by impurities can be reduced, the autofluorescence of the microspheres can be reduced, 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 fluorescent 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 18, 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 propose a fluorescent magnetic bead and a method for making the same, which can improve the magnetic response of the fluorescent magnetic bead, reduce the interference signal caused by impurities, reduce the autofluorescence of the microsphere, and improve the detection sensitivity.

A first aspect of the present application provides a fluorescent magnetic bead, which includes: a microsphere; a water-soluble magnetic nanoparticle coupled to the outer surface of the microsphere; a lipid-soluble fluorescent dye, at least dispersed inside the microsphere; Soluble magnetic nanoparticles, at least dispersed in the interior of the microspheres.

A second aspect of the present application provides a method for making fluorescent magnetic beads, the method comprising: combining microspheres and water-soluble magnetic nanoparticles using adsorption technology, so that the water-soluble magnetic nanoparticles are coupled to the outer surface of the microspheres; The outer surface of the microspheres with water-soluble magnetic nanoparticles is coated with a gel layer, and the gel layer coats at least part of the water-soluble magnetic nanoparticles; wherein, the adsorption technology is used to combine the microspheres and the water-soluble magnetic nanoparticles, so that the Before the step of coupling the water-soluble magnetic nanoparticles to the outer surface of the microspheres, or, after the step of combining the microspheres and the water-soluble magnetic nanoparticles using an adsorption technique, so that the water-soluble magnetic nanoparticles are coupled to the outer surface of the microspheres , the method further includes: combining the microspheres and the fat-soluble fluorescent dye by using a swelling technology, so that the fat-soluble fluorescent dye is dispersed and distributed in the interior of the microspheres and/or the interior of the gel layer; before the step of coupling the water-soluble magnetic nanoparticles to the outer surface of the microspheres, or combining the microspheres with the water-soluble magnetic nanoparticles using adsorption techniques, so that the water-soluble magnetic nanoparticles are coupled to the microspheres After the step of forming the outer surface of the microsphere, or, using the swelling technology to combine the microsphere and the lipid-soluble fluorescent dye, so that the lipid-soluble fluorescent dye is dispersed and distributed in the interior of the microsphere, the method also includes: using the swelling technology to combine the microsphere and the lipid-soluble fluorescent dye. The soluble magnetic nanoparticles make the lipid-soluble magnetic nanoparticles dispersed in the interior of the microspheres.

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, and lipid-soluble fluorescent dyes and lipid-soluble magnetic nanoparticles dispersed at least in the interior of the microspheres. Under the premise of avoiding excessive adsorption of magnetic nanoparticles on the surface of fluorescent microspheres, the magnetic response signal of fluorescent magnetic beads can be enhanced by dispersing lipid-soluble magnetic nanoparticles inside and coupling water-soluble magnetic nanoparticles outside. group signal), and reduce the interference signal caused by impurities, and can reduce the autofluorescence of the microspheres, 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 structural diagram of a fluorescent magnetic bead proposed in this application;

Fig. 15 is the first structural schematic diagram of the microsphere 11 in Fig. 14;

Fig. 16 is the second structural schematic diagram of the microsphere 11 in Fig. 14;

Fig. 17 is the third structural schematic diagram of the microsphere 11 in Fig. 14;

Fig. 18 is the magnetic response signal diagram when the magnetic nanoparticles are excessively adsorbed on the surface of the microsphere in the prior art;

Figure 19 is a graph of the magnetic response signal of the fluorescent magnetic beads of the present application;

FIG. 20 is another schematic structural diagram of the fluorescent magnetic beads 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 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.

S20: A gel layer is coated on the outer surface of the microspheres coupled with the water-soluble magnetic nanoparticles, and the gel layer coats at least part of the water-soluble magnetic nanoparticles.

Mixing the microspheres prepared in step S10 with water-soluble magnetic nanoparticles coupled on the outer surface with the gel material, adding a cross-linking agent, stirring and reacting, after the obtained product is allowed to stand, magnetic separation is performed to remove the residual gel material to obtain an external gel material. Microspheres coated with a gel layer.

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

S30: Combining the microspheres and the liposoluble fluorescent dye using swelling technology, so that the liposoluble fluorescent dye is 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 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/or the interior of the gel layer, and the supernatant is removed by magnetic separation to obtain microspheres with lipid-soluble magnetic nanoparticles 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 fat-soluble magnetic nanoparticles, but also swell the microspheres, for example, chloroform, n-butanol, isobutanol , n-hexane, cyclohexane, tetrahydrofuran, one or more combinations, but not limited to the above-mentioned substances.

Wherein, the microspheres may be at least one of non-crosslinked microspheres, crosslinked porous microspheres or hollow mesoporous microspheres. The cross-linked porous microspheres and hollow mesoporous microspheres 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 magnetic responsiveness of the fluorescent magnetic beads.

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

Specifically, the microspheres are dispersed in the first medium, a third medium containing a lipid-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 lipid-soluble fluorescent dye is packaged Buried in the interior of the microspheres and/or the gel layer, and magnetically separated the supernatant 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 the fat-soluble magnetic nanoparticles, but also swell the microspheres, for example, chloroform, n-butanol, isobutanol , n-hexane, cyclohexane, tetrahydrofuran, one or more combinations, but not limited to the above-mentioned 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.

Further, step S40 may be performed multiple times, respectively combining lipid-soluble fluorescent dyes and microspheres with different fluorescence characteristics, or combining lipid-soluble fluorescent dyes and microspheres with different concentrations, respectively, to obtain a plurality of microspheres with different fluorescence intensities. , namely fluorescently encoded microspheres. Wherein, different lipid-soluble fluorescent dyes can be mixed in different proportions to prepare different encoded microspheres. It is precisely because of the different ratios of different lipid-soluble fluorescent dyes that the prepared fluorescently encoded microspheres have different fluorescent characteristics.

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.

S20: A gel layer is coated on the outer surface of the microspheres coupled with the water-soluble magnetic nanoparticles, and the gel layer coats 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: Combining the microspheres and the liposoluble fluorescent dye using swelling technology, so that the liposoluble fluorescent dye is 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.

S20: A gel layer is coated on the outer surface of the microspheres coupled with the water-soluble magnetic nanoparticles, and the gel layer coats at least part of the water-soluble magnetic nanoparticles.

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

Specifically, the microspheres are dispersed in the 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 is performed (vortex dispersion is uniform). , rotation reaction preset time), lipid-soluble magnetic nanoparticles and lipid-soluble fluorescent dyes are simultaneously embedded in the interior of the microspheres, and the supernatant liquid is removed by magnetic separation to obtain the fluorescence with lipid-soluble magnetic nanoparticles and lipid-soluble fluorescent dyes distributed inside. magnetic beads.

Further, step S50 can be performed multiple times, respectively combining lipid-soluble fluorescent dyes and microspheres with different fluorescence characteristics, or, respectively combining lipid-soluble fluorescent dyes and microspheres with different concentrations, and combining lipid-soluble magnetic nanoparticles and microspheres simultaneously. , to obtain multiple fluorescent magnetic beads with different fluorescence intensities, that is, fluorescently encoded fluorescent magnetic beads. Wherein, different liposoluble fluorescent dyes can be mixed in different proportions to prepare different encoded fluorescent magnetic beads. It is precisely because of the different ratios of different lipid-soluble fluorescent dyes that the prepared fluorescently encoded fluorescent magnetic beads have different fluorescent characteristics.

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

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:

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: Use swelling technology to combine microspheres and lipid-soluble fluorescent dyes, so that the lipid-soluble fluorescent dyes 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.

S20: A gel layer is coated on the outer surface of the microspheres coupled with the water-soluble magnetic nanoparticles, and the gel layer coats at least part of the water-soluble magnetic nanoparticles.

Different from the first embodiment, the swelling of the magnetic nanoparticles and the lipid-soluble fluorescent dyes in the fourth embodiment is before the coating of the gel layer. Therefore, the lipid-soluble magnetic nanoparticles and the lipid-soluble fluorescent dyes can more easily enter the interior of the microspheres. The embedding capacity of lipid-soluble fluorescent dyes and lipid-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.

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: Use swelling technology to combine microspheres and lipid-soluble fluorescent dyes, so that the lipid-soluble fluorescent dyes are dispersed in the interior of the microspheres.

S20: A gel layer is coated on the outer surface of the microspheres coupled with the water-soluble magnetic nanoparticles, and the gel layer coats at least part of the water-soluble magnetic nanoparticles.

Different from the first embodiment, the swelling of the magnetic nanoparticles and the liposoluble fluorescent dyes in the fifth embodiment is before the coating of the gel layer. Therefore, the liposoluble magnetic nanoparticles and the liposoluble fluorescent dyes are more likely to enter the interior of the microspheres. The embedding capacity of lipid-soluble fluorescent dyes and lipid-soluble magnetic nanoparticles in the microspheres is greatly improved.

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.

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

S20: A gel layer is coated on the outer surface of the microspheres coupled with the water-soluble magnetic nanoparticles, and the gel layer coats at least part of the water-soluble magnetic nanoparticles.

Different from the first embodiment, the swelling of the sixth embodiment is before the coating of the gel layer. Therefore, the lipid-soluble magnetic nanoparticles and the lipid-soluble fluorescent dyes can more easily enter the interior of the microspheres, thereby enhancing the lipid content in the microspheres. Embedding capacity of soluble fluorescent dyes and liposoluble magnetic nanoparticles. In addition, in the sixth embodiment, the liposoluble magnetic nanoparticles and the liposoluble fluorescent dye are simultaneously embedded into the microspheres through the swelling technology, which can simplify the operation steps and save the amount of reagents.

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:

S30: Use swelling technology to combine microspheres and lipid-soluble fluorescent dyes, so that the lipid-soluble fluorescent dyes 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.

S20: A gel layer is coated on the outer surface of the microspheres coupled with the water-soluble magnetic nanoparticles, and the gel layer coats at least part of the water-soluble magnetic nanoparticles.

Different from the first embodiment, the swelling of the lipid-soluble magnetic nanoparticles and the lipid-soluble fluorescent dyes in the seventh embodiment is before the coating of the gel layer. Therefore, the lipid-soluble magnetic nanoparticles and the lipid-soluble fluorescent dyes are more likely to enter the microspheres. Internally, the embedding capacity of lipid-soluble fluorescent dyes and lipid-soluble magnetic nanoparticles in the microspheres is greatly improved.

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:

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: Use swelling technology to combine microspheres and lipid-soluble fluorescent dyes, so that the lipid-soluble fluorescent dyes 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.

S20: A gel layer is coated on the outer surface of the microspheres coupled with the water-soluble magnetic nanoparticles, and the gel layer coats at least part of the water-soluble magnetic nanoparticles.

Different from the first embodiment, the swelling of the eighth embodiment is before the adsorption of the water-soluble magnetic nanoparticles, so that the lipid-soluble magnetic nanoparticles can be avoided due to the hydrophilicity (ie, oleophobicity) of the water-soluble magnetic nanoparticles. It is difficult for the lipid-soluble magnetic nanoparticles and lipid-soluble fluorescent dyes to enter the interior of the microspheres, which greatly improves the embedding capacity of the lipid-soluble magnetic nanoparticles and lipid-soluble fluorescent dyes in the microspheres.

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:

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

S20: A gel layer is coated on the outer surface of the microspheres coupled with the water-soluble magnetic nanoparticles, and the gel layer coats at least part of the water-soluble magnetic nanoparticles.

Different from the first embodiment, the swelling of the ninth embodiment is before the adsorption of the water-soluble magnetic nanoparticles, so that the lipid-soluble magnetic nanoparticles can be avoided due to the hydrophilicity (ie, oleophobicity) of the water-soluble magnetic nanoparticles. It is difficult for the lipid-soluble magnetic nanoparticles and lipid-soluble fluorescent dyes to enter the interior of the microspheres, which greatly improves the embedding capacity of the lipid-soluble magnetic nanoparticles and lipid-soluble fluorescent dyes in the microspheres. In addition, in the ninth embodiment, the liposoluble magnetic nanoparticles and the liposoluble fluorescent dye are simultaneously embedded into the microspheres through the swelling technology, which can simplify the operation steps and save the amount of reagents.

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

S30: Use swelling technology to combine microspheres and lipid-soluble fluorescent dyes, so that the lipid-soluble fluorescent dyes 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.

S20: A gel layer is coated on the outer surface of the microspheres coupled with the water-soluble magnetic nanoparticles, and the gel layer coats at least part of the water-soluble magnetic nanoparticles.

Different from the first embodiment, the swelling of the fat-soluble fluorescent dye in the tenth embodiment is before the adsorption of the water-soluble magnetic nanoparticles, so that the lipid caused by the hydrophilicity (ie oleophobicity) of the water-soluble magnetic nanoparticles can be avoided. It is difficult for soluble fluorescent dyes to enter the interior of the microspheres, which greatly improves the embedding capacity of the lipid-soluble fluorescent dyes in the microspheres. In the ninth embodiment, the fat-soluble magnetic nanoparticles are swollen before the gel layer is coated. Therefore, the fat-soluble magnetic nanoparticles can more easily enter the inside of the microspheres, and the embedding capacity of the fat-soluble magnetic nanoparticles in the microspheres is greatly improved.

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.

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: Use swelling technology to combine microspheres and lipid-soluble fluorescent dyes, so that the lipid-soluble fluorescent dyes are dispersed in the interior of the microspheres.

S20: A gel layer is coated on the outer surface of the microspheres coupled with the water-soluble magnetic nanoparticles, and the gel layer coats at least part of the water-soluble magnetic nanoparticles.

Different from the first embodiment, the swelling of the fat-soluble magnetic nanoparticles in the eleventh embodiment is before the adsorption of the water-soluble magnetic nanoparticles, so the hydrophilicity (ie oleophobicity) of the water-soluble magnetic nanoparticles can avoid As a result, it is difficult for the lipid-soluble magnetic nanoparticles to enter the interior of the microspheres, and the embedding capacity of the lipid-soluble magnetic nanoparticles in the microspheres is greatly improved. The swelling of the fat-soluble fluorescent dye in the ninth embodiment is before the coating of the gel layer. Therefore, the fat-soluble fluorescent dye can more easily enter the interior of the microspheres, and the embedding capacity of the fat-soluble fluorescent dye in the microspheres is greatly improved.

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: Use swelling technology to combine microspheres and lipid-soluble fluorescent dyes, so that the lipid-soluble fluorescent dyes 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.

S20: A gel layer is coated on the outer surface of the microspheres coupled with the water-soluble magnetic nanoparticles, and the gel layer coats 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.

Different from the first embodiment, the swelling of the liposoluble fluorescent dye in the twelfth embodiment is before the adsorption of the water-soluble magnetic nanoparticles, so the hydrophilicity (ie oleophobicity) of the water-soluble magnetic nanoparticles can be avoided. It is difficult for the lipid-soluble fluorescent dyes to enter the interior of the microspheres, which greatly improves the embedding capacity of the lipid-soluble fluorescent dyes in the microspheres.

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:

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.

S20: A gel layer is coated on the outer surface of the microspheres coupled with the water-soluble magnetic nanoparticles, and the gel layer coats at least part of the water-soluble magnetic nanoparticles.

S30: Combining the microspheres and the liposoluble fluorescent dye using swelling technology, so that the liposoluble fluorescent dye is dispersed and distributed in the interior of the microspheres and/or the interior of the gel layer.

Different from the first embodiment, the swelling of the fat-soluble magnetic nanoparticles in the thirteenth embodiment is before the adsorption of the water-soluble magnetic nanoparticles. As a result, it is difficult for the lipid-soluble magnetic nanoparticles to enter the interior of the microspheres, and the embedding capacity of the lipid-soluble magnetic nanoparticles in the microspheres is greatly improved.

Optionally, the gel layer in step S20 of the first embodiment to the thirteenth embodiment can be replaced with a polymer coating layer, and the material of the polymer coating layer is polystyrene, polymethyl methacrylate, At least one of polyethylene-methyl methacrylate, polyacrylonitrile, polyethylene, polypropylene, and polyethyl acrylate.

Thus, the structure of the fluorescent magnetic beads 10 prepared by the above-mentioned first to thirteenth embodiments or their alternatives is shown in FIG. 14 , and the fluorescent magnetic beads 10 include: microspheres 11 , lipid-soluble fluorescent dyes 12 , lipid-soluble fluorescent dyes The water-soluble magnetic nanoparticles 13 , the water-soluble magnetic nanoparticles 14 and the coating layer 15 . Among them, the lipid-soluble fluorescent dye 12 and the lipid-soluble magnetic nanoparticles 13 are dispersed in the interior of the microsphere 11. It is understood that, after entering the interior of the microsphere 11 through swelling, the lipid-soluble fluorescent dye 12, the lipid-soluble magnetic nanoparticles 13 Embedded inside the microspheres 11 . Water-soluble magnetic nanoparticles 14 can be coupled to the outer surface of the microspheres 11 . The coating layer 15 coats the outer surface of the microspheres 11 , and the coating layer 15 coats at least part of the water-soluble magnetic nanoparticles 14 . Optionally, the coating layer 15 covers all the water-soluble magnetic nanoparticles 14, and the outer surface of the fluorescent magnetic beads 10 is a smooth surface. Wherein, the above-mentioned coating layer 15 may be a gel layer or a polymer coating layer. It can be understood that the liposoluble fluorescent dye 12 and the liposoluble magnetic nanoparticles 13 can be dispersed and distributed in the interior of the gel layer, and are limited by the material of the polymer coating layer, the liposoluble fluorescent dye 12 and the liposoluble magnetic nanoparticles 13 13 is not dispersible in the interior of the polymer coating.

Further, steps S20 of the first embodiment to the thirteenth embodiment can be deleted, and the structure of the magnetic bead 10 thus obtained is shown in FIG. 20 . The fluorescent magnetic bead 10 includes: microspheres 11 , lipid-soluble fluorescent dyes 12. Fat-soluble magnetic nanoparticles 13 and water-soluble magnetic nanoparticles 14. Among them, the lipid-soluble fluorescent dye 12 and the lipid-soluble magnetic nanoparticles 13 are dispersed in the interior of the microsphere 11. It is understood that, after entering the interior of the microsphere 11 through swelling, the lipid-soluble fluorescent dye 12, the lipid-soluble magnetic nanoparticles 13 Embedded inside the microspheres 11 . Water-soluble magnetic nanoparticles 14 can be coupled to the outer surface of the microspheres 11 .

In some embodiments, the material of the microspheres 11 is a polymer, and the polymer includes polystyrene, polymethyl methacrylate, polyethylene-methyl methacrylate, polyacrylonitrile, polyethylene, polypropylene, polyacrylic acid At least one of ethyl esters. The outer surface of the microspheres 11 contains charged functional groups, and the charged functional groups include at least one of charged carboxyl groups, amino groups, sulfonic acid groups or mercapto groups.

In some embodiments, the water-soluble magnetic nanoparticles 14 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 fat-soluble magnetic nanoparticles 13 are nanoparticles with paramagnetic properties, wherein the nanoparticles with paramagnetic properties can be selected from at least one of triiron tetroxide or ferric oxide. In addition, the fat-soluble magnetic nanoparticles 13 contain a fat-soluble ligand of at least one of unsaturated fatty acid, saturated fatty acid, unsaturated fatty amine, or saturated fatty amine. The above-mentioned lipid-soluble ligand can bind to the surface of the lipid-soluble magnetic nanoparticles 13 , thereby stabilizing the lipid-soluble magnetic nanoparticles 13 . 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 11 is 1 μm˜50 μm (eg 1 μm, 5 μm, 10 μm, 20 μm, 50 μm), and the particle size of the fat-soluble magnetic nanoparticles 13 is 1 nm˜200 nm (eg 1 nm, 5 nm, 50 nm, 100 nm, 200 nm), and the particle size of the water-soluble magnetic nanoparticles 14 is 1 nm˜200 nm (for example, 1 nm, 5 nm, 50 nm, 100 nm, 200 nm).

Further, according to the sequence of the preparation steps, when the microspheres 11 are coated with the coating layer 15, during the process of embedding the lipid-soluble magnetic nanoparticles 13 and/or the lipid-soluble fluorescent dyes 12 into the interior of the microspheres 11, The liposoluble magnetic nanoparticles 13 and/or the liposoluble fluorescent dye 12 may be embedded in the inside of the coating layer 15 .

In some embodiments, the microspheres 11 may be at least one of magnetic microspheres or non-magnetic microspheres.

In some embodiments, as shown in FIG. 15 , the magnetic microspheres 11 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 11 shown in FIG. 15 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. 16 , the magnetic microspheres at least include: the magnetic microspheres 11 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 11 shown in FIG. 16 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 11 .

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 the magnetic microspheres 11 , 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 to 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 11 .

In some embodiments, as shown in FIG. 17 , the magnetic microspheres 11 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 11 shown in FIG. 17 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. The embedding capacity of magnetic nanoparticles increases the fluorescence intensity and magnetic responsivity of the microspheres.

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, and lipid-soluble fluorescent dyes and lipid-soluble magnetic nanoparticles dispersed at least in the interior of the microspheres. Under the premise of avoiding excessive adsorption of magnetic nanoparticles on the surface of fluorescent microspheres, as shown in Figure 19, the internal dispersion of lipid-soluble magnetic nanoparticles and the external coupling of water-soluble magnetic nanoparticles can enhance the performance of fluorescent magnetic beads. The magnetic 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.

In the above embodiment, in terms of mass percentage, the fluorescent magnetic beads include: microspheres: 50% to 99.5% of fat-soluble fluorescent dyes, 0.1% to 10.0% of fat-soluble magnetic nanoparticles, and 0.1% to 49.5% of water-soluble magnetic nanoparticles. Nanoparticles are.

Further, in the above embodiment, step S20 specifically includes the following steps:

S21: Mix the microspheres coupled with water-soluble magnetic nanoparticles and the gel material, 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 coated with gel Floor.

When the gel material is chitosan, the cross-linking agent can be glutaraldehyde, wherein the mass ratio of chitosan and microspheres is: 0.01-20%, and chitosan chemically interacts with glutaraldehyde through electrostatic interaction. The reaction produces 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.

Further, the operating temperature of step S10 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 S20 is 20-65°C ( For example, 20°C, 25°C, 30°C, 50°C, 65°C), the operating temperature of step S30 is 0-65°C (for example, 0°C, 25°C, 30°C, 50°C, 65°C), and the operating temperature of step S30 It is 0-65 degreeC (for example, 0 degreeC, 25 degreeC, 30 degreeC, 50 degreeC, 65 degreeC).

Preferably, the operating temperature of step S10 in the above embodiment is 0-30°C (eg 0°C, 15°C, 30°C), and the operating temperature of step S20 is 20-30°C (eg 20°C, 25°C, 30°C) ), the operating temperature of step S30 is 0-30°C (eg 0°C, 15°C, 30°C), and the operating temperature of step S40 is 0-30°C (eg 0°C, 15°C, 30°C). Within the above operating temperature range, the autofluorescence increase of the microspheres caused by heating can be effectively avoided, thereby improving the detection sensitivity.

More preferably, the operating temperature of step S10 in the above-mentioned embodiment is room temperature (23 ℃ ± 2 ℃), the operating temperature of step S20 is room temperature (23 ℃ ± 2 ℃), and the operating temperature of step S30 is room temperature (23 ℃ ± 2 ℃). 2° C.), and the operating temperature of step S40 is room temperature (23° C.±2° C.). Within the above operating temperature range, the autofluorescence increase 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 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 displayed 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 to other related technologies Fields are similarly included within the scope of patent protection of this application.

Claims (16)

1. a fluorescent magnetic bead, is characterized in that, described fluorescent magnetic bead comprises: Microspheres; water-soluble magnetic nanoparticles coupled to the outer surface of the microspheres; A fat-soluble fluorescent dye, at least dispersed in the interior of the microspheres; The fat-soluble magnetic nanoparticles are at least dispersed in the interior of the microspheres. 2. The fluorescent magnetic bead according to claim 1, wherein the fluorescent magnetic bead further comprises: The coating layer is coated on the outer surface of the microspheres, and the coating layer covers at least part of the water-soluble magnetic nanoparticles. 3. The magnetic bead according to claim 2, characterized in that, The coating layer is a gel layer or a polymer coating layer; Wherein, the fat-soluble fluorescent dye is dispersed and distributed inside the gel layer; The fat-soluble magnetic nanoparticles are dispersed and distributed inside the gel layer. 4. The magnetic bead according to claim 3, 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; The material of the polymer coating layer is at least one of polystyrene, polymethyl methacrylate, polyethylene-methyl methacrylate, polyacrylonitrile, polyethylene, polypropylene, and polyethyl acrylate. 5 . The fluorescent magnetic beads according to claim 1 , wherein the microspheres are at least one of magnetic microspheres or non-magnetic microspheres. 6 . 6. The fluorescent magnetic beads according to claim 5, wherein the magnetic microspheres comprise at least: A magnetic nanoparticle layer, and a polymer coating layer covering the outer surface of the water-soluble magnetic nanoparticle layer. 7 . The fluorescent magnetic beads according to claim 5 , wherein the magnetic microspheres at least comprise: polymer core microspheres and magnetic nanoparticles distributed inside the polymer core microspheres. 8 . 8. The fluorescent magnetic beads according to claim 5, wherein the magnetic microspheres at least comprise: A magnetic nanoparticle layer, a polymer coating layer coated on the outer surface of the water-soluble magnetic nanoparticle layer, and a fat-soluble magnetic nanoparticle layer distributed inside the polymer coating layer. 9. The fluorescent magnetic beads according to claim 5, 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. 10 . The fluorescent magnetic bead according to claim 1 , wherein the outer surface of the microsphere contains charged functional groups. 11 . 11. The fluorescent magnetic bead according to claim 1, wherein, The fat-soluble magnetic nanoparticles are paramagnetic nanoparticles, 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; The water-soluble magnetic nanoparticles are paramagnetic nanoparticles. 12. The fluorescent magnetic bead according to claim 1, wherein, In terms of mass percentage, the fluorescent magnetic beads include: the microspheres: 50%-99.5% of the fat-soluble fluorescent dye, 0.1%-10.0% of the fat-soluble magnetic nanoparticles, 0.1%-49.5% of the fat-soluble magnetic nanoparticles The water-soluble magnetic nanoparticles are: 13. 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; Wherein, before the step of combining microspheres and water-soluble magnetic nanoparticles by adsorption technology, so that the water-soluble magnetic nanoparticles are coupled to the outer surface of the microspheres, or, before the step of combining microspheres by adsorption technology balls and water-soluble magnetic nanoparticles, after the step of coupling the water-soluble magnetic nanoparticles to the outer surface of the microspheres, the method further comprises: combining the microspheres and the lipid-soluble fluorescent dye by using a swelling technique, so that the lipid-soluble fluorescent dye is dispersed and distributed in the interior of the microspheres; Wherein, before the step of combining microspheres and water-soluble magnetic nanoparticles by adsorption technology, so that the water-soluble magnetic nanoparticles are coupled to the outer surface of the microspheres, or, before the step of combining microspheres by adsorption technology balls and water-soluble magnetic nanoparticles, after the step of coupling the water-soluble magnetic nanoparticles to the outer surface of the microspheres, or, after the combination of the microspheres and the lipid-soluble fluorescent dye using the swelling technique, so that While the fat-soluble fluorescent dye is dispersed and distributed in the interior of the microsphere, the method further includes: combining the microsphere and the fat-soluble magnetic nanoparticle using a swelling technique, so that the fat-soluble magnetic nanoparticle is dispersed and distributed in the microsphere. the inside of the microspheres. 14. The method according to claim 13, characterized in that, in the combination of microspheres and water-soluble magnetic nanoparticles using adsorption technology, the water-soluble magnetic nanoparticles are coupled to the outer surface of the microspheres. After the step, the method further includes: The outer surfaces of the microspheres to which the water-soluble magnetic nanoparticles are coupled are coated with a polymer coating layer, and the polymer coating layer coats at least part of the water-soluble magnetic nanoparticles. 15. The method according to claim 13, characterized in that, in the combination of microspheres and water-soluble magnetic nanoparticles using adsorption technology, the water-soluble magnetic nanoparticles are coupled to the outer surface of the microspheres. After the step, the method further includes: A gel layer is coated on the outer surface of the microspheres to which the water-soluble magnetic nanoparticles are coupled, and the gel layer coats at least part of the water-soluble magnetic nanoparticles. 16. The method of claim 15, 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 gel layer is coated on the outer surface of the microspheres coupled with the water-soluble magnetic nanoparticles, and the operating temperature of the gel layer coating at least part of the water-soluble magnetic nanoparticles is 20 °C. ~65℃; The swelling technology is used to combine the microspheres and the lipid-soluble fluorescent dye, so that the lipid-soluble fluorescent dye is dispersed and distributed in the interior of the microspheres and/or the gel layer, and the operating temperature is 0-65° C. ; The use of swelling technology to combine the microspheres and the lipid-soluble magnetic nanoparticles, so that the lipid-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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