CN115127971A - Magnetic beads and methods of making the same - Google Patents

Abstract

The application relates to the technical field of magnetic bead modification, and particularly discloses a magnetic bead and a manufacturing method thereof, wherein the magnetic bead comprises: non-magnetic microspheres; the water-soluble magnetic nano-particles are coupled on the outer surface of the non-magnetic microspheres; and the fat-soluble magnetic nano particles are at least dispersed and distributed in the non-magnetic microspheres. By the mode, the magnetic response of the magnetic beads is improved, meanwhile, interference signals caused by impurities are reduced, autofluorescence of the non-magnetic microspheres can be reduced, and detection sensitivity is improved.

Description

Magnetic beads and methods of making the same

technical field

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

Background technique

Magnetic microspheres are a kind of spherical composite materials with a diameter of nanometer or micrometer. 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 14, 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, an object of the present application is to propose a magnetic bead and a method for making the same, which can reduce the interference signal caused by impurities while improving the magnetic response of the magnetic bead, and can reduce the autofluorescence of non-magnetic microspheres and improve detection. sensitivity.

A first aspect of the present application provides a magnetic bead, the magnetic bead includes: non-magnetic microspheres; water-soluble magnetic nanoparticles coupled to the outer surface of the non-magnetic microspheres; and a gel layer coated on the outer surface of the non-magnetic microspheres surface, and the gel layer coats at least part of the water-soluble magnetic nanoparticles. Lipid-soluble magnetic nanoparticles, at least dispersed in the interior of non-magnetic microspheres.

A second aspect of the present application provides a method for making magnetic beads, the method comprising: combining non-magnetic microspheres and water-soluble magnetic nanoparticles using adsorption technology, so that the water-soluble magnetic nanoparticles are coupled to the outer surface of the non-magnetic microspheres; Wherein, before the step of combining the non-magnetic 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 non-magnetic microspheres, or, before using the adsorption technology to combine the non-magnetic microspheres with the water-soluble magnetic nanoparticles After the step of coupling the water-soluble magnetic nanoparticles to the outer surface of the non-magnetic microspheres, the method further includes: combining the non-magnetic microspheres and the lipid-soluble magnetic nanoparticles by using a swelling technique, so that the lipid-soluble magnetic nanoparticles are combined with the water-soluble magnetic nanoparticles. Nanoparticles are dispersed inside the non-magnetic microspheres.

Different from the situation in the prior art, the magnetic beads of the present application include: water-soluble magnetic nanoparticles coupled to the outer surface of the non-magnetic microspheres, and lipid-soluble magnetic nanoparticles dispersed at least in the interior of the non-magnetic microspheres, As shown in FIG. 15 , under the premise of avoiding excessive adsorption of magnetic nanoparticles on the surface of the microspheres, the magnetic beads of the present application can enhance the magnetic properties of the magnetic beads by swollen internal fat-soluble magnetic nanoparticles and externally coupled water-soluble magnetic nanoparticles. The response signal (ie the main group signal), and the interference signal caused by impurities can be reduced, the autofluorescence of the non-magnetic microspheres can be reduced, and the detection sensitivity can be improved.

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 making magnetic beads according to the first embodiment of the present application;

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

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

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

FIG. 5 is a schematic flowchart of a method for making magnetic beads according to the fifth embodiment of the present application;

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

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

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

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

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

Figure 11 is a schematic flowchart of step S20 in Figures 1-10;

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

13 is a schematic diagram of the second structure of the magnetic bead proposed in the present application;

Fig. 14 is the magnetic response signal diagram when the magnetic nanoparticles are excessively adsorbed on the surface of the microsphere in the prior art;

Fig. 15 is the magnetic response signal diagram of the magnetic beads of the present application;

Fig. 16 is the third structural schematic diagram of the magnetic bead proposed by the present application;

FIG. 17 is a schematic diagram of the fourth structure of the magnetic bead proposed in the present application.

Detailed ways

The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the 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.

The first embodiment of the present application proposes a method for making magnetic beads, and the method includes the following steps:

S10: The non-magnetic microspheres and the water-soluble magnetic nanoparticles are combined by adsorption technology, so that the water-soluble magnetic nanoparticles are coupled to the outer surface of the non-magnetic microspheres.

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

Optionally, the outer surface of the non-magnetic microspheres can be chemically modified with required charged functional groups, wherein the charged functional groups include at least one of charged carboxyl groups, amino groups, sulfonic acid groups or sulfhydryl groups, which are used for coupling. Water-soluble magnetic nanoparticles offer the possibility.

The water-soluble magnetic nanoparticles are dissolved in deionized water, non-magnetic microspheres are added, and the water-soluble magnetic nanoparticles and the non-magnetic microspheres are cross-linked by a rotation reaction to obtain microspheres with water-soluble magnetic nanoparticles coupled on the outer surface.

S20: A gel layer is coated on the outer surface of the non-magnetic microspheres coupled with 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: The non-magnetic microspheres and the lipid-soluble magnetic nanoparticles are combined by swelling technology, so that the lipid-soluble magnetic nanoparticles are dispersed and distributed in the interior of the non-magnetic microspheres and/or the interior of the gel layer.

Specifically, the non-magnetic 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 (the vortex dispersion is uniform, and the rotation reaction time is preset), and the lipid-soluble The magnetic nanoparticles are embedded in the interior of the non-magnetic 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 non-magnetic microspheres, for example, the swelling media can be chloroform, chlorine One or a combination of methane, ethanol, methanol, hexanediol, n-butanol, isobutanol, n-hexane, cyclohexane, and tetrahydrofuran, but not limited to the above-mentioned substances.

Wherein, the non-magnetic microspheres may be at least one of non-crosslinked non-magnetic microspheres, cross-linked porous non-magnetic microspheres or hollow mesoporous non-magnetic microspheres. The cross-linked porous non-magnetic microspheres and hollow mesoporous non-magnetic microspheres have the characteristics of high porosity and high specific surface area, therefore, the embedding capacity of the lipid-soluble magnetic nanoparticles in the non-magnetic microspheres can be improved, thereby improving the magnetic beads. magnetic responsiveness.

The second embodiment of the present application proposes a method for making magnetic beads, and the method includes the following steps:

S10: The non-magnetic microspheres and the water-soluble magnetic nanoparticles are combined by adsorption technology, so that the water-soluble magnetic nanoparticles are coupled to the outer surface of the non-magnetic microspheres.

S30: The non-magnetic microspheres and the fat-soluble magnetic nanoparticles are combined by the swelling technology, so that the fat-soluble magnetic nanoparticles are dispersed in the interior of the non-magnetic microspheres.

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

Different from the first embodiment, the second embodiment swells before the gel layer is coated. Therefore, the fat-soluble magnetic nanoparticles can more easily enter the interior of the non-magnetic microspheres, and the fat-soluble magnetic properties in the non-magnetic microspheres are greatly improved. Embedding capacity of nanoparticles.

The third embodiment of the present application proposes a method for making magnetic beads, the method comprising the following steps:

S30: The non-magnetic microspheres and the fat-soluble magnetic nanoparticles are combined by the swelling technology, so that the fat-soluble magnetic nanoparticles are dispersed in the interior of the non-magnetic microspheres.

S10: The non-magnetic microspheres and the water-soluble magnetic nanoparticles are combined by adsorption technology, so that the water-soluble magnetic nanoparticles are coupled to the outer surface of the non-magnetic microspheres.

S20: A gel layer is coated on the outer surface of the non-magnetic microspheres coupled with 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 third 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 to enter the interior of the non-magnetic microspheres, which greatly improves the embedding capacity of the fat-soluble magnetic nanoparticles in the non-magnetic microspheres.

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

Thus, the structures of the magnetic beads 10 prepared by the above-mentioned first to third embodiments or their alternatives are shown in FIG. 12 , and the magnetic beads 10 include: non-magnetic microspheres 11 , fat-soluble magnetic nanoparticles 12 , water-soluble The magnetic nanoparticles 13 and the coating layer 14 are formed. The fat-soluble magnetic nanoparticles 12 are dispersed in the interior of the non-magnetic microspheres 11 . It can be understood that, after entering the interior of the non-magnetic microspheres 11 through swelling, the fat-soluble magnetic nanoparticles 12 are embedded in the non-magnetic microspheres 11 . internal. Water-soluble magnetic nanoparticles 13 can be coupled to the outer surface of the non-magnetic microspheres 11 . The coating layer 14 coats the outer surface of the non-magnetic microspheres 11 , and the coating layer 14 coats at least part of the water-soluble magnetic nanoparticles 13 . Optionally, the coating layer 14 covers all the water-soluble magnetic nanoparticles 13, and the outer surface of the magnetic beads 10 is a smooth surface. Wherein, the above-mentioned coating layer 14 may be a gel layer or a polymer coating layer. It can be understood that the fat-soluble magnetic nanoparticles 12 can be dispersed and distributed in the interior of the gel layer, but limited by the material of the polymer coating layer, the fat-soluble magnetic nanoparticles 12 cannot be dispersed in the interior of the polymer coating layer. .

Further, step S20 in the above-mentioned first embodiment to third embodiment can be deleted, and the structure of the magnetic bead 10 thus obtained is shown in FIG. 16 . The magnetic bead 10 includes: Magnetic nanoparticles 12 and water-soluble magnetic nanoparticles 13 . The fat-soluble magnetic nanoparticles 12 are dispersed in the interior of the non-magnetic microspheres 11 . It can be understood that, after entering the interior of the non-magnetic microspheres 11 through swelling, the fat-soluble magnetic nanoparticles 12 are embedded in the non-magnetic microspheres 11 . internal. Water-soluble magnetic nanoparticles 13 can be coupled to the outer surface of the non-magnetic microspheres 11 .

Different from the situation in the prior art, the magnetic beads of the present application include: water-soluble magnetic nanoparticles coupled to the outer surface of the non-magnetic microspheres, and lipid-soluble magnetic nanoparticles dispersed at least in the interior of the non-magnetic microspheres, Under the premise of avoiding excessive adsorption of magnetic nanoparticles on the surface of the microspheres, the magnetic response signal (i.e. the main group signal) of the magnetic beads can be enhanced by dispersing lipid-soluble magnetic nanoparticles inside and coupling water-soluble magnetic nanoparticles outside. And reduce the interference signal caused by impurities, and can reduce the autofluorescence of non-magnetic microspheres, and improve the detection sensitivity.

In some embodiments, the material of the non-magnetic microspheres 11 is a polymer, and the polymer includes polystyrene, polymethyl methacrylate, polyethylene-methyl methacrylate, polyacrylonitrile, polyethylene, polypropylene, At least one of polyethyl acrylate. The outer surface of the non-magnetic 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 13 are paramagnetic nanoparticles, wherein the paramagnetic nanoparticles can be selected from triiron tetroxide, ferric oxide, and alloy-type paramagnetic particles containing nickel or cobalt at least one of the magnetic particles.

In some embodiments, the fat-soluble magnetic nanoparticles 12 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 12 contain a fat-soluble ligand of at least one of unsaturated fatty acids, saturated fatty acids, unsaturated fatty amines, or saturated fatty amines. The above-mentioned lipid-soluble ligands can bind to the surface of the lipid-soluble magnetic nanoparticles 12 , thereby stabilizing the lipid-soluble magnetic nanoparticles 12 . The fat-soluble ligand may include at least one of oleic acid, oleylamine, or stearic acid.

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

In some embodiments, by mass percentage, the magnetic beads 10 include: 50%-99.5% non-magnetic microspheres 11, 0.1%-49.9% fat-soluble magnetic nanoparticles 12, 0.1%-49.9% water-soluble magnetic nanoparticles Granules 13.

The fourth embodiment of the present application proposes a method for making magnetic beads, and the method includes the following steps:

S40: The non-magnetic microspheres and the lipid-soluble fluorescent dyes are combined by the swelling technology, so that the lipid-soluble fluorescent dyes are dispersed and distributed inside the non-magnetic microspheres.

Specifically, the non-magnetic 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 fluorescence The dye is embedded in the non-magnetic microspheres, and the supernatant is magnetically separated to obtain fluorescent microspheres with lipid-soluble fluorescent dyes distributed inside. Wherein, the first medium and the third medium are both swelling media, specifically a single or mixed solvent that can not only disperse and distribute fat-soluble magnetic nanoparticles, but also swell non-magnetic microspheres, such as chloroform, n-butanol, isopropyl alcohol, etc. One or a combination of butanol, n-hexane, cyclohexane, and tetrahydrofuran, but not limited to the above-mentioned substances.

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.

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, it can improve the liposoluble fluorescent dyes in the non-magnetic microspheres. Embedding capacity, thereby increasing the fluorescence intensity of the microspheres.

Further, step S40 may be performed multiple times, respectively combining lipid-soluble fluorescent dyes and non-magnetic microspheres with different fluorescence characteristics, or combining lipid-soluble fluorescent dyes and non-magnetic microspheres with different concentrations, respectively, to obtain multiple different fluorescent dyes. Intensity of the microspheres, that is, 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.

S30: The non-magnetic microspheres and the fat-soluble magnetic nanoparticles are combined by the swelling technology, so that the fat-soluble magnetic nanoparticles are dispersed in the interior of the non-magnetic microspheres.

S10: The non-magnetic microspheres and the water-soluble magnetic nanoparticles are combined by adsorption technology, so that the water-soluble magnetic nanoparticles are coupled to the outer surface of the non-magnetic microspheres.

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

The fifth embodiment of the present application proposes a method for making magnetic beads, and the method includes the following steps:

S40: The non-magnetic microspheres and the lipid-soluble fluorescent dyes are combined by the swelling technology, so that the lipid-soluble fluorescent dyes are dispersed and distributed inside the non-magnetic microspheres.

S10: The non-magnetic microspheres and the water-soluble magnetic nanoparticles are combined by adsorption technology, so that the water-soluble magnetic nanoparticles are coupled to the outer surface of the non-magnetic microspheres.

S30: The non-magnetic microspheres and the fat-soluble magnetic nanoparticles are combined by the swelling technology, so that the fat-soluble magnetic nanoparticles are dispersed in the interior of the non-magnetic microspheres.

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

The sixth embodiment of the present application proposes a method for making magnetic beads, and the method includes the following steps:

S40: The non-magnetic microspheres and the lipid-soluble fluorescent dyes are combined by the swelling technology, so that the lipid-soluble fluorescent dyes are dispersed and distributed inside the non-magnetic microspheres.

S10: The non-magnetic microspheres and the water-soluble magnetic nanoparticles are combined by adsorption technology, so that the water-soluble magnetic nanoparticles are coupled to the outer surface of the non-magnetic microspheres.

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

S30: The non-magnetic microspheres and the lipid-soluble magnetic nanoparticles are combined by swelling technology, so that the lipid-soluble magnetic nanoparticles are dispersed and distributed in the interior of the non-magnetic microspheres and/or the interior of the gel layer.

The seventh embodiment of the present application proposes a method for making magnetic beads, and the method includes the following steps:

S50: The non-magnetic microspheres and lipid-soluble fluorescent dyes, the non-magnetic microspheres and the lipid-soluble magnetic nanoparticles are simultaneously combined by swelling technology, so that the lipid-soluble fluorescent dyes and lipid-soluble magnetic nanoparticles are dispersed and distributed inside the non-magnetic microspheres.

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

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

S10: The non-magnetic microspheres and the water-soluble magnetic nanoparticles are combined by adsorption technology, so that the water-soluble magnetic nanoparticles are coupled to the outer surface of the non-magnetic microspheres.

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

The eighth embodiment of the present application proposes a method for manufacturing a magnetic bead, and the method includes the following steps:

S10: The non-magnetic microspheres and the water-soluble magnetic nanoparticles are combined by adsorption technology, so that the water-soluble magnetic nanoparticles are coupled to the outer surface of the non-magnetic microspheres.

S50: The non-magnetic microspheres and lipid-soluble fluorescent dyes, the non-magnetic microspheres and the lipid-soluble magnetic nanoparticles are simultaneously combined by swelling technology, so that the lipid-soluble fluorescent dyes and lipid-soluble magnetic nanoparticles are dispersed and distributed inside the non-magnetic microspheres.

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

The ninth embodiment of the present application proposes a method for making magnetic beads, and the method includes the following steps:

S10: The non-magnetic microspheres and the water-soluble magnetic nanoparticles are combined by adsorption technology, so that the water-soluble magnetic nanoparticles are coupled to the outer surface of the non-magnetic microspheres.

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

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

The tenth embodiment of the present application proposes a method for manufacturing a magnetic bead, and the method includes the following steps:

S30: The non-magnetic microspheres and the fat-soluble magnetic nanoparticles are combined by the swelling technology, so that the fat-soluble magnetic nanoparticles are dispersed in the interior of the non-magnetic microspheres.

S10: The non-magnetic microspheres and the water-soluble magnetic nanoparticles are combined by adsorption technology, so that the water-soluble magnetic nanoparticles are coupled to the outer surface of the non-magnetic microspheres.

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

S40: The non-magnetic microspheres and the liposoluble fluorescent dye are combined by the swelling technology, so that the liposoluble fluorescent dye is dispersed and distributed in the interior of the non-magnetic microspheres and/or the interior of the gel layer.

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

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

Further, step S20 in the above-mentioned fourth embodiment to tenth embodiment can be deleted, and the structure of the magnetic bead 10 obtained therefrom is shown in FIG. 17 . The magnetic bead 10 includes: Fluorescent dye 15 , lipid-soluble magnetic nanoparticles 12 and water-soluble magnetic nanoparticles 13 . Among them, the lipid-soluble fluorescent dye 15 and the lipid-soluble magnetic nanoparticles 12 are dispersed in the interior of the non-magnetic microspheres 11 . The magnetic nanoparticles 12 are embedded inside the non-magnetic microspheres 11 . Water-soluble magnetic nanoparticles 13 can be coupled to the outer surface of the non-magnetic microspheres 11 .

Further, in the above-mentioned first to tenth embodiments, 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 increase of the autofluorescence of the non-magnetic 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, 30°C, 50°C, 65°C), the operating temperature of step S30 is 0～65°C (eg 0°C, 25°C, 30°C, 50°C, 65°C), and the operating temperature of step S30 is 0～65°C 65°C (eg 0°C, 25°C, 30°C, 50°C, 65°C).

Preferably, the operating temperature of step S10 in the above embodiment is 0-30°C (eg 0°C, 25°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, 25°C, 30°C), and the operating temperature of step S40 is 0-30°C (eg 0°C, 25°C, 30°C). Within the above operating temperature range, the increase of autofluorescence of the non-magnetic microspheres 11 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 increase of autofluorescence of the non-magnetic microspheres 11 caused by heating can be effectively avoided, thereby improving the detection sensitivity.

The magnetic beads 10 of the present application include: water-soluble magnetic nanoparticles 13 coupled to the outer surface of the non-magnetic microspheres 11 ; The coating layer 14 and the fat-soluble magnetic nanoparticles 12 dispersed in the interior of the non-magnetic microspheres 11 and/or the coating layer 14, under the premise of avoiding excessive adsorption of the magnetic nanoparticles on the surface of the microspheres, through the internal dispersion Distributed with lipid-soluble magnetic nanoparticles and externally coupled with water-soluble magnetic nanoparticles, the magnetic response signal (ie the main group signal) of the magnetic beads 10 can be enhanced, the interference signal caused by impurities can be reduced, and the non-magnetic microspheres can be reduced. The autofluorescence of 11 increases 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 (17)

1. a magnetic bead, is characterized in that, described magnetic bead comprises: non-magnetic microspheres; water-soluble magnetic nanoparticles, coupled to the outer surface of the non-magnetic microspheres; The fat-soluble magnetic nanoparticles are at least dispersed in the interior of the non-magnetic microspheres. 2. The magnetic bead according to claim 1, wherein the magnetic bead further comprises: A coating layer is coated on the outer surface of the non-magnetic 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 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 magnetic beads according to claim 1, wherein the non-magnetic microspheres are at least one of non-crosslinked non-magnetic microspheres, cross-linked porous non-magnetic microspheres or hollow mesoporous non-magnetic microspheres kind. 6 . The magnetic bead according to claim 1 , wherein the material of the non-magnetic microspheres is a polymer, and the outer surfaces of the non-magnetic microspheres contain charged functional groups. 7 . 7. The magnetic bead according to claim 1, wherein, 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. 8. The magnetic bead according to claim 1, wherein, The water-soluble magnetic nanoparticles are paramagnetic nanoparticles. 9. The magnetic bead according to claim 1, wherein the magnetic bead further comprises: The liposoluble fluorescent dye is at least dispersed in the interior of the non-magnetic microspheres. 10. The magnetic bead according to claim 3, wherein the magnetic bead further comprises: The fat-soluble fluorescent dye is dispersed and distributed at least inside the gel layer. 11. The magnetic bead according to claim 1, wherein, The particle size of the non-magnetic microspheres is 1 μm˜50 μm; The particle size of the fat-soluble magnetic nanoparticles is 1 nm to 200 nm; The particle size of the water-soluble magnetic nanoparticles is 1 nm˜200 nm. 12. The magnetic bead according to claim 1, wherein, By mass percentage, the magnetic beads include: 50%-99.5% of the non-magnetic microspheres, 0.1%-49.9% of the fat-soluble magnetic nanoparticles, 0.1%-49.9% of the water-soluble magnetic nanoparticles particles. 13. A method for making magnetic beads, wherein the method comprises: Using adsorption technology to combine non-magnetic microspheres and water-soluble magnetic nanoparticles, so that the water-soluble magnetic nanoparticles are coupled to the outer surface of the non-magnetic microspheres; Wherein, before the step of combining the non-magnetic 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 non-magnetic microspheres, or, in the After the step of combining the non-magnetic 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 non-magnetic microspheres, the method further comprises: using swelling The technology combines the non-magnetic microspheres and the lipid-soluble magnetic nanoparticles, so that the lipid-soluble magnetic nanoparticles are dispersed and distributed inside the non-magnetic microspheres. 14. The method of claim 13, wherein After the step of combining the non-magnetic 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 non-magnetic microspheres, the method further includes: A polymer coating layer is coated on the outer surface of the non-magnetic microspheres to which the water-soluble magnetic nanoparticles are coupled, and the polymer coating layer coats at least part of the water-soluble magnetic nanoparticles . 15. The method of claim 14, wherein: After the step of combining the non-magnetic 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 non-magnetic microspheres, 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 fat-soluble magnetic nanoparticles, so that the operating temperature of the fat-soluble magnetic nanoparticles dispersed in the microspheres is 0-65°C. 17. The method of claim 13, wherein the method further comprises: The non-magnetic microspheres and the liposoluble fluorescent dye are combined by a swelling technique, so that the liposoluble fluorescent dye is dispersed and distributed at least inside the non-magnetic microspheres.

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