CN115127973A - Fluorescent magnetic beads and method of making the same - Google Patents

Abstract

The application relates to the technical field of magnetic bead modification, and particularly discloses a fluorescent magnetic bead and a manufacturing method thereof, wherein the fluorescent magnetic bead comprises the following components: microspheres; water-soluble magnetic nanoparticles coupled to the outer surface of the microspheres; the gel layer is coated on the outer surface of the microsphere and coats at least part of the water-soluble magnetic nanoparticles; water-soluble fluorescent dye molecules bonded to the outer surface of the gel layer; and the fat-soluble magnetic nano particles are dispersed in the microspheres and/or the gel layer. By the mode, the magnetic response of the fluorescent magnetic beads is improved, meanwhile, interference signals caused by impurities are reduced, the autofluorescence of the microspheres can be reduced, and the detection sensitivity is improved.

Description

Fluorescent 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 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.

During 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 9, 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 gel layer covering the outer surface of the microsphere and condensing The gel layer coats at least part of the water-soluble magnetic nanoparticles, wherein, water-soluble fluorescent dye molecules are combined in the gel layer; the fat-soluble magnetic nanoparticles are dispersed and distributed in the interior of the microspheres and/or the interior of the gel layer.

A second aspect of the present application provides a method for manufacturing fluorescent magnetic beads, the method includes: providing a method for manufacturing fluorescent magnetic beads, the method includes: combining microspheres and water-soluble magnetic nanoparticles by using adsorption technology, so that the water-soluble magnetic nanoparticles The particles are coupled to the outer surface of the microspheres; the water-soluble fluorescent dye molecules are combined with the gel layer material to obtain the gel molecules; the gel molecules are coated on the outer surface of the microspheres coupled with the water-soluble magnetic nanoparticles to form a gel layer with water-soluble fluorescent dye molecules combined inside, wherein 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 to make the water-soluble magnetic nanoparticles Before the step of coupling the magnetic nanoparticles to the outer surface of the microspheres, or, before coating the gel molecules on the outer surface of the microspheres coupled with the water-soluble magnetic nanoparticles, to form a water-soluble fluorescent Before the step of gel layer of dye molecules, or, before coating the gel molecules on the outer surface of microspheres coupled with water-soluble magnetic nanoparticles, to form a gel layer with water-soluble fluorescent dye molecules bound inside After the step of , the method further includes: using swelling technology to combine the microspheres and the liposoluble magnetic nanoparticles, so that the liposoluble magnetic nanoparticles are dispersed in the inside of the microspheres and/or the inside of the gel layer.

Different from the situation in the prior art, the fluorescent magnetic beads of the present application include: water-soluble magnetic nanoparticles coupled to the outer surface of the microspheres, and water-soluble magnetic nanoparticles coated on the outer surface of the microspheres and at least partially coated with the water-soluble magnetic nanoparticles. The gel layer, the water-soluble fluorescent dye molecules bound to the outer surface of the gel layer, and the lipid-soluble magnetic nanoparticles dispersed in the interior of the microspheres and/or the gel layer, on the premise of avoiding excessive adsorption of magnetic nanoparticles on the surface of the microspheres Through the internal dispersion of lipid-soluble magnetic nanoparticles and the external coupling of water-soluble magnetic nanoparticles, the magnetic response signal of the fluorescent magnetic beads can be enhanced, the interference signal caused by impurities can be reduced, and the autofluorescence of the microspheres can be reduced. , to 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;

Fig. 4 is a schematic flowchart of step S20 in Figs. 1-3;

5 is a schematic structural diagram of a fluorescent magnetic bead proposed in the present application;

Fig. 6 is the first structural representation of the microsphere 11 in Fig. 5;

Fig. 7 is the second structural representation of the microsphere 11 in Fig. 5;

Fig. 8 is the third structural schematic diagram of the microsphere 11 in Fig. 5;

Fig. 9 is the magnetic response signal diagram when the magnetic nanoparticles are excessively adsorbed on the surface of the microsphere in the prior art;

FIG. 10 is a graph of the magnetic response signal of the fluorescent magnetic beads of the present application.

Detailed ways

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

Throughout the specification, unless specifically stated otherwise, terms used herein are to be understood as commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. In case of conflict, the present specification takes precedence.

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

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

Referring to FIG. 1, the first 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.

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 water-soluble magnetic nanoparticles in deionized water, adding microspheres, and rotating the water-soluble magnetic nanoparticles to couple with the microspheres to obtain 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 and gel molecules, adding a cross-linking agent, stirring the reaction, and after the obtained product is allowed to stand, magnetic separation is performed to remove residual gel molecules to obtain an external gel molecule. Microspheres coated with a gel layer.

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

S30: Combining microspheres and water-soluble fluorescent dye molecules using adsorption technology, so that the water-soluble fluorescent dye molecules are bound to the outer surface of the gel layer.

Wherein, the outer surface of the gel layer can be chemically modified with required charged functional groups, wherein the charged functional groups can include at least one of charged carboxyl groups, amino groups, sulfonic acid groups or sulfhydryl groups, which are water-soluble for subsequent coupling. Sexual fluorescent dye molecules offer the possibility.

The water-soluble fluorescent dye molecules may have active groups, and the water-soluble fluorescent dye molecules are combined with the gel layer through the active groups. The active group includes at least one of N-hydroxysuccinimide group, carboxyl group, mercapto group, epoxy group or tosyl group, which provides the possibility for water-soluble fluorescent dye molecules to combine with the gel layer material.

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 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 the supernatant is magnetically separated 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, the swelling media can be chloroform, dichloromethane , ethanol, methanol, n-butanol, isobutanol, n-hexane, cyclohexane, tetrahydrofuran, but not limited to the above substances.

Wherein, the microspheres may be at least one of non-crosslinked microspheres, crosslinked porous microspheres or hollow mesoporous microspheres. 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, step S30 may be performed multiple times, respectively combining water-soluble fluorescent dye molecules with different fluorescent characteristics and gel layer materials, or combining water-soluble fluorescent dye molecules and gel layer materials with different concentrations, respectively, to obtain a plurality of Gel layers with different fluorescence intensities are then obtained to obtain fluorescent magnetic beads with different fluorescence intensities, that is, fluorescently encoded magnetic beads. Wherein, different water-soluble fluorescent dye molecules can be mixed in different proportions to prepare different encoded microspheres. It is precisely due to the different proportions of different water-soluble fluorescent dye molecules that endow the prepared fluorescently encoded magnetic beads with different fluorescent characteristics.

Referring to FIG. 2 , a second embodiment of the present application proposes a method for manufacturing fluorescent magnetic beads. The method includes the following steps:

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

S40: 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.

S30: Combining microspheres and water-soluble fluorescent dye molecules using adsorption technology, so that the water-soluble fluorescent dye molecules are bound to the outer surface of the gel layer.

Different from the first embodiment, the second embodiment "combining microspheres and water-soluble magnetic nanoparticles by adsorption technology" and "combining microspheres and lipid-soluble magnetic nanoparticles by swelling technology" occurs before the coating of the gel layer. , therefore, the water-soluble magnetic nanoparticles and the lipid-soluble magnetic nanoparticles are more likely to enter the interior of the microspheres, and the embedding capacity of the water-soluble magnetic nanoparticles and the lipid-soluble magnetic nanoparticles on the microspheres is greatly improved.

Referring to FIG. 3 , a third embodiment of the present application proposes a method for manufacturing fluorescent magnetic beads, which includes the following steps:

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

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

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 microspheres and water-soluble fluorescent dye molecules using adsorption technology, so that the water-soluble fluorescent dye molecules are bound to the outer surface of the gel layer.

Different from the first embodiment, in the third embodiment, "combining microspheres and lipid-soluble magnetic nanoparticles by swelling technology" occurs before "combining microspheres and water-soluble magnetic nanoparticles by adsorption technology", so it can avoid the The hydrophilicity (ie oleophobicity) of the water-soluble magnetic nanoparticles makes it difficult for the fat-soluble magnetic nanoparticles to enter the interior of the microspheres, which greatly increases the number of entrapped fat-soluble magnetic nanoparticles in the microspheres.

The structures of the fluorescent magnetic beads 10 prepared by the above-mentioned first to third embodiments are shown in FIG. 5 . The fluorescent magnetic beads 10 include: microspheres 11 , water-soluble fluorescent dye molecules 15 , lipid-soluble magnetic nanoparticles 14 , and water-soluble magnetic nanoparticles 14 . The magnetic nanoparticles 12 and the gel layer 13 are formed. Among them, the water-soluble fluorescent dye molecules 15 can be combined with the outer surface of the gel layer 13, and the lipid-soluble magnetic nanoparticles 14 can be dispersed in the interior of the microspheres 11. It can be understood that after entering the interior of the microspheres 11 through swelling, the lipid The soluble magnetic nanoparticles 14 are embedded inside the microspheres 11 . The water-soluble magnetic nanoparticles 12 may be coupled to the outer surface of the microspheres 11 . The gel layer 13 covers the outer surface of the microspheres 11 , and the gel layer 13 covers at least part of the water-soluble magnetic nanoparticles 12 . Optionally, the gel layer 13 covers all the water-soluble magnetic nanoparticles 12, and the outer surface of the fluorescent magnetic beads 10 is a smooth surface.

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

In some embodiments, as shown in FIG. 6 , 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. 6 is as follows: adopt the adsorption technology to combine the silica core microspheres 101 and the 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. 7 , the magnetic microspheres 11 at least include: polymer core microspheres 104 and magnetic nanoparticles 105 distributed inside the polymer core microspheres 104 .

Specifically, the material of the polymer core microspheres 104 may include polystyrene, polymethyl methacrylate, polyethylene-methyl methacrylate, polyacrylonitrile, polyethylene, polypropylene, and polyethyl acrylate. at least one of.

Specifically, the manufacturing method of the magnetic microspheres 11 shown in FIG. 7 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. 8 , 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. 8 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 .

The material of the above-mentioned polymer core microspheres 106 may include at least one of polystyrene, polymethyl methacrylate, polyethylene-methyl methacrylate, polyacrylonitrile, polyethylene, polypropylene, and polyethyl acrylate . The outer surface of the microsphere 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. 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 11 are at least one of cross-linked non-magnetic microspheres 11 , non-cross-linked non-magnetic microspheres 11 or hollow mesoporous non-magnetic microspheres 11 .

When the non-magnetic microspheres 11 are cross-linked porous non-magnetic microspheres 11 or hollow mesoporous non-magnetic microspheres 11 , they have the characteristics of high porosity and high specific surface area, therefore, the lipid-soluble magnetic properties in the microspheres 11 can be improved. The embedding capacity of the nanoparticles 14 further improves the fluorescence intensity and magnetic responsivity of the microspheres 11 .

In some embodiments, the material of the water-soluble magnetic nanoparticles 12 is paramagnetic nanoparticles, wherein the paramagnetic nanoparticles can be selected from ferric oxide, ferric oxide, alloys containing nickel or cobalt At least one of the paramagnetic magnetic particles.

In some embodiments, the material of the fat-soluble magnetic nanoparticles 14 is paramagnetic nanoparticles, wherein the paramagnetic nanoparticles can be selected from at least one of ferric oxide or ferric oxide. In addition, the outer surface of the fat-soluble magnetic nanoparticles 14 contains a fat-soluble ligand having at least one of unsaturated fatty acid, saturated fatty acid, unsaturated fatty amine, or saturated fatty amine. The above-mentioned lipid-soluble ligands can bind to the surface of the lipid-soluble magnetic nanoparticles 14 , thereby stabilizing the lipid-soluble magnetic nanoparticles 14 . 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 lipid-soluble magnetic nanoparticles 14 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 12 is 1 nm˜200 nm (for example, 1 nm, 5 nm, 50 nm, 100 nm, 200 nm).

In the above embodiment, in terms of mass percentage, the fluorescent magnetic beads include: 50%-99.5% of the microspheres, 0.1%-10.0% of the water-soluble fluorescent dye molecules, 0.1%-49.5% of the Fat-soluble magnetic nanoparticles, 0.1% to 49.5% of the water-soluble magnetic nanoparticles.

Further, according to the sequence of the preparation steps, when the microspheres 11 are coated with the gel layer 13, during the process of embedding the lipid-soluble magnetic nanoparticles 14 into the interior of the microspheres 11, the lipid-soluble magnetic nanoparticles 14 can be coated with the gel layer 13. Buried inside the gel layer 13 .

Different from the situation in the prior art, the fluorescent magnetic beads 10 of the present application include: water-soluble magnetic nanoparticles 12 coupled to the outer surface of the microspheres 11 , coated on the outer surface of the microspheres 11 and coated at least partially water-soluble The gel layer 13 of the magnetic nanoparticles 12, the water-soluble fluorescent dye molecules 15 bound to the outer surface of the gel layer 13, and the lipid-soluble magnetic nanoparticles 14 dispersed and distributed in the interior of the microspheres 11 and/or the interior of the gel layer 13, such as As shown in FIG. 10 , under the premise of avoiding excessive adsorption of magnetic nanoparticles on the surface of the microspheres 11 , by dispersing the lipid-soluble magnetic nanoparticles 14 inside and coupling with the water-soluble magnetic nanoparticles 12 on the outside, the fluorescence magnetic beads 10 can be enhanced. The magnetic response signal (ie the main group signal), and the interference signal caused by impurities can be reduced, and the autofluorescence of the microspheres 11 can be reduced, and the detection sensitivity can be improved.

In some embodiments, in the fluorescent magnetic beads, microspheres: water-soluble fluorescent dye molecules: lipid-soluble magnetic nanoparticles: water-soluble magnetic nanoparticles are (50%-99.5%): (0.1%- 10.0%): (0.1%-49.5%): (0.1%-49.5%).

Referring to FIG. 4, further, in the above embodiment, step S20 specifically includes the following steps:

S21: Mix the microspheres coupled with water-soluble magnetic nanoparticles and gel molecules, 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 molecule 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, in the above embodiment, the operating temperature of step S10 is 0-100°C, the operating temperature of step S20 is 20-65°C, the operating temperature of step S30 is 0-100°C, and the operating temperature of step S40 is 0-65°C °C.

Preferably, in the above embodiment, the operating temperature of step S10 is 0-30°C, the operating temperature of step S20 is 20-30°C, the operating temperature of step S30 is 0-65°C, and the operating temperature of step S40 is 0-30°C °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 modules or units is only a logical function division. In actual implementation, there may be other divisions, for example, multiple units or components may be combined or Can be integrated into another system, or some features can be ignored, or not implemented.

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

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

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

Claims (12)

1. A fluorescent magnetic bead, comprising:

microspheres;

water-soluble magnetic nanoparticles coupled to the outer surface of the microspheres;

the gel layer is coated on the outer surface of the microsphere, and at least part of the water-soluble magnetic nanoparticles are coated by the gel layer;

water-soluble fluorescent dye molecules bonded to an outer surface of the gel layer;

and the fat-soluble magnetic nano particles are dispersed in the microspheres and/or the gel layer.

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

the water-soluble fluorescent dye molecules have a reactive group, and the water-soluble fluorescent dye molecules are bonded to the gel layer through the reactive group.

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

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

4. The fluorescent magnetic bead of claim 1, wherein the microsphere is at least one of a magnetic microsphere or a non-magnetic microsphere.

5. The fluorescent magnetic bead of claim 4, wherein the magnetic microsphere comprises at least: the magnetic nanoparticle comprises a silica core microsphere, a water-soluble magnetic nanoparticle layer coupled to the outer surface of the silica core microsphere, and a polymer coating layer coated on the outer surface of the water-soluble magnetic nanoparticle layer.

6. The fluorescent magnetic bead of claim 4, wherein the magnetic microsphere comprises at least: the magnetic microsphere comprises a polymer core microsphere and magnetic nanoparticles distributed inside the polymer core microsphere.

7. The fluorescent magnetic bead of claim 4, wherein the magnetic microsphere comprises at least: the magnetic nanoparticle composite material comprises a polymer core microsphere, a water-soluble magnetic nanoparticle layer coupled to the outer surface of the polymer core microsphere, a polymer coating layer coated on the outer surface of the water-soluble magnetic nanoparticle layer, and fat-soluble magnetic nanoparticles distributed inside the polymer core microsphere and/or inside the polymer coating layer.

8. The fluorescent magnetic bead of claim 4, wherein the nonmagnetic microspheres are at least one of cross-linked nonmagnetic microspheres, non-cross-linked nonmagnetic microspheres, or hollow mesoporous nonmagnetic microspheres.

9. Fluorescent magnetic bead according to claim 1,

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

the water-soluble magnetic nanoparticles are paramagnetic nanoparticles.

10. Fluorescent magnetic bead according to claim 1,

the fluorescent magnetic bead comprises the following components in percentage by mass: 50 to 99.5 percent of the microsphere, 0.1 to 10.0 percent of the water-soluble fluorescent dye molecule, 0.1 to 49.5 percent of the fat-soluble magnetic nano-particle and 0.1 to 49.5 percent of the water-soluble magnetic nano-particle.

11. A method for manufacturing fluorescent magnetic beads is characterized by comprising the following steps:

combining microspheres and water-soluble magnetic nanoparticles by adopting an adsorption technology so that the water-soluble magnetic nanoparticles are coupled to the outer surfaces of the microspheres;

coating a gel layer on the outer surface of the microsphere coupled with the water-soluble magnetic nanoparticles, wherein the gel layer coats at least part of the water-soluble magnetic nanoparticles;

combining microspheres and water-soluble fluorescent dye molecules by adopting an adsorption technology so that the water-soluble fluorescent dye molecules are combined on the outer surface of the gel layer;

wherein the method further comprises, prior to the step of combining microspheres with water-soluble magnetic nanoparticles using an adsorption technique such that the water-soluble magnetic nanoparticles are coupled to the outer surface of the microspheres, or prior to the step of combining microspheres with water-soluble fluorescent dye molecules using an adsorption technique such that the water-soluble fluorescent dye molecules are bound to the outer surface of the gel layer, or subsequent to the step of combining microspheres with water-soluble fluorescent dye molecules using an adsorption technique such that the water-soluble fluorescent dye molecules are bound to the outer surface of the gel layer: and combining the microspheres and the fat-soluble magnetic nanoparticles by adopting a swelling technology, so that the fat-soluble magnetic nanoparticles are dispersed in the microspheres and/or the gel layer.

12. The method of claim 11,

combining microspheres and water-soluble magnetic nanoparticles by adopting an adsorption technology, wherein the operation temperature of coupling the water-soluble magnetic nanoparticles to the outer surfaces of the microspheres is 0-100 ℃;

the operation temperature of coating the outer surface of the microsphere coupled with the water-soluble magnetic nanoparticles with a gel layer is 20-65 ℃;

combining microspheres and water-soluble fluorescent dye molecules by adopting an adsorption technology, so that the operation temperature of the water-soluble fluorescent dye molecules combined on the outer surface of the gel layer is 0-100 ℃;

the microspheres and the fat-soluble magnetic nanoparticles are combined by adopting a swelling technology, so that the operating temperature of the fat-soluble magnetic nanoparticles dispersed and distributed in the microspheres and/or the gel layer is 0-65 ℃.

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