High-magnetic-response micron magnetic bead and preparation method and application thereof

Abstract

The invention belongs to the technical field of micron magnetic beads, and relates to a micron magnetic bead with high magnetic response as well as a preparation method and application thereof. The invention discloses a high magnetic response micron magnetic bead preparation method, which comprises: S1, uniformly mixing non-porous silica microspheres, triethanolamine, hexadecyl trimethyl ammonium bromide, sodium salicylate and water, and adding a silane coupling agent to carry out a reaction to obtain non-porous silica and dendritic mesoporous silicon composite microspheres; s2, in a nitrogen atmosphere, mixing ferric acetylacetonate, 2-pyrrolidone and the non-porous silicon dioxide and dendritic mesoporous silicon composite microspheres to obtain an iron precursor mixture; performing thermal decomposition reaction on the iron precursor mixture, and then performing magnetic separation and cleaning to obtain micron magnetic beads with high magnetic response; the high-magnetic-response micron magnetic bead is of a core-shell structure, the inner core of the high-magnetic-response micron magnetic bead is a non-porous silicon dioxide microsphere, the middle layer is a dendritic mesoporous silicon layer, and the outer shell is a ferroferric oxide layer; and ferroferric oxide is loaded in mesopores of the dendritic mesoporous silicon layer.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of micron magnetic beads, and relates to a micron magnetic bead with high magnetic response and a preparation method and application thereof. BACKGROUND

[0002] As a new type of nanomaterial, the magnetic bead is a core raw material most widely used in the field of in vitro diagnosis, and is usually a spherical magnetic material with a particle size of several hundred nanometers to several microns, which is combined by inorganic magnetic material and organic polymer. The magnetic bead can be quickly separated from a substrate liquid under the action of an external magnetic field in combination with immunodetection, molecular diagnosis and biochemical analysis in in vitro diagnosis. For the magnetic bead used as a carrier or signal marking material for in vitro separation and disease diagnosis, only the magnetic bead with good spherical homogeneous structure, high specific saturation magnetization, good biocompatibility and dispersion stability can ensure the sensitivity and consistency of detection.

[0003] At present, the preparation methods of the magnetic bead mainly include an interface deposition method, a polymer embedding method, a seed polymerization method, a suspension polymerization method, a dispersion polymerization method and an emulsion polymerization method. The seed polymerization method is most widely used, and the method uses a polymer microsphere with a porous structure as a template, performs sulfonation (-SO3) or nitration (-NO2) on the inside and outside of the microsphere hole to make the microsphere hole have a hydrophilic interface, then the microsphere is soaked in an iron salt aqueous solution, and under appropriate reaction conditions, superparamagnetic Fe3O4 or gamma-Fe3O4 is generated in the hole, and finally a monomer containing an active functional group is selected to swell, polymerize and coat the microsphere, so that the microsphere hole is closed and the surface is functionalized. However, due to the incompatibility between the inorganic magnetic nanoparticles and the organic olefin monomer, the seed polymerization method has a complex preparation process, and the prepared magnetic beads mostly have uneven particle size distribution, low magnetic content and poor biocompatibility. Therefore, it is imperative to develop a new magnetic bead preparation method to prepare a magnetic bead with uniform particle size distribution, high magnetic content and good biocompatibility. SUMMARY

[0004] The application aims to solve the above problems in the prior art, and provides a micron magnetic bead with a non-porous silica microsphere as a core, a dendritic mesoporous silica layer and a ferroferric oxide layer coated in sequence, and the micron magnetic bead has high magnetic response performance.

[0005] One object of the application can be achieved by the following technical solutions.

[0006] A preparation method of a micron magnetic bead with high magnetic response, comprising:

[0007] S1, mixing the non-porous silica microspheres, triethanolamine, cetyltrimethylammonium bromide, sodium salicylate and water, then adding the silane coupling agent drop by drop, and reacting under mechanical stirring to obtain non-porous silica@dendritic mesoporous silica composite microspheres nSiO2@nSiO2@dSiO2;

[0008] S2, mixing and stirring acetylacetone iron, 2-pyrrolidone and the non-porous silica@dendritic mesoporous silica composite microspheres under a nitrogen atmosphere to obtain an iron precursor mixture; performing thermal decomposition reaction on the iron precursor mixture, then performing magnetic separation and washing to obtain the high-magnetic-response micron magnetic beads nSiO2@nSiO2@dSiO2@Fe3O4;

[0009] The high-magnetic-response micron magnetic beads have a core-shell structure, the inner core is the non-porous silica microspheres, the intermediate layer is the dendritic mesoporous silica layer, and the outer shell is the magnetite layer; and the magnetite is loaded in the mesopores of the dendritic mesoporous silica layer.

[0010] Preferably, the average particle size of the high-magnetic-response micron magnetic beads is 0.8-3 μm.

[0011] Further preferably, the average particle size of the high-magnetic-response micron magnetic beads is 1-1.5 μm.

[0012] Preferably, the average particle size of the non-porous silica microspheres in step S1 is 600-800 nm.

[0013] Preferably, the non-porous silica microspheres in step S1 are layer-by-layer assembled non-porous silica microspheres nSiO2@nSiO2.

[0014] Further preferably, the layer-by-layer assembled non-porous silica microspheres nSiO2@nSiO2 are prepared by assembling a silica layer on the surface of the non-porous silica microsphere single core nSiO2.

[0015] Further preferably, the average particle size of the layer-by-layer assembled non-porous silica microspheres nSiO2@nSiO2 is 600-800 nm.

[0016] Preferably, the preparation method of the non-porous silica microspheres in step S1 comprises:

[0017] S11, mixing ethanol, ammonia water and water, then adding the silane coupling agent drop by drop to obtain a first mixed solution, and reacting under mechanical stirring to obtain a seed solution containing non-porous silica microsphere single core nSiO2;

[0018] S12, mixing ethanol, ammonia water and water, then adding the silane coupling agent drop by drop to obtain a second mixed solution; adding the seed solution containing non-porous silica microsphere single core nSiO2, and reacting under mechanical stirring for 1-20 h.

[0019] S13, repeating step (2) 0-10 times to obtain layer-by-layer assembled nonporous silica microspheres nSiO2@nSiO2.

[0020] Further preferably, in steps S11 and S12, the silane coupling agent comprises one or more of tetraethyl orthosilicate, acetyltrimethylsilane, methacryloyloxysilane, and tetramethyl orthosilicate.

[0021] Further preferably, in step S12, the seed solution containing nonporous silica microspheres mononuclear nSiO2 is added to the second mixed solution in a volume of 1 / 8-1 / 2.

[0022] Further preferably, in step S12, the seed solution containing nonporous silica microspheres mononuclear nSiO2 is added to the second mixed solution in a volume of 1 / 4-1 / 2.

[0023] Further preferably, in step S11, the volume ratio of water, ethanol, ammonia, and silane coupling agent is (1-50):(10-100):(1-50):(1-10).

[0024] In step S12, the volume ratio of water, ethanol, ammonia, and silane coupling agent is (1-50):(10-100):(1-50):(1-10).

[0025] Further preferably, in step S11, the volume ratio of water, ethanol, ammonia, and silane coupling agent is (5-20):(20-50):(1-5):(1-5).

[0026] In step S12, the volume ratio of water, ethanol, ammonia, and silane coupling agent is (5-20):(20-50):(1-5):(1-5).

[0027] Further preferably, the first mixed solution is the same as the second mixed solution.

[0028] As a preference, the thickness of the dendritic mesoporous silica layer is 200-450 nm.

[0029] As a preference, in step S1, the mass ratio of nonporous silica microspheres, triethanolamine, cetyltrimethylammonium bromide, and sodium salicylate is (100-1000):(10-200):(200-600):(100-500).

[0030] Further preferably, the mass ratio of nonporous silica microspheres, triethanolamine, cetyltrimethylammonium bromide, and sodium salicylate is 500:(40-100):(300-500):(200-300).

[0031] Further preferably, the mass ratio of the non-porous silica microspheres, triethanolamine, cetyltrimethylammonium bromide, and sodium salicylate is 500:68:380:218.

[0032] Preferably, the stirring time in step S1 is 1-600 min.

[0033] Preferably, after mechanical stirring in step S1, post-treatment is performed, which includes centrifugal collection of the precipitate, washing with ethanol, and then dispersing in a mixed solution of concentrated hydrochloric acid and ethanol, heating and refluxing, and centrifugal collection of the precipitate.

[0034] Further preferably, the heating and refluxing temperature is 50-80℃, and the time is 12-48 h.

[0035] Preferably, the mass-volume ratio of the iron acetylacetonate, 2-pyrrolidone, and non-porous silica@dendritic mesoporous silica composite microspheres in step S2 is (0.1-2.0) g:(10-50) ml:(0.01-1.0) g.

[0036] Further preferably, the mass-volume ratio of the iron acetylacetonate, 2-pyrrolidone, and non-porous silica@dendritic mesoporous silica composite microspheres in step S2 is (0.5-1.0) g:(15-30) ml:0.025 g.

[0037] Preferably, the stirring time in step S2 is 1-240 min.

[0038] Preferably, the temperature of the thermal decomposition reaction in step S2 is 180-300℃, and the time is 1-300 min.

[0039] Further preferably, the temperature of the thermal decomposition reaction in step S2 is 210-265℃, and the time is 1-60 min.

[0040] In the thermal decomposition process, the iron precursor filled in the dendritic mesoporous silica layer and on the outer surface of the non-porous silica@dendritic mesoporous silica composite microspheres is converted into ferroferric oxide.

[0041] The second object of the application can be achieved by the following technical solutions:

[0042] A high-magnetic-response micron magnetic bead is prepared by the above preparation method.

[0043] Preferably, the high-magnetic-response micron magnetic bead is of a core-shell structure, the inner core is a non-porous silica microsphere, the intermediate layer is a dendritic mesoporous silica layer, and the outer shell is a ferroferric oxide layer; and the dendritic mesoporous silica layer has ferroferric oxide loaded inside the mesopores.

[0044] Further preferably, the average particle size of the non-porous silica microspheres is 600-800 nm, the average thickness of the dendritic mesoporous silica layer is 200-600 nm, and the average thickness of the ferroferric oxide layer is 100-300 nm.

[0045] Further preferably, the average thickness of the dendritic mesoporous silica layer in the high-magnetic-response micro-magnetic beads accounts for 15-50% of the average particle size of the high-magnetic-response micro-magnetic beads.

[0046] More preferably, the average thickness of the dendritic mesoporous silica layer in the high-magnetic-response micro-magnetic beads accounts for 25-35% of the average particle size of the high-magnetic-response micro-magnetic beads.

[0047] Preferably, the high-magnetic-response micro-magnetic beads have a sedimentation rate of <5% after being left to stand in ultrapure water at room temperature for 12-48 hours.

[0048] Further preferably, the high-magnetic-response micro-magnetic beads have a concentration of 1-30 mg / mL in ultrapure water.

[0049] More preferably, the high-magnetic-response micro-magnetic beads have a concentration of 10 mg / mL in ultrapure water.

[0050] Preferably, the high-magnetic-response micro-magnetic beads are functionalized with Tosyl modification groups with a circumscribed value of 100-500 μmol / g, and the functionalized micro-magnetic beads have a sedimentation rate of <5% after being left to stand in ultrapure water at room temperature for 12-48 hours at a concentration of 1-20 mg / mL.

[0051] Further preferably, the functionalized micro-magnetic beads are used in a Tosyl magnetic bead coupling kit, and the antibody coupling rate is 60-95%.

[0052] More preferably, the functionalized micro-magnetic beads are used in a Tosyl magnetic bead coupling kit, and the antibody coupling rate is 80-95%.

[0053] The third object of the present application can be achieved by the following technical solution:

[0054] A high-magnetic-response micro-magnetic bead in the field of chemiluminescence detection.

[0055] Preferably, the application comprises functionalizing the high-magnetic-response micro-magnetic beads with Tosyl modification groups, and the antibody coupling rate of the functionalized micro-magnetic beads is 80-95%.

[0056] Further preferably, the concentration of the Tosyl modification groups is 100-500 μmol / g.

[0057] Further preferably, the antibody is an IgG antibody.

[0058] Compared with the prior art, the present application has the following beneficial effects:

[0059] 1、The high-magnetic-response micron magnetic beads of the present application take non-porous silica microspheres as the inner core, and are coated with a dendritic mesoporous silica layer and a ferroferric oxide layer in sequence. The non-porous silica microsphere inner core provides stable skeleton support for the magnetic beads, ensuring the rigidity and morphological uniformity of the overall structure; the dendritic mesoporous silica layer in the middle provides sufficient and uniform attachment sites for the loading of the outermost ferroferric oxide layer, and reduces the stress conflict between the inner core and the outer layer material through the buffering effect of the pores; the outer ferroferric oxide layer directly imparts efficient magnetic response capability to the magnetic beads. This structure enables the magnetic beads to maintain structural integrity while achieving efficient balance between magnetism and stability.

[0060] 2、The high specific surface area and open pores of the dendritic mesoporous silica layer in the high-magnetic-response micron magnetic beads of the present application maximize the loading capacity of ferroferric oxide, and in combination with the characteristic of the outer magnetic material directly interacting with the magnetic field, the response speed and separation efficiency of the magnetic beads to the external magnetic field are far superior to those of traditional core-shell structure magnetic beads; and the non-porous silica inner core greatly improves the suspension and dispersibility of the magnetic beads in solution, reduces the sedimentation phenomenon during the reaction process, and ensures the sufficiency of contact with target substances.

[0061] 3、The outer ferroferric oxide layer of the high-magnetic-response micron magnetic beads of the present application can be directly surface-modified, facilitating the coupling of specific recognition molecules and expanding the application field. DETAILED DESCRIPTION

[0062] Figure 1 TEM image and particle size distribution graph of the layer-by-layer assembled non-porous silica microspheres nSiO2@nSiO2 in Example 1 of the present application.

[0063] Figure 2 TEM image and particle size distribution graph of the layer-by-layer assembled non-porous silica / dendritic mesoporous silica composite microspheres nSiO2@nSiO2@dSiO2 in Example 1 of the present application.

[0064] Figure 3 TEM image and particle size distribution graph of the layer-by-layer assembled non-porous silica / dendritic mesoporous silica / ferroferric oxide micron magnetic beads nSiO2@nSiO2@dSiO2@Fe3O4 in Example 1 of the present application.

[0065] Figure 4Zeta potential diagram of the layer-by-layer assembled nonporous silica microspheres nSiO2@nSiO2, the layer-by-layer assembled nonporous silica / dendritic mesoporous silica composite microspheres nSiO2@nSiO2@dSiO2, and the layer-by-layer assembled nonporous silica / dendritic mesoporous silica / ferroferric oxide micro magnetic beads nSiO2@nSiO2@dSiO2@Fe3O4 in Example 1 of the present application.

[0066] Figure 5 Room temperature hysteresis regression curve diagram of the layer-by-layer assembled nonporous silica / dendritic mesoporous silica / ferroferric oxide micro magnetic beads nSiO2@nSiO2@dSiO2@Fe3O4 in Example 1 of the present application. DETAILED DESCRIPTION

[0067] The following are specific embodiments of the present application, which further describe the technical solutions of the present application, but the present application is not limited to these embodiments.

[0068] Unless otherwise specified, the materials used in the present application are conventional commercially available products, and the methods used are conventional technical means.

[0069] In this context, the preparation method of the high-magnetic-response micro magnetic beads comprises:

[0070] S1, mixing nonporous silica microspheres, triethanolamine, cetyltrimethylammonium bromide, sodium salicylate, and water in a mass ratio of 500:(40-100):(300-500):(200-300), and then adding silane coupling agent dropwise, and reacting under mechanical stirring to obtain nonporous silica@dendritic mesoporous silica composite microspheres nSiO2@nSiO2@dSiO2;

[0071] S2, mixing and stirring acetylacetone iron, 2-pyrrolidone, and nonporous silica@dendritic mesoporous silica composite microspheres in a mass-volume ratio of (0.5-1.0) g:(15-30) ml:0.025 g under a nitrogen atmosphere to obtain an iron precursor mixture; after thermal decomposition reaction of the iron precursor mixture at 180-300℃ for 1-300 min, performing magnetic separation and washing to obtain high-magnetic-response micro magnetic beads nSiO2@nSiO2@dSiO2@Fe3O4;

[0072] The high-magnetic-response micro magnetic beads are of a core-shell structure, the inner core is nonporous silica microspheres, the middle layer is a dendritic mesoporous silica layer, and the outer shell is a ferroferric oxide layer; and the ferroferric oxide is loaded in the mesopores of the dendritic mesoporous silica layer.

[0073] In the present text, only when the dendritic mesoporous silica layer and the ferroferric oxide layer are sequentially loaded on the surface of the mononuclear nSiO2 or the layer-by-layer assembled nonporous silica microspheres nSiO2@nSiO2 can the stable magnetic performance described in the present application be achieved, and the magnetic response is fast and the suspension is good; if the dendritic mesoporous silica microspheres are directly used as the inner core, the batch magnetic content and the suspension of the micron magnetic beads will be affected; if the dendritic mesoporous silica intermediate layer is removed and the ferroferric oxide layer is directly loaded on the surface of the layer-by-layer assembled nonporous silica microspheres nSiO2@nSiO2, the magnetic content will be reduced.

[0074] In the present text, the particle size of the layer-by-layer assembled nonporous silica / dendritic mesoporous silica / ferroferric oxide micron magnetic beads is 0.85-1.25 μm, and after functional modification, it can be applied to chemiluminescence detection.

[0075] Example 1

[0076] (1) Layer-by-layer assembled nonporous silica microspheres:

[0077] Take 9.9 mL of water, 33.4 mL of ethanol, and 3.3 mL of ammonia water, mix them uniformly at room temperature under mechanical stirring, dropwise add 3.4 mL of tetraethyl orthosilicate, and stir for 6 h; obtain a solution containing nonporous silica microsphere mononuclear, take 1 / 8 volume of the solution containing nonporous silica microsphere mononuclear as a seed, add 9.9 mL of water, 33.4 mL of ethanol, and 3.3 mL of ammonia water, mix them uniformly at room temperature under mechanical stirring, dropwise add 3.4 mL of tetraethyl orthosilicate, and stir for 6 h; wash several times with ethanol to obtain layer-by-layer assembled nonporous silica microspheres nSiO2@nSiO2.

[0078] Figure 1 The TEM image of the layer-by-layer assembled nonporous silica microspheres nSiO2@nSiO2 in the present example is shown; it can be seen that the average particle size is 695 nm.

[0079] (2) Layer-by-layer assembled nonporous silica / dendritic mesoporous silica composite microspheres:

[0080] Disperse 500 mg of layer-by-layer assembled nonporous silica microspheres and 68 mg of triethanolamine in 25 mL of deionized water, and stir at 80°C under magnetic force for 15 min; add 380 mg of cetyltrimethylammonium bromide and 218 mg of sodium salicylate, and stir at 80°C under magnetic force for 15 min; add 4 mL of tetraethyl orthosilicate, and stir at 80°C under magnetic force for 3 h; cool the product to room temperature, centrifuge to collect the precipitate, and wash several times with ethanol; disperse the product in 3 mL of concentrated hydrochloric acid and 50 mL of ethanol, reflux at 60°C for 24 h, centrifuge to collect the precipitate, and obtain nonporous silica / dendritic mesoporous silica composite microspheres nSiO2@nSiO2@dSiO2.

[0081] Figure 2This is a TEM image of the layer-by-layer assembled non-porous silica / dendritic mesoporous silica composite microspheres nSiO2@nSiO2@dSiO2 in this embodiment. The average particle size is 1.1 μm.

[0082] (3) Layer-by-layer assembly of non-porous silica / dendritic mesoporous silica / iron oxide micron magnetic beads:

[0083] Under a nitrogen atmosphere, 0.7063 g of acetylacetone iron, 20 mL of 2-pyrrolidone, and 0.025 g of layer-by-layer assembled non-porous silica / dendritic mesoporous silica microsphere template were stirred for 30 min to mix evenly. Then, the mixture was stirred at 245 °C for 30 min. After the temperature dropped to room temperature, magnetic separation was performed using a magnetic rack and the product was collected. The product was washed several times with ethanol to obtain layer-by-layer assembled non-porous silica / dendritic mesoporous silica / iron oxide micron magnetic beads nSiO2@nSiO2@dSiO2@Fe3O4.

[0084] Figure 3 The TEM image shows the layer-by-layer assembly of non-porous silica / dendritic mesoporous silica / iron oxide micron magnetic beads nSiO2@nSiO2@dSiO2@Fe3O4 in this embodiment, which shows that the average particle size is 1.25 μm.

[0085] according to Figure 4 It can be seen that the layer-by-layer assembled non-porous silica microspheres nSiO2@nSiO2, the non-porous silica / dendritic mesoporous silica composite microspheres nSiO2@nSiO2@dSiO2, and the layer-by-layer assembled non-porous silica / dendritic mesoporous silica / iron oxide micro-magnetic beads nSiO2@nSiO2@dSiO2@Fe3O4 all have good dispersion stability, and the Zeta potential of nSiO2@nSiO2@dSiO2@Fe3O4 is positive.

[0086] according to Figure 5 It can be seen that the layer-by-layer assembled non-porous silica / dendritic mesoporous silica / iron oxide micron magnetic beads nSiO2@nSiO2@dSiO2@Fe3O4 in this embodiment has strong magnetism at room temperature and exhibits obvious magnetic hysteresis.

[0087] Example 2

[0088] (1) Layer-by-layer assembly of non-porous silica microspheres:

[0089] Take 9.9 mL of water, 33.4 mL of ethanol, and 3.3 mL of ammonia water, mix them mechanically at room temperature, and add 3.4 mL of tetraethyl orthosilicate dropwise. Stir the reaction for 6 h to obtain a solution containing mononuclear nonporous silica microspheres. Take 1 / 5 volume of the solution containing nonporous silica microspheres and wash it with ethanol to obtain mononuclear nonporous silica microspheres. Add 9.9 mL of water, 33.4 mL of ethanol, and 3.3 mL of ammonia water again, mix them mechanically at room temperature, and add 3.4 mL of tetraethyl orthosilicate dropwise. Stir the reaction for 6 h. Wash several times with ethanol to obtain layer-by-layer assembled nonporous silica microspheres nSiO2@nSiO2.

[0090] (2) Same as step (2) in Example 1.

[0091] (3) is the same as step (3) in Example 1.

[0092] The layer-by-layer assembled non-porous silica / dendritic mesoporous silica / iron oxide micron magnetic beads (nSiO2@nSiO2@dSiO2@Fe3O4) obtained in this embodiment have an average particle size of 1.18 μm.

[0093] Example 3

[0094] (1) Layer-by-layer assembly of non-porous silica microspheres:

[0095] Take 9.9 mL of water, 33.4 mL of ethanol, and 3.3 mL of ammonia water, mix them mechanically at room temperature, and add 3.4 mL of tetraethyl orthosilicate dropwise. Stir the reaction for 6 h to obtain a solution containing mononuclear nonporous silica microspheres. Take 1 / 4 volume of the solution containing mononuclear nonporous silica microspheres as seeds, add 9.9 mL of water, 33.4 mL of ethanol, and 3.3 mL of ammonia water, mix them mechanically at room temperature, and add 3.4 mL of tetraethyl orthosilicate dropwise. Stir the reaction for 6 h to obtain layer-by-layer assembled nonporous silica microspheres nSiO2@nSiO2.

[0096] (2) Same as step (2) in Example 1.

[0097] (3) is the same as step (3) in Example 1.

[0098] The average particle size of the layer-by-layer assembled non-porous silica / dendritic mesoporous silica / iron oxide micron magnetic beads (nSiO2@nSiO2@dSiO2@Fe3O4) obtained in this embodiment is 0.95 μm.

[0099] Example 4

[0100] (1) Layer-by-layer assembly of non-porous silica microspheres:

[0101] Take 9.9 mL of water, 33.4 mL of ethanol, and 3.3 mL of ammonia water, mix them mechanically at room temperature, and add 3.4 mL of tetraethyl orthosilicate dropwise. Stir the reaction for 6 h to obtain a solution containing non-porous silica microspheres. Take half the volume of the solution containing non-porous silica microspheres as seeds, add 9.9 mL of water, 33.4 mL of ethanol, and 3.3 mL of ammonia water, mix them mechanically at room temperature, and add 3.4 mL of tetraethyl orthosilicate dropwise. Stir the reaction for 6 h to obtain layer-by-layer assembled non-porous silica microspheres nSiO2@nSiO2.

[0102] (2) Same as step (2) in Example 1.

[0103] (3) is the same as step (3) in Example 1.

[0104] The average particle size of the layer-by-layer assembled non-porous silica / dendritic mesoporous silica / iron oxide micron magnetic beads (nSiO2@nSiO2@dSiO2@Fe3O4) obtained in this embodiment is 0.85 μm.

[0105] Example 5

[0106] (1) Non-porous silica microspheres:

[0107] Take 9.9 mL of water, 33.4 mL of ethanol, and 3.3 mL of ammonia water, mix them mechanically at room temperature, add 3.4 mL of tetraethyl orthosilicate dropwise, and stir the reaction for 6 h to obtain a solution containing mononuclear nonporous silica microspheres; take 1 / 8 volume of the solution containing mononuclear nonporous silica microspheres and wash it with ethanol to obtain mononuclear nonporous silica microspheres nSiO2; its average particle size is 350 nm.

[0108] (2) Proceed as in step (2) of Example 1, except that the non-porous silica microspheres obtained in step S1 of this example are replaced by the layer-by-layer assembly of non-porous silica microspheres in step S1 of Example 1.

[0109] (3) is the same as step (3) in Example 1.

[0110] The average particle size of the non-porous silica / dendritic mesoporous silica / iron oxide micron magnetic beads nSiO2@dSiO2@Fe3O4 obtained in this embodiment is 0.81 μm.

[0111] Example 6

[0112] (1) Same as step (1) in Example 1.

[0113] (2) 300 mg of layer-by-layer assembled non-porous silica microspheres and 68 mg of triethanolamine were dispersed in 25 mL of deionized water and magnetically stirred at 80 °C for 15 min; 380 mg of cetyltrimethylammonium bromide and 218 mg of sodium salicylate were added and magnetically stirred at 80 °C for 15 min; 4 mL of tetraethyl orthosilicate was added and magnetically stirred at 80 °C for 3 h; the product was cooled to room temperature, the precipitate was collected by centrifugation and washed several times with ethanol; it was then dispersed in 3 mL of concentrated hydrochloric acid and 50 mL of ethanol, refluxed at 60 °C for 24 h, and the precipitate was collected by centrifugation to obtain non-porous silica / dendritic mesoporous silica composite microspheres nSiO2@nSiO2@dSiO2.

[0114] (3) is the same as step (3) in Example 1.

[0115] The average particle size of the non-porous silica / dendritic mesoporous silica / iron oxide micron magnetic beads nSiO2@dSiO2@Fe3O4 obtained in this embodiment is 1.42 μm.

[0116] Example 7

[0117] (1) Same as step (1) in Example 1.

[0118] (2) 800 mg of layer-by-layer assembled non-porous silica microspheres and 68 mg of triethanolamine were dispersed in 25 mL of deionized water and magnetically stirred at 80 °C for 15 min; 380 mg of cetyltrimethylammonium bromide and 218 mg of sodium salicylate were added and magnetically stirred at 80 °C for 15 min; 4 mL of tetraethyl orthosilicate was added and magnetically stirred at 80 °C for 3 h; the product was cooled to room temperature, the precipitate was collected by centrifugation and washed several times with ethanol; it was then dispersed in 3 mL of concentrated hydrochloric acid and 50 mL of ethanol, refluxed at 60 °C for 24 h, and the precipitate was collected by centrifugation to obtain non-porous silica / dendritic mesoporous silica composite microspheres nSiO2@nSiO2@dSiO2.

[0119] (3) is the same as step (3) in Example 1.

[0120] The average particle size of the non-porous silica / dendritic mesoporous silica / iron oxide micron magnetic beads nSiO2@dSiO2@Fe3O4 obtained in this embodiment is 1.03 μm.

[0121] Comparative Example 1

[0122] In this comparative example, the micron-sized magnetic beads dSiO2@Fe3O4 use dendritic mesoporous silicon microspheres as the core, and then coat the surface of the core with a layer of iron oxide.

[0123] (1) Dendritic mesoporous silica microspheres:

[0124] 68 mg of triethanolamine was dispersed in 25 mL of deionized water, and then 380 mg of cetyltrimethylammonium bromide and 218 mg of sodium salicylate were added. The mixture was magnetically stirred at 80 °C for 15 min. 4 mL of tetraethyl orthosilicate was added, and the mixture was magnetically stirred at 80 °C for 3 h. The product was cooled to room temperature, and the precipitate was collected by centrifugation. The precipitate was washed several times with ethanol, and the process was repeated several times until the average particle size was the same as that of the non-porous silica / dendritic mesoporous silica composite microspheres in Example 1. The precipitate was then dispersed in 3 mL of concentrated hydrochloric acid and 50 mL of ethanol, refluxed at 60 °C for 24 h, and the precipitate was collected by centrifugation to obtain dendritic mesoporous silica microspheres dSiO2.

[0125] (2) Same as step (3) in Example 1, except that the dendritic mesoporous silicon microspheres dSiO2 obtained in step S1 of this comparative example are used instead of nSiO2@nSiO2@dSiO2 in step S1 of Example 1.

[0126] The average particle size of the micron-sized magnetic beads dSiO2@Fe3O4 prepared in this comparative example is 1.25 μm.

[0127] The performance of the above-mentioned micron magnetic beads was tested. The layer-by-layer assembled non-porous silica / dendritic mesoporous silica / iron oxide micron magnetic beads (nSiO2@nSiO2@dSiO2@Fe3O4) obtained in Examples 1-4 of this invention all have good magnetic properties, and their sedimentation rate is <5% after standing in ultrapure water for 24 hours, which can be applied to chemiluminescence detection.

[0128] In Example 5, the micron magnetic beads with a non-porous silica microsphere mononuclear core have similar magnetism, but the sedimentation rate is 29% after standing in ultrapure water for 24 hours.

[0129] Compared to Example 1, the magnetism of the micron magnetic beads in Example 6 decreased slightly, and the sedimentation rate was 13% after standing in ultrapure water for 24 hours; the magnetism of the micron magnetic beads in Example 7 increased, and the sedimentation rate was 21% after standing in ultrapure water for 24 hours.

[0130] Although the dendritic mesoporous silicon / iron oxide micron magnetic beads dSiO2@Fe3O4 in Comparative Example 1 have the same average particle size as nSiO2@nSiO2@dSiO2@Fe3O4 in Example 1, their sedimentation rate is 36% after standing in ultrapure water for 24 hours.

[0131] Functionalized micro-magnetic beads were prepared by externally attaching 100 μmol / g Tosyl modification groups to the highly magnetically responsive micro-beads from Examples 1, 5, and Comparative Example 1. The beads were then used in ultrapure water at a concentration of 10 mg / mL and applied to a Tosyl magnetic bead conjugation kit (commercially available; model: Beyotime, product number: P1640S). The antibody conjugation rates were 85%, 64%, and 52%, respectively.

[0132] In summary, the high magnetic response micron-sized magnetic beads of the present invention use non-porous silica microspheres as the core, with a dendritic mesoporous silica layer and a magnetite layer sequentially coated on the surface of the core. The non-porous silica microsphere core provides a stable skeletal support for the magnetic beads, ensuring the rigidity and morphological uniformity of the overall structure. The middle dendritic mesoporous silica layer, with its abundant dendritic pore structure, provides sufficient and uniform attachment sites for the load of the outermost magnetite layer, and reduces stress conflict between the core and the outer layer material through the buffering effect of the pores. The outer magnetite layer directly endows the magnetic beads with efficient magnetic response capability. This structure allows the magnetic beads to achieve an efficient balance between magnetism and stability while maintaining structural integrity.

[0133] All aspects, embodiments, and features of this invention should be considered illustrative in all respects and not limiting of the invention; the scope of the invention is defined only by the claims. Other embodiments, modifications, and uses will become apparent to those skilled in the art without departing from the spirit and scope of the invention as claimed.

[0134] In the preparation method of this invention, the order of the steps is not limited to the listed order. For those skilled in the art, variations in the order of the steps without creative effort are also within the scope of protection of this invention. Furthermore, two or more steps or actions can be performed simultaneously.

[0135] Finally, it should be noted that the specific embodiments described herein are merely illustrative examples of the invention and are not intended to limit the implementation of the invention. Those skilled in the art can make various modifications or additions to the described specific embodiments or use similar methods to replace them; it is neither necessary nor possible to exemplify all embodiments here. However, these obvious variations or modifications derived from the essential spirit of the invention still fall within the scope of protection of the invention, and interpreting them as any additional limitation would contradict the spirit of the invention.

Claims

1. A method for preparing micron-sized magnetic beads with high magnetic response, characterized in that, The preparation method includes: S1. Mix non-porous silica microspheres, triethanolamine, hexadecyltrimethylammonium bromide, sodium salicylate and water, then add silane coupling agent dropwise and react under mechanical stirring to obtain non-porous silica@dendritic mesoporous silica composite microspheres nSiO2@nSiO2@dSiO2. S2. Under a nitrogen atmosphere, acetylacetone iron, 2-pyrrolidone, and non-porous silica@dendritic mesoporous silica composite microspheres are mixed and stirred to obtain an iron precursor mixture; after thermal decomposition of the iron precursor mixture, magnetic separation and washing are performed to obtain micron magnetic beads with high magnetic response nSiO2@nSiO2@dSiO2@Fe3O4. The high magnetic response micron magnetic beads have a core-shell structure, with a core of non-porous silica microspheres, an intermediate layer of dendritic mesoporous silica, and an outer shell of iron oxide; and the mesopores of the dendritic mesoporous silica layer are loaded with iron oxide.

2. The method for preparing high magnetic response micron-sized magnetic beads according to claim 1, characterized in that, The average particle size of the highly magnetically responsive micron magnetic beads is 0.8–3 μm.

3. The method for preparing high magnetic response micron-sized magnetic beads according to claim 1, characterized in that, The layer-by-layer assembled non-porous silica microspheres nSiO2@nSiO2 are prepared by assembling a silica layer on the surface of the non-porous silica microsphere mononuclear nSiO2.

4. The method for preparing high magnetic response micron-sized magnetic beads according to claim 1, characterized in that, The method for preparing non-porous silica microspheres in step S1 includes: S11. Mix ethanol, ammonia and water, then add silane coupling agent dropwise to obtain the first mixture. React under mechanical stirring to obtain a seed solution containing mononuclear nSiO2 nonporous silica microspheres. S12. Mix ethanol, ammonia and water, then add silane coupling agent dropwise to obtain a second mixture; add seed liquid containing mononuclear nSiO2 nonporous silica microspheres, and react for 1-20 hours under mechanical stirring. S13. Repeat step (2) 0 to 10 times to obtain layer-by-layer assembled non-porous silica microspheres nSiO2@nSiO2.

5. The method for preparing high magnetic response micron-sized magnetic beads according to claim 1, characterized in that, In step S1, the mass ratio of non-porous silica microspheres, triethanolamine, hexadecyltrimethylammonium bromide, and sodium salicylate is (100-1000):(10-200):(200-600):(100-500).

6. The method for preparing high magnetic response micron-sized magnetic beads according to claim 1, characterized in that, In step S2, the mass-to-volume ratio of acetylacetone iron, 2-pyrrolidone, and non-porous silica@dendritic mesoporous silica composite microspheres is (0.1-2.0) g : (10-50) ml : (0.01-1.0) g.

7. The method for preparing high magnetic response micron-sized magnetic beads according to claim 1, characterized in that, The temperature of the thermal decomposition reaction in step S2 is 180–300℃, and the time is 1–300 min.

8. A micron-sized magnetic bead with high magnetic response, characterized in that, It is prepared by the preparation method of the high magnetic response micron magnetic beads as described in any one of claims 1 to 7.

9. The high magnetic response micron magnetic bead according to claim 8, characterized in that, The high magnetic response micron magnetic beads have a core-shell structure, with a core of non-porous silica microspheres, an intermediate layer of dendritic mesoporous silica, and an outer shell of magnetite; and magnetite is loaded inside the mesopores of the dendritic mesoporous silica layer; the high magnetic response micron magnetic beads have a sedimentation rate of <5% after standing in ultrapure water at room temperature for 12 to 48 hours.

10. The application of a high magnetic response micron-sized magnetic bead in the field of chemiluminescence detection, characterized in that, The application includes: preparing highly magnetically responsive micro-magnetic beads by the preparation method of the highly magnetically responsive micro-magnetic beads as described in any one of claims 1 to 7, or by externally attaching a Tosyl modification group to highly magnetically responsive micro-magnetic beads as described in any one of claims 8 to 9, to obtain functionalized micro-magnetic beads, wherein the antibody conjugation rate of the functionalized micro-magnetic beads is 80 to 95%.

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