A magnetic microsphere and a preparation method thereof

Abstract

This application belongs to the technical field of microsphere preparation, and specifically relates to a magnetic microsphere and a preparation method thereof. In this application, a polystyrene microsphere with good swelling and shrinking properties is provided through the cooperation of a crosslinking agent and a monomer; during the swelling process, divalent and trivalent iron ions are introduced, which can coordinate with the carboxyl groups of the microspheres and be uniformly dispersed in the microsphere structure; then, by adjusting the pH value, these iron ions react to form magnetite distributed inside and on the surface of the microspheres. During the shrinking process of the microspheres, the spatial occupation of magnetite reduces the density of aromatic rings, thereby reducing the background fluorescence and improving the signal-to-noise ratio and sensitivity of fluorescence detection. In addition, magnetite has an optical shielding effect and can cooperate with the coating layer arranged outside the microspheres to jointly reduce the fluorescence intensity of the magnetic microspheres made of polystyrene material.

Description

Technical Field

[0001] This application belongs to the technical field of microsphere preparation, and specifically relates to a magnetic microsphere with a low fluorescence background. Background Art

[0002] Magnetic microspheres are composite micron-sized spheres formed by combining organic polymer materials with inorganic magnetic particles. They not only have good biocompatibility and the property of being easily functionalized on the surface, but also can respond to an external magnetic field, which makes magnetic microspheres play an important role in fields such as in vitro diagnosis, cell sorting, targeted drug delivery, and gene delivery. Especially in antigen recognition and detection, the application of magnetic microspheres demonstrates its unique advantages.

[0003] To achieve high-sensitivity and high-specificity recognition and quantification of target antigens, a detection technique that combines magnetic microspheres modified with specific antibodies and fluorescent microspheres is usually adopted. This process includes several key steps: First, magnetic microspheres with specific antibodies modified on the surface are added to the sample to be tested. These magnetic microspheres can specifically bind to the target antigen in the sample to form an antigen-antibody-magnetic bead complex. Then, by applying an external magnetic field, the magnetic microspheres bound to the antigen can be effectively separated from the sample, and the unbound target components are removed. This step not only achieves the purification of the complex but also provides a pure sample for subsequent analysis, reducing the interference of non-specific binders. Subsequently, fluorescent microspheres modified with secondary antibodies are added to the purified complex. The secondary antibodies on these fluorescent microspheres can specifically recognize and bind to the primary antibodies on the magnetic microspheres and bind to the antigens on the surface of the magnetic microspheres. This step ensures that only the magnetic microspheres that have successfully captured the antigen will be fluorescently labeled, forming positive magnetic microspheres. If the magnetic microspheres fail to capture the antigen, they will not bind to the fluorescent microspheres and will appear as negative magnetic microspheres without obvious fluorescence signals.

[0004] Therefore, by subsequently detecting the intensity of the fluorescence signal, it is possible to effectively determine whether the target antigen exists in the above sample and its content. Since the intensity of the fluorescence signal is positively correlated with the number of target antigens in the sample, the fluorescence signal plays a crucial role in the accuracy and reliability of the detection results. Given the importance of the fluorescence signal, current research on magnetic microspheres mainly focuses on improving their fluorescence intensity and expanding the types of fluorescence coding. Summary of the Invention

[0005] The applicant has found through research that in currently common polystyrene magnetic microspheres on the market, the high-density benzene ring structure will cause more electron cloud overlap, resulting in a certain background fluorescence, interfering with the results of fluorescence detection and analysis, and affecting the accuracy and sensitivity of the detection results. Based on this, the invention object of this application is to provide a magnetic microsphere with a low fluorescence background, which is specifically achieved through the following technical solutions:

[0006] In a first aspect, the present application provides a method for preparing magnetic microspheres, comprising the following steps: S1. Reacting styrene monomer, crosslinking agent, initiator and carboxyl functional monomer to obtain polystyrene microspheres; S2. Swelling the polystyrene microspheres and adding iron salt, such that divalent and trivalent iron ions in the iron salt coordinate with carboxyl groups in the polystyrene microspheres to obtain a pretreated microsphere solution; S3. Adjusting the pH value of the pretreated microsphere solution to 8-9, such that magnetite is formed inside and on the surface of the microspheres to obtain magnetized microspheres; S4. Shrinking the magnetized microspheres to obtain magnetic microsphere precursors; S5. Providing a coating layer on the surface of the magnetic microsphere precursors to obtain magnetic microspheres.

[0007] Preferably, the carboxyl functional monomer includes one or more of acrylic acid, hydroxyethyl acrylate, and hydroxyethyl methacrylate; the initiator is one or both of potassium persulfate and azobisisobutyronitrile.

[0008] Preferably, in step S1, the molar ratio of styrene monomer, crosslinking agent and carboxyl functional monomer is 1:(1.5-2.5):(1-1.2). Further preferably, the total monomer concentration is 10%-20%; the molar ratio of initiator to monomer is (1-2):200.

[0009] Preferably, in step S1, the reaction conditions are: temperature 70-80°C, time 3-6 h.

[0010] Preferably, in step S2, the swelling treatment step is: dissolving the polystyrene microspheres in a mixed solution of water, ethanol and acetonitrile, and heating and raising the temperature to 70-90°C under inert gas protection.

[0011] Preferably, in step S2, the molar ratio of divalent and trivalent iron ions is 1:(2-3).

[0012] Preferably, in step S2, the total amount ratio of divalent and trivalent iron ions to the carboxyl functional monomer does not exceed 3:8.

[0013] Preferably, in step S5, a hydrophilic polymer layer is formed on the surface of the magnetic microsphere precursors by seed polymerization, and the hydrophilic polymer layer is a silica layer. The steps for providing the silica layer are: adding ammonia water and tetraethyl orthosilicate (TEOS) to the magnetic microsphere dispersion to form a silica layer. The ratio of ammonia water (30%) to TEOS (10%) is (0.7-0.8):2.

[0014] Preferably, the method for preparing magnetic microspheres further includes introducing amino or carboxyl groups to the surface of the magnetic microspheres.

[0015] In a second aspect, the present application also provides a magnetic microsphere prepared by any of the preparation methods described above.

[0016] Compared with the prior art, this application has the following beneficial effects:

[0017] The present application prepares a polystyrene microsphere with good swelling and shrinking performance by the coordination between a cross-linking agent and a monomer; During the swelling process, by adding iron salts, divalent iron ions and trivalent iron ions are coordinated with the carboxyl groups on the surface or inside of the polystyrene microspheres, and are introduced into the skeleton structure to achieve the orderly dispersion of divalent iron ions and trivalent iron ions; Subsequently, by adjusting the pH, the adjacent divalent and trivalent iron ions inside and on the surface of the microspheres react rapidly with each other, forming ferroferric oxide with superparamagnetism at a specific position of the polystyrene microspheres, which not only gives the polystyrene microspheres a higher magnetic response performance, but more importantly, in the subsequent contraction process, the ferroferric oxide formed can occupy the space originally occupied by the aromatic ring structure, effectively reducing the density of the aromatic ring in the polystyrene skeleton. Since the aromatic ring is one of the main sources of background fluorescence, reducing its density can significantly reduce the generation of background fluorescence, thereby improving the signal-to-noise ratio and sensitivity of fluorescence detection. In addition, the ferroferric oxide nanoparticles themselves do not produce fluorescence, and have a certain optical shielding effect, which can further suppress the interference of background fluorescence and ensure the purity of the fluorescence signal. In order to prevent the ferroferric oxide nanoparticles formed on the surface of the microspheres from falling off during the subsequent treatment process, resulting in a weakened fluorescence shielding effect, the present application coats the surface of the magnetic microsphere precursor with a coating layer such as silicon dioxide, which can not only enhance the stability and biocompatibility of the magnetic microspheres, but also firmly fix the ferroferric oxide nanoparticles to prevent the problem of poor fluorescence shielding caused by their escape. The performance test results show that the fluorescence intensity is reduced by 42% when the ferroferric oxide in-situ formation process is used alone; the fluorescence intensity is reduced by 24.5% when the ferroferric oxide impregnation process is used alone; and the present application utilizes the swelling properties of special polystyrene microspheres, and combines the in-situ formation process of ferroferric oxide, and further uses the fixed covering process of coating layers such as silicon dioxide. Multiple processes can cooperate with each other to jointly solve the technical problem of excessive fluorescence of polystyrene material magnetic microspheres. Compared with polystyrene magnetic microspheres with swelling properties, the fluorescence intensity of the magnetic microspheres of the present application is reduced by about 88%, reflecting unexpected technical effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The synthetic route of polystyrene microspheres is shown in Figure 2.

[0019] Figure 2 Schematic diagram of magnetizing polystyrene microspheres;

[0020] Figure 3Schematic diagram for avoiding modification of magnetic microspheres. Detailed implementation mode

[0021] The present application will be further described below by way of specific embodiments. Those of ordinary skill in the art will be able to implement the present application based on these descriptions. In addition, the embodiments of the present application involved in the following description are usually only a part of the embodiments of the present application, rather than all the embodiments. Therefore, all other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present application without creative efforts shall fall within the scope of protection of the present application.

[0022] Example 1

[0023] This example discloses a preparation method of magnetic microspheres, which specifically includes the following steps:

[0024] S1. Mix styrene (0.9 mL), crosslinking agent divinylbenzene (2.75 mL, 80%) and functional monomer acrylic acid containing carboxyl group (1.0 mL) together to form a monomer solution; weigh (0.02 g) azobisisobutyronitrile as an initiator; (10 mL) toluene and 40 mL acetonitrile are blended as solvents. Magnetically stir and mix the above monomer solution, initiator and solvent evenly in a three-necked flask and seal it. Introduce an inert gas to remove oxygen in the flask, and then slowly heat it to 80 °C by an oil bath stirrer, stir and react for 3 h. After the reaction is completed, slowly stir and cool down and separate by centrifugation. Wash the microspheres with ethanol and water to obtain a white polystyrene microsphere suspension. Of course, it can be understood that in other embodiments, the molar ratio of styrene monomer, crosslinking agent and carboxyl functional monomer can be selected from any ratio in 1:(1.5~2.5):(1~1.2); the total monomer concentration can be 10%~20%; the molar ratio of initiator to monomer can be (1~2):200; the reaction conditions can be any parameter in: temperature 70~80 °C, time 3~6 h.

[0025] S2. Disperse polystyrene microspheres (0.5 g) in a mixed solution of water, ethanol, and acetonitrile. After completely dispersing the cross-linked polystyrene microspheres by ultrasonic treatment, add them to a semi-sealed three-necked flask. Under the protection of an inert gas, mechanically stir and slowly heat up to 80 °C to obtain swollen polystyrene microspheres. Another 162 mg of ferric chloride hexahydrate and 111 mg of ferrous sulfate heptahydrate with a molar ratio of 2:1 (the amount of polystyrene microspheres is 0.5 g) are weighed, mixed and dissolved in water, and quickly added to the above three-necked flask. After stirring and reacting for 1 h, add diluted ammonia water and adjust the pH value to 8 - 9. After continuing to react for 1 h, slowly stir and cool down to room temperature. Then, slowly add deionized water with the same volume as the reaction mixture to dilute the mixed solution, and then use magnetic separation to remove the supernatant. Then, repeatedly add deionized water and stir for washing to achieve the purpose of shrinking and washing, and obtain the magnetic microsphere precursor. Of course, it can be understood that in other embodiments, the temperature of the swelling treatment can be any parameter in the range of 70 - 90 °C; the molar ratio of ferric chloride hexahydrate to ferrous sulfate heptahydrate can be 1:(2 - 3), and the total amount of divalent and trivalent iron ions and the molar ratio of the carboxyl functional monomer do not exceed 3:8.

[0026] S3. Take (200 mg) of the magnetic microsphere precursor, disperse it in a PVP-K30 (polyvinylpyrrolidone) ethanol (stabilizer and dispersant to prevent aggregation) solution, stir overnight on a shaker, and then add ammonia water (90 μL, 30% aqueous solution) and TEOS (tetraethyl orthosilicate) (125 μL, 10% ethanol solution) to the mixed solution. After reacting at room temperature for 12 h, add the same volume of TEOS ethanol solution again and continue to react for 12 h to obtain magnetic microspheres coated with silica. Further, in this embodiment, by adding the same volume of APTES ethanol solution and reacting overnight, amino groups are introduced on the surface of the magnetic microspheres coated with silica, and magnetic microspheres are obtained after separation and purification. Of course, it can be understood that in other embodiments, the ratio of ammonia water (30%) to TEOS (10%) can be any parameter in the range of (0.7 - 0.8):2.

[0027] Comparative Example 1

[0028] The difference between the preparation method of the magnetic microspheres in this Comparative Example 1 and that in Example 1 is only that: it does not include step S3, and in step S2, the magnetic endowment process of first adding ferric chloride hexahydrate and ferrous sulfate heptahydrate and then adding ammonia water is not adopted, but a magnetic endowment process of directly adding magnetite is adopted, that is, adding magnetite magnetic fluid to the swollen polystyrene microsphere solution, and then performing shrinkage washing treatment to obtain the magnetic microsphere precursor.

[0029] Comparative Example 2

[0030] The preparation method of the magnetic microspheres in Comparative Example 2 is only different from that in Example 1 in that: it does not include step S2, that is, the polystyrene microspheres are not subjected to swelling and shrinking treatment, and at the same time, the magnetic imparting process is not carried out after swelling. Only a coating layer containing amino groups in S3 is provided on the surface of the polystyrene microspheres.

[0031] Comparative Example 3

[0032] The preparation method of the magnetic microspheres in Comparative Example 3 is only different from that in Example 1 in that: it does not include step S3, that is, the surface of the magnetic microsphere precursor is not coated with silica, and only the in-situ magnetic imparting process in S2 is adopted.

[0033] Comparative Example 4

[0034] The preparation method of the magnetic microspheres in Comparative Example 4 is only different from that in Example 1 in that: it does not include steps S2 and S3.

[0035] Performance Test

[0036] The fluorescence intensity of the magnetic microspheres prepared in Example 1 and Comparative Examples 1-4 was detected in this performance test, and the detection results are shown as follows:

[0037] Table 1. Detection Results of Fluorescence Intensity of Each Group

[0038] Group Example 1 Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 Fluorescence intensity (5000 / 2^16 mV / cell) 28 176 32 137 233

[0039] It can be found by observing the above table that the present application provides a magnetic microsphere with significantly reduced fluorescence intensity by organically combining the preparation technology of polystyrene microspheres with special swelling properties, the swelling and shrinking treatment process, the in-situ formation process of iron tetroxide (Fe3O4), and the coating process. Compared with the polystyrene microspheres with only special swelling properties in Comparative Example 4, the magnetic microspheres prepared in the present application achieved an 88% reduction in fluorescence intensity. This result not only reflects the synergistic effect between the processes but also demonstrates the relevant breakthroughs achieved in reducing background fluorescence.

[0040] Please refer to the two kinds of magnetic microspheres disclosed in Comparative Examples 1 and 3 and the corresponding fluorescence intensity detection results. Comparative Example 1 is a magnetic microsphere obtained by treating the swollen microspheres with a pure ferroferric oxide magnetic fluid impregnation process without using silica coating. Compared with the swollen microspheres of Comparative Example 4, it can achieve a 24.5% reduction in fluorescence intensity; Comparative Example 3 is a magnetic microsphere obtained by treating the swollen microspheres with the in-situ formation process of ferroferric oxide of the present application without using silica coating. Compared with the swollen microspheres of Comparative Example 4, it can achieve a 42% reduction in fluorescence intensity, which is 17.5% lower than that of Comparative Example 1. This indicates that although the impregnation process can introduce magnetic substances to a certain extent, due to the lack of cooperation with the swelling and shrinking treatment process, it cannot effectively solve the problem of high background fluorescence caused by the aromatic ring structure inside the microspheres. Therefore, its effect in reducing fluorescence intensity is limited. The in-situ formation process of ferroferric oxide of the present application can better block fluorescence.

[0041] Please refer to the two kinds of magnetic microspheres disclosed in Example 1 and Comparative Example 3 and the corresponding fluorescence intensity detection results. Comparative Example 3 solely relies on the in-situ formation process of ferroferric oxide, that is, directly generating Fe3O4 nanoparticles inside or on the surface of the microspheres without any coating treatment. In this case, the fluorescence intensity does decrease, reaching 42%, but it is still far from the effect of reducing the fluorescence intensity of the present application, with a difference of 46% from the present application. On the one hand, because the uncoated Fe3O4 nanoparticles are easily detached from the surface of the magnetic microspheres, resulting in their inability to stably play the role of blocking fluorescence. Therefore, even though Fe3O4 itself does not emit light and can shield part of the background fluorescence, its effect is greatly reduced due to the detachment problem. On the other hand, please combine the fluorescence intensity detection results obtained by separately using silica for microsphere coating in Comparative Example 2. When using silica alone as the outermost coating material, although silica can block part of the fluorescence, its effect is limited and cannot achieve the effect of the synergistic action of multiple processes in the present application.

[0042] In summary, through a series of process steps - starting from the preparation of special polystyrene microspheres, introducing Fe3O4 using their swelling characteristics, and then performing shrinkage and coating treatments, the present application has successfully solved the problem of excessively high fluorescence in traditional methods. The combination of these processes not only greatly reduces the fluorescence intensity of the magnetic microspheres but also enhances the stability and biocompatibility of the microspheres, providing a more pure and reliable signal source for applications such as fluorescence detection. Experimental data clearly show that the method of the present application performs excellently in reducing background fluorescence and has obvious superiority and innovation.

Claims

1. A method for preparing magnetic microspheres, characterized in that: The following steps are involved: S1, reacting styrene monomer, crosslinking agent, initiator and carboxyl functional monomer to obtain polystyrene microspheres; S2, performing swelling treatment on the polystyrene microspheres, and adding iron salt, so that the divalent and trivalent iron ions in the iron salt react with the carboxyl groups in the polystyrene microspheres to obtain a pre-treated microsphere solution; S3, adjusting the pH value of the pre-treated microsphere solution to 8-9, so that ferroferric oxide is formed in situ inside and on the surface of the microspheres to obtain magnetized microspheres; S4, shrinking the magnetized microspheres to obtain magnetic microsphere precursors; S5. Disposing a silicon dioxide coating layer on the surface of the magnetic microsphere precursor to obtain magnetic microspheres.

2. The method for preparing magnetic microspheres according to claim 1, characterized in that: In step S1, the molar ratio of the styrene monomer, the crosslinking agent and the carboxyl functional monomer is 1:(1.5-2.5):(1-1.2).

3. The method for preparing magnetic microspheres according to claim 1, characterized in that: In step S1, the reaction conditions are: temperature 70-80°C, time 3-6h.

4. The method for preparing magnetic microspheres according to claim 1, characterized in that: In step S2, the swelling treatment step is: dissolving the polystyrene microspheres in a mixture of water, ethanol and acetonitrile, and heating the mixture to 70-90° C. under the protection of an inert gas.

5. The method for preparing magnetic microspheres according to claim 1, characterized in that: In step S2, the ratio of the amount of divalent iron ions to the amount of trivalent iron ions is 1:(2-3).

6. The method for preparing magnetic microspheres according to claim 1, characterized in that: In step S2, the total amount of divalent and trivalent iron ions and the amount of carboxyl functional monomer material is no more than 3:

8.

7. The method for preparing magnetic microspheres according to claim 1, characterized in that: In step S5, a hydrophilic polymer layer is formed on the surface of the magnetic microsphere precursor by using a seed polymerization method, and the hydrophilic polymer layer is a silicon dioxide layer.

8. The method for preparing magnetic microspheres according to claim 1, characterized in that: The method further comprises step S6 of introducing amino groups or carboxyl groups onto the surface of the magnetic microspheres.

9. A magnetic microsphere, characterized in that: The method is prepared by any one of claims 1 to 8.

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