Magnetic microsphere and preparation method thereof

Abstract

The invention belongs to the technical field of microsphere preparation, and particularly relates to a magnetic microsphere and a preparation method thereof. Through cooperation of the cross-linking agent and the monomer, the polystyrene microsphere with good swelling and shrinking performance is provided; in the swelling process, introduced divalent and trivalent iron ions can be coordinated with carboxyl groups of the microspheres and are uniformly dispersed in the microsphere structure; then, by adjusting the pH value, the iron ions react to generate ferroferric oxide distributed in the microspheres and on the surfaces of the microspheres, and in the shrinkage process of the microspheres, the density of aromatic rings is reduced by the ferroferric oxide based on space occupation of the ferroferric oxide, so that background fluorescence is reduced, and the signal-to-noise ratio and sensitivity of fluorescence detection are improved. In addition, ferroferric oxide has an optical shielding effect and can be mutually matched with a coating layer arranged outside the microsphere, so that the fluorescence intensity of the polystyrene material magnetic microsphere is jointly reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of microsphere preparation, and in particular to a magnetic microsphere with low fluorescence background. Background Art

[0002] Magnetic microspheres are a type of composite micron-sized spheres formed by combining organic polymer materials with inorganic magnetic particles. They not only have good biocompatibility and easy surface functionalization, but also can respond to external magnetic fields, which makes them play an important role in in vitro diagnosis, cell sorting, targeted drug delivery, gene delivery, etc. Especially in antigen recognition and detection, the application of magnetic microspheres has shown its unique advantages.

[0003] In order to achieve high sensitivity and high specificity recognition and quantification of target antigens, the detection technology of magnetic microspheres modified with specific antibodies combined with fluorescent microspheres is usually used. 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 binding substances. 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 successfully capture the antigen will be fluorescently labeled to form positive magnetic microspheres. If the magnetic microspheres fail to capture the antigen, they will not bind to the fluorescent microspheres, showing negative magnetic microspheres with no obvious fluorescent signals.

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

[0005] The applicant has found through research that the high-density benzene ring structure in the polystyrene magnetic microspheres currently available on the market will lead to more electron cloud overlap, thereby generating a certain background fluorescence, interfering with the fluorescence detection and analysis results, 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 low fluorescence background, which is specifically achieved through the following technical solutions: In a first aspect, the present application provides a method for preparing magnetic microspheres, comprising the following steps: S1, reacting a styrene monomer, a cross-linking agent, an initiator and a carboxyl functional monomer to obtain polystyrene microspheres; S2, swelling the polystyrene microspheres, and adding iron salts, so that the divalent and trivalent iron ions in the iron salts 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 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 precursor to obtain magnetic microspheres.

[0006] Preferably, the carboxyl functional monomer includes one or more of acrylic acid, hydroxyethyl acrylic acid, and hydroxyethyl methacrylic acid; and the initiator is one or two of potassium persulfate and azobisisobutyronitrile.

[0007] 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). More preferably, the total monomer concentration is 10%-20%; the molar ratio of initiator to monomer is (1-2):200.

[0008] Preferably, in step S1, the reaction conditions are: temperature 70-80° C., time 3-6 h.

[0009] Preferably, 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.

[0010] Preferably, in step S2, the ratio of the amount of divalent iron ions to the amount of trivalent iron ions is 1:(2-3).

[0011] Preferably, in step S2, the ratio of the total amount of divalent and trivalent iron ions to the amount of carboxyl functional monomer substance is no more than 3:8.

[0012] Preferably, in step S5, a hydrophilic polymer layer is formed on the surface of the magnetic microsphere precursor by seed polymerization, and the hydrophilic polymer layer is a silicon dioxide layer. The step of setting the silicon dioxide layer is: adding ammonia water and tetraethyl orthosilicate (TEOS) to the magnetic microsphere dispersion to form a silicon dioxide layer. The ratio of ammonia water (30%) to TEOS (10%) is (0.7-0.8):2.

[0013] Preferably, the method for preparing the magnetic microspheres further comprises introducing amino groups or carboxyl groups into the surfaces of the magnetic microspheres.

[0014] In a second aspect, the present application also provides a magnetic microsphere prepared by any of the preparation methods described above.

[0015] Compared with the prior art, this application has the following beneficial effects: 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

[0016] Figure 1 The synthetic route of polystyrene microspheres is shown in Figure 2. Figure 2 Schematic diagram of magnetizing polystyrene microspheres; Figure 3 Schematic diagram of magnetic microspheres to avoid modification. DETAILED DESCRIPTION

[0017] The present application will be further described below in the form 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 generally only embodiments of a part of the present application, rather than all embodiments. Therefore, based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without making creative work should fall within the scope of protection of the present application.

[0018] Example 1 This embodiment discloses a method for preparing magnetic microspheres, which specifically comprises the following steps: S1. Styrene (0.9 mL), crosslinker divinylbenzene (2.75 mL, 80%) and functional monomer acrylic acid containing carboxyl group (1.0 mL) were mixed together to form a monomer solution; (0.02 g) azobisisobutyronitrile was weighed as an initiator; (10 mL) toluene and 40 mL acetonitrile were mixed as solvent. The above monomer solution, initiator and solvent were mixed evenly in a three-necked flask with magnetic stirring and sealed, and inert gas was introduced to remove oxygen in the flask, and then slowly heated to 80°C with an oil bath stirrer, stirred and reacted for 3 hours. After the reaction was completed, the temperature was slowly cooled with stirring and separated by centrifugation, and the microspheres were washed 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 of 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 of temperature 70~80°C and time 3~6h.

[0019] S2. Disperse polystyrene microspheres (0.5 g) in a mixture of water, ethanol and acetonitrile. After the cross-linked polystyrene microspheres are completely dispersed by ultrasound, they are added to a semi-sealed three-necked flask, mechanically stirred and slowly heated to 80°C under the protection of inert gas to obtain swollen polystyrene microspheres. Separately weigh 2:1 molar ratio of ferric chloride hydrate and ferrous sulfate hydrate (162 mg ferric chloride hexahydrate, 111 mg ferrous sulfate heptahydrate, and 0.5 g of polystyrene microspheres), mix and dissolve them in water and quickly add them to the above three-necked flask, stir and react for 1 hour, add diluted ammonia water, and adjust the pH value to 8-9. After continuing the reaction for 1 hour, slowly stir and cool to room temperature, and then slowly add the same volume of deionized water as the reaction mixture to dilute the mixed solution, and then remove the supernatant by magnetic separation, and then repeatedly add deionized water to stir and wash to achieve the purpose of shrinking and washing, and obtain a 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 to 90° C.; the ratio of the amount of hydrated ferric chloride to the amount of hydrated ferrous sulfate can be 1:(2 to 3), and the total amount of divalent and trivalent iron ions to the amount of carboxyl functional monomer material does not exceed 3:8.

[0020] S3, take (200 mg) of magnetic microsphere precursor, disperse it in PVP-K30 (polyvinyl pyrrolidone) ethanol (stabilizer, dispersant, to prevent aggregation) solution, stir on a shaker overnight, add ammonia (90 μL, 30% aqueous solution) and TEOS (tetraethyl orthosilicate) (125 μL, 10% ethanol solution) ethanol solution to the mixture, react at room temperature for 12 hours, then drop the same volume of TEOS ethanol solution again, continue to react for 12 hours, and obtain magnetic microspheres coated with silica. Further, this embodiment also introduces amino groups on the surface of magnetic microspheres coated with silica by adding the same volume of APTES ethanol solution, reacting overnight, and obtaining magnetic microspheres after separation and purification. Of course, it can be understood that in other embodiments, the ratio of ammonia (30%) to TEOS (10%) can be any parameter in (0.7~0.8):2.

[0021] Comparative Example 1 The preparation method of magnetic microspheres in this comparative example 1 is different from that in Example 1 only in that: step S3 is not included, and in step S2, the magnetic imparting process of first adding hydrated ferric chloride and hydrated ferrous sulfate and then adding ammonia water is not adopted, but the magnetic imparting process of directly adding ferrosoferric oxide is adopted, that is, ferrosoferric oxide magnetic fluid is added to the swollen polystyrene microsphere solution, and then shrinkage washing treatment is performed to obtain a magnetic microsphere precursor.

[0022] Comparative Example 2 The preparation method of magnetic microspheres in this comparative example 2 differs from that in example 1 only in that step S2 is not included, i.e., the polystyrene microspheres are not subjected to swelling and shrinking treatment, and no magnetic imparting process is performed after swelling. Only a coating layer containing amino groups is provided on the surface of the polystyrene microspheres in step S3.

[0023] Comparative Example 3 The only difference between the preparation method of magnetic microspheres in Comparative Example 3 and Example 1 is that step S3 is not included, that is, the surface of the magnetic microsphere precursor is not coated with silicon dioxide, and only the in-situ magnetism imparting process in S2 is used.

[0024] Comparative Example 4 The only difference between the preparation method of magnetic microspheres in Comparative Example 4 and Example 1 is that it does not include steps S2 and S3.

[0025] Performance Testing This performance test was performed on the fluorescence intensity of the magnetic microspheres prepared in Example 1 and Comparative Examples 1 to 4, and the test results are as follows: Table 1. Fluorescence intensity test results of each group Group Example 1 Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 Fluorescence intensity (5000 / 2^16 mV / piece) 28 176 32 137 233 By observing the above table, it can be found 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 shrinkage treatment process, the in-situ formation process of ferroferric oxide (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 various processes, but also demonstrates the relevant breakthroughs in reducing background fluorescence.

[0026] Please refer to the two 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 spheres with a 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 spheres with a ferroferric oxide in-situ formation process 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 shows that although the impregnation process can introduce magnetic substances to a certain extent, due to the lack of cooperation with the swelling and shrinkage treatment process, it is impossible to effectively solve the high background fluorescence problem caused by the aromatic ring structure inside the microspheres, so the effect of reducing fluorescence intensity is limited. The in-situ formation process of ferroferric oxide in the present application can better achieve fluorescence blocking.

[0027] Please refer to the two magnetic microspheres disclosed in Example 1 and Comparative Example 3 and the corresponding fluorescence intensity detection results. Comparative Example 3 simply 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 to 42%, but it is still far less than the effect of reducing the fluorescence intensity of the present application, which differs by 46% from the present application. On the one hand, it is because the uncoated Fe3O4 nanoparticles easily fall off from the surface of the magnetic microspheres, causing them to be unable to stably play the role of blocking fluorescence. Therefore, even if Fe3O4 itself does not emit light and can shield part of the background fluorescence, its effect is greatly reduced due to the problem of falling off. On the other hand, please combine the fluorescence intensity detection results obtained by using silica alone for microsphere coating in Comparative Example 2. Silica is used 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 effect of multiple processes in the present application.

[0028] In summary, this application successfully solves the problem of excessive fluorescence in traditional methods through a series of process steps - starting from the preparation of special polystyrene microspheres, introducing Fe3O4 by utilizing its swelling properties, and then shrinking and coating treatment. The combination of these processes not only greatly reduces the fluorescence intensity of magnetic microspheres, but also enhances the stability and biocompatibility of the microspheres, providing a purer and more reliable signal source for applications such as fluorescence detection. Experimental data clearly show that the method of this application performs well 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. Arranging a 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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