Application of trifluoromethanesulfonyl magnetic nanospheres in chemiluminescence immunoassay

Abstract

This invention provides the application of trifluoromethanesulfonyl magnetic nanospheres in chemiluminescent immunoassay. The trifluoromethanesulfonyl magnetic nanospheres are used for direct covalent coupling with antigens or antibodies. The specific preparation method includes first forming epoxy magnetic nanobead micelles, then adding trifluoromethanesulfonyl chloride as a modifier in batches under conditions of 0-25°C to obtain a crude product of trifluoromethanesulfonyl magnetic nanospheres. Then, the particles are screened by column chromatography and centrifugation to obtain the final product of trifluoromethanesulfonyl magnetic nanospheres. The trifluoromethanesulfonyl magnetic nanospheres have stronger thermal stability than p-toluenesulfonyl magnetic nanobeads, eliminating the need for an activation step. Under the action of ammonium sulfate catalyst, they are directly covalently coupled with amino-containing biomacromolecules, reducing antibody cross-linking and magnetic bead aggregation caused by activators such as EDC or glutaraldehyde.

Description

Technical Field

[0001] This invention relates to the field of in vitro diagnostics, specifically to the application of trifluoromethanesulfonyl magnetic nanospheres in chemiluminescent immunoassay. Background Technology

[0002] Magnetic nanospheres not only possess the excellent high specific surface area of ​​nanomaterials, but also have special magnetic field induction characteristics. As a solid-phase carrier, they are widely used in biomedical fields such as nucleic acid separation and purification, chemiluminescence immunoassay, immobilization of special enzymes, or targeted drug delivery. In the IVD field, whether it is Roche's electrochemiluminescence, Abbott's direct chemiluminescence, or Beckman's enzyme immunoassay chemiluminescence, magnetic nanospheres are a key raw material. As a solid-phase carrier for magnetic separation, they are used to couple streptavidin, antigens, or antibodies to form immune complexes, and finally for separation and detection. A good magnetic nanosphere has the following 3-5 characteristics: (1) Low background and less passive adsorption. (2) Strong magnetism, facilitating rapid separation. (3) Good suspension properties, resulting in a more complete immune reaction. (4) Uniform particle size and narrow size distribution range. (5) Simple coupling steps, with no magnetic bead aggregation during the coupling process. (6) Good thermal stability and low requirements for storage conditions.

[0003] Magnetic nanospheres, as a functionalized solid-phase carrier, are typically modified with carboxyl, amino, hydroxyl, epoxy, chloromethyl, p-toluenesulfonyl, or aldehyde groups. In chemiluminescent immunoassay, carboxyl or amino magnetic beads are the most commonly used. Before use, they must be pretreated with activators such as EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide) or glutaraldehyde, making the coupling process cumbersome. When using EDC or glutaraldehyde for activation, it often leads to magnetic bead aggregation and protein self-crosslinking, affecting the coupling operation and chemiluminescent immunoassay.

[0004] While epoxy magnetic nanospheres can be directly coupled to proteins, the epoxy groups in these nanospheres are easily degraded into hydroxyl groups, resulting in poor stability. Dynabeads, a commercially available pre-activated p-toluenesulfonyl magnetic microsphere, is modified with p-toluenesulfonyl groups and is primarily used for the capture and separation of proteins (including antigens, antibodies, and enzymes), nucleic acids, or cells. Patent CN112034164A discloses a technique for preparing p-toluenesulfonyl magnetic beads: epoxy magnetic beads react with mercaptoethanol to obtain hydroxyl magnetic beads; then, the hydroxyl magnetic beads are modified by reacting p-toluenesulfonyl chloride in acetone solution to obtain p-toluenesulfonyl magnetic beads. The p-toluenesulfonyl magnetic beads prepared by this method show decreased activity after 5-7 days of accelerated reaction at 37°C, resulting in a lower coupling rate when reacted with proteins. Patent CN111569844A provided by Nanjing University of Science and Technology in China discloses a technical scheme for preparing p-toluenesulfonyl magnetic beads: first, a hydrophilic polymer primary hydroxyl group is prepared and reacted with p-toluenesulfonyl chloride to prepare a p-toluenesulfonyl hydrophilic polymer; then, the p-toluenesulfonyl hydrophilic polymer is reacted with amino magnetic beads to prepare p-toluenesulfonyl magnetic nanospheres. The advantage of this method is that the reaction steps are simple, but the p-toluenesulfonyl hydrophilic polymer is prone to react with multiple amino magnetic beads and easily agglomerates, which leads to the p-toluenesulfonyl group reacting with amino-containing proteins, resulting in very low efficiency. Summary of the Invention

[0005] The purpose of this invention is to provide a pre-activated magnetic nanosphere for chemiluminescent immunoassay, the surface of which contains trifluoromethanesulfonyl groups.

[0006] The technical solution adopted in this invention is as follows: the application of trifluoromethanesulfonyl magnetic nanospheres in chemiluminescent immunoassay, wherein the trifluoromethanesulfonyl magnetic nanospheres are used to conjugate with antigens or antibodies.

[0007] In one embodiment of the present invention, the trifluoromethanesulfonyl magnetic nanospheres are obtained by reacting the modifier trifluoromethanesulfonyl chloride with epoxy-based magnetic nanospheres.

[0008] Furthermore, in order to improve the preparation process of trifluoromethanesulfonyl nanospheres by addressing agglomeration and thermal stability issues, the specific preparation process of the trifluoromethanesulfonyl magnetic nanospheres preferably includes the following steps:

[0009] 1) Formation of epoxy-based magnetic nanosphere micelles;

[0010] 2) Trifluoromethanesulfonyl chloride, a modifier, was added dropwise to the epoxy-based magnetic nanosphere micelles prepared in step 1) at 0-25℃ to obtain crude trifluoromethanesulfonyl magnetic nanospheres.

[0011] 3) The crude product of trifluoromethanesulfonyl magnetic nanospheres was subjected to particle size screening by column chromatography and centrifugation to obtain trifluoromethanesulfonyl magnetic nanospheres.

[0012] Preferably, the mass-to-volume ratio of the epoxy magnetic nanospheres to trifluoromethanesulfonyl chloride is 4 mg: 5-15 μL.

[0013] During the preparation process, epoxy-based magnetic nanosphere micelles are first formed, which greatly reduces the aggregation of trifluoromethanesulfonyl nanospheres during the preparation process.

[0014] More preferably, the specific preparation process of epoxy-based magnetic nanosphere micelles is as follows: sodium dodecyl sulfate is added to an organic solvent, and after emulsification by stirring under alkaline conditions, epoxy-based magnetic nanospheres are added, and epoxy-based magnetic nanosphere micelles are formed under stirring conditions.

[0015] In the specific preparation process of epoxy-based magnetic nanosphere micelles, the mass-to-volume ratio of sodium dodecyl sulfate to organic solvent is 5 mg: 4-5 mL; preferably, the organic solvent is dichloromethane or tetrahydrofuran.

[0016] In the specific preparation process of epoxy-based magnetic nanosphere micelles, potassium hydroxide solution or triethylamine is added to form alkaline conditions; preferably, the mass ratio of sodium dodecyl sulfate to potassium hydroxide is 0.04-0.06:1; and the volume ratio of sodium dodecyl sulfate to triethylamine is 5-10 mg:1 mL.

[0017] Preferably, the trifluoromethanesulfonyl magnetic nanospheres have a diameter of 1-2 μm.

[0018] The beneficial effects of this invention are as follows:

[0019] This invention develops a pre-activated magnetic nanosphere containing trifluoromethanesulfonyl groups on its surface, which has stronger thermal stability than p-toluenesulfonyl magnetic nanospheres. This eliminates the need for an activation step, and the nanospheres are directly covalently coupled with amino-containing biomolecules (such as antigens and antibodies) under the action of ammonium sulfate catalyst, reducing antibody crosslinking and magnetic bead aggregation caused by activators such as EDC or glutaraldehyde. Attached Figure Description

[0020] To more clearly illustrate the technical solution of the present invention, the drawings used in the embodiments will be briefly introduced below. Obviously, those skilled in the art can obtain other drawings based on these drawings without creative effort.

[0021] Figure 1 This is a SEM image of the trifluoromethanesulfonyl magnetic nanospheres of the present invention. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with specific embodiments and corresponding drawings. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention. The technical solutions provided by various embodiments of this invention will be described in detail below with reference to the accompanying drawings.

[0023] 1. Preparation of crude product of trifluoromethanesulfonyl magnetic nanospheres

[0024] Example 1:

[0025] In a fume hood, 40 mL of THF (Aladdin), 20 mL of 9 mol / L (504 g / L) KOH (Aladdin) aqueous solution, and 50 mg of sodium dodecyl sulfate (SDS) were added to a three-necked flask, and a mechanical stirrer was installed at 400 rpm. Then, 40 mL of epoxy magnetic nanospheres (10 mg / mL, water, Nanjing Jidan Pharmaceutical Co., Ltd.) were slowly added dropwise to a 250 mL three-necked flask, and the mixture was stirred thoroughly for 1 hour to emulsify. The three-necked flask was placed in a water bath with ice to form an ice-water bath. Every 2 hours, 500 μL of trifluoromethanesulfonyl chloride was slowly added dropwise, for a total of 3 times. Then, the temperature of the constant temperature water bath was raised to 25 °C, and the reaction was continued for 2 hours. After the reaction was completed, the reaction mixture was transferred to a 250 mL PP plastic bottle and separated using a 1T magnetic separator until the liquid in the plastic bottle became clear. The waste liquid was discarded to obtain the crude product of trifluoromethanesulfonyl magnetic nanospheres.

[0026] Example 2:

[0027] In a fume hood, 40 mL of THF (Aladdin), 10 mL of triethylamine, and 50 mg of sodium dodecyl sulfate (SDS) were added to a three-necked flask, and a mechanical stirrer was installed at 400 rpm. Then, 40 mL of epoxy magnetic nanospheres (10 mg / mL, water) were slowly added dropwise to a 250 mL three-necked flask, and the mixture was stirred thoroughly for 1 h to emulsify. The three-necked flask was placed in a water bath with ice to form an ice-water bath, and 400 μL of trifluoromethanesulfonyl chloride was slowly added dropwise. Every 2 h, another 400 μL of trifluoromethanesulfonyl chloride was added dropwise, for a total of 3 times. Then, the temperature of the constant temperature water bath was raised to 25 °C, and the reaction was continued for 2 h. After the reaction was completed, the reaction mixture was transferred to a 250 mL PP plastic bottle and separated using a 1T magnetic separator until the liquid in the plastic bottle was clear. The waste liquid was discarded to obtain the crude product of trifluoromethanesulfonyl magnetic nanospheres.

[0028] Example 3:

[0029] In a fume hood, 40 mL of dichloromethane (Aladdin), 20 mL of 9 mol / L KOH (Aladdin) aqueous solution, and 50 mg of sodium dodecyl sulfate (SDS) were added to a three-necked flask, and a mechanical stirrer was installed at 400 rpm. Then, 40 mL of epoxy magnetic nanospheres (10 mg / mL, water) were slowly added dropwise to a 250 mL three-necked flask, and the mixture was stirred thoroughly for 1 h to emulsify. The three-necked flask was placed in a water bath with ice to form an ice-water bath. Every 2 h, 500 μL of trifluoromethanesulfonyl chloride was slowly added dropwise, for a total of 3 times. The temperature of the water bath was then raised to 25 °C, and the reaction was continued for 2 h. After the reaction was completed, the reaction mixture was transferred to a 250 mL PP plastic bottle and separated using a 1T magnetic separator until the liquid in the plastic bottle became clear. The waste liquid was then discarded, yielding the crude product of trifluoromethanesulfonyl magnetic nanospheres.

[0030] Example 4:

[0031] In a fume hood, 40 mL of dichloromethane (Aladdin), 10 mL of triethylamine, and 50 mg of sodium dodecyl sulfate (SDS) were added to a three-necked flask, and a mechanical stirrer was installed at 400 rpm. Then, 40 mL of epoxy magnetic nanospheres (10 mg / mL, water) were slowly added dropwise to a 250 mL three-necked flask, and the mixture was stirred thoroughly for 1 hour to emulsify. The three-necked flask was placed in a water bath with ice to form an ice-water bath, and 400 μL of trifluoromethanesulfonyl chloride was slowly added dropwise. Every 2 hours, another 400 μL of trifluoromethanesulfonyl chloride was added dropwise, for a total of 3 times. The temperature of the water bath was then raised to 25 °C, and the reaction was continued for 2 hours. After the reaction was completed, the reaction mixture was transferred to a 250 mL PP plastic bottle and separated using a 1T magnetic separator until the liquid in the plastic bottle became clear. The waste liquid was then discarded, yielding the crude product of trifluoromethanesulfonyl magnetic nanospheres.

[0032] Comparative Examples 1-4

[0033] Comparative Examples 1-4 correspond to the same operating steps as Examples 1-4, except that sodium dodecyl sulfate (SDS) was not added.

[0034] 2. Particle size screening and post-treatment of trifluoromethanesulfonyl magnetic nanospheres

[0035] The crude trifluoromethanesulfonyl magnetic nanospheres obtained in Examples 1-4 were washed three times with methanol or ethanol to remove unreacted reactants. The magnetic microspheres were then resuspended in deionized water by ultrasound and centrifuged five times to remove excess surfactants from the reaction. The uniformly dispersed magnetic microspheres were then passed through a column containing agarose or dextran microspheres to remove impurities or small agglomerates generated during the reaction. Finally, after repeated centrifugation, the pH of the supernatant was measured to be less than 8 or the conductivity less than 10, indicating the completion of the trifluoromethanesulfonyl magnetic nanosphere treatment. The solid content was determined, and the final volume was adjusted with purified water to a final concentration of 10 mg / mL. The nanospheres were then stored at 2-8°C.

[0036] Table 1 shows a comparison of the particle sizes of the trifluoromethanesulfonyl magnetic nanospheres prepared in Examples 1-4 and Comparative Examples 1-4.

[0037] Table 1. Effect of preemulsification micelle formation on the average particle size of trifluoromethanesulfonyl magnetic beads

[0038]

[0039]

[0040] As shown in Table 1, the particle size of the epoxy magnetic beads before preparation was 1094-1238 nm. After adding SDS emulsification, the average particle size did not increase significantly, while the average particle size increased without adding SDS. This indicates that during the preparation process, epoxy-based magnetic nanosphere micelles are formed first, which greatly reduces the aggregation of trifluoromethanesulfonyl nanospheres during the preparation process.

[0041] 3. Application of trifluoromethanesulfonyl magnetic nanospheres

[0042] The trifluoromethanesulfonyl magnetic nanospheres prepared in Examples 1-4 were coated with myoglobin antibody 1 (Nanjing GeneDan Pharmaceutical Biotechnology Co., Ltd.) according to the following scheme. The buffer formulations involved in this experiment are as follows:

[0043] Formula 1: 0.05mol / L MES (pH=5.5): Weigh 1.06g of morpholine ethanesulfonic acid monohydrate, add 80mL of purified water, stir well, adjust the pH to 5.5 with 0.5mol / L sodium hydroxide solution, and bring the volume to 100mL. Stir well.

[0044] Formula 2: 0.05mol / L PBS (pH=7.5): Weigh 1.45g Na2HPO4·12H2O, 0.13g (KH2PO4) and 4g NaCl, make up to 100mL and stir well.

[0045] Formula 3: 0.05mol / L CB (pH=9.5): Weigh 0.126g sodium bicarbonate and 0.371g sodium carbonate, make up to 100mL, and stir well.

[0046] Formula 4: 3mol / L ammonium sulfate: Weigh 39.6g of ammonium sulfate, add purified water to a final volume of 100mL, and stir well.

[0047] 3.1 Trifluoromethanesulfonyl magnetic nanospheres coated with myoglobin antibodies

[0048] (1) Take 25 mg of the above trifluoroformyl magnetic nanospheres and place them in a 10 mL centrifuge tube. Sonicate for 1 min, magnetically separate for 2 min, and keep the buffer solution. Then add 1 mL of 0.05 mol / L CB (pH 9.5), magnetically separate for 2 min, and discard the carbonate buffer solution.

[0049] (2) Add 600 μL of 0.05 mol / L carbonate buffer (pH 9.5) and 400 μL of 3 mol / L ammonium sulfate respectively, and then add 208 μL of myoglobin antibody with a concentration of 12 mg / mL (the myoglobin antibody is derived from Hytest, and the mass ratio of trifluoroformyl magnetic nanospheres to myoglobin antibody coating is 1:0.1), vortex to mix well; incubate in a constant temperature shaker at 220 r / min and 37 °C for 16 h.

[0050] (3) Magnetically separate the reaction solution for 2 min, discard the supernatant buffer, and add 5 mL of 0.1 mol / L PBS (pH 5.5).

[0051] 7.2) + 0.05% Tween - 20 + 0.1% P300) for cleaning, ultrasonic for 1 minute, repeat 5 times.

[0052] (4) Add 10 mL of storage buffer (0.1 mol / L TrisHCl (pH 7.2) + 2% BSA + 0.05% Tween-20 + 0.05% sodium azide) to a final concentration of 2.5 mg / mL.

[0053] 3.2 Preparation of acridine esterified myoglobin antibody component

[0054] (1) Turn on the power switch of the digital display constant temperature water bath and set the temperature to 37.0℃;

[0055] (2) Take 1 mg of antibody (antibody is from Hytest), add 6 μL of acridine sulfonamide (1 mg / mL, DMF), then add CB (see formula 3), the final volume is 1000 μL; shake to mix, and let stand at 37℃ for 60 min;

[0056] (3) After the reaction is complete, add 100 μL of blocking solution 0.05 mol / L TrisHCl (pH 7.2) to the above reaction tube and mix. Then, react at 37℃ for 30 min to obtain intermediate product 1.

[0057] (4) Add intermediate 1 to the dialysis bag, then place it in a 2L beaker, add 1.5L 0.1mol / L PBS (pH 7.2) for dialysis, change the solution every 4 hours until the luminescence value of the dialysis solution is less than 5000, and measure the protein concentration of the prepared acridine esterified myoglobin antibody using a micro-protein analyzer.

[0058] (5) The prepared acridine esterified myoglobin antibody was diluted to 5-10 μg / mL with storage buffer (0.1 mol / L TrisHCl (pH 7.2) + 2% BSA + 0.05% Tween-20 + 0.05% sodium azide) to obtain the acridine esterified myoglobin antibody component.

[0059] Examples 1-4: Myoglobin antibody was coated onto trifluoromethanesulfonyl magnetic nanospheres to evaluate myoglobin detection. Specifically, myoglobin control samples were detected using a fully automated chemiluminescence analyzer (MAGICL 6800). The sample loading method for myoglobin detection was: 25 μL sample + 95 μL acridine esterified myoglobin antibody component + 10 μL trifluoromethanesulfonyl magnetic nanospheres coated with myoglobin antibody or Thermofisher p-toluenesulfonyl magnetic nanospheres coated with myoglobin antibody (the specific preparation method is described in 3.1, only the Thermofisher p-toluenesulfonyl magnetic nanospheres were used instead of trifluoromethanesulfonyl magnetic nanospheres). The mixture was incubated for 10 min and washed 3 times. The measurement results are shown in Table 1.

[0060] 4. Performance testing of trifluoromethanesulfonyl magnetic microspheres

[0061] 4.1 Signal value and signal-to-noise ratio test after coating trifluoromethanesulfonyl magnetic microspheres with myoglobin antibody

[0062] As shown in Table 2, the background test results and signal values ​​and signal-to-noise ratio of the myoglobin protein control samples of Examples 1-3 are basically the same as those of the p-toluenesulfonyl magnetic nanospheres (purchased from Thermofisher). This indicates that the performance of the microspheres in Examples 1 and 3 is similar to that of the Thermofisher p-toluenesulfonyl magnetic nanospheres. Furthermore, the KOH solution used in this invention is better than triethylamine, and 50 wt / v% KOH is preferred.

[0063] Table 2. Signal-to-noise ratio test of the trifluoromethanesulfonyl magnetic microspheres coated with myoglobin antibody according to the present invention.

[0064]

[0065] Note: Baseline, control 1, control 2, and control 3 were all tested in 10 parallel samples.

[0066] 4.2 Repeatability test of trifluoromethanesulfonyl magnetic microspheres coated with myoglobin antibody

[0067] As shown in Table 3, the repeatability of the trifluoromethanesulfonyl magnetic microspheres of the present invention in the determination of myoglobin was compared with that of a commercially available p-toluenesulfonyl magnetic microsphere. The repeatability of both was 2.5-7.5%, with no significant difference.

[0068] Table 3. Repeatability test of the trifluoromethanesulfonyl magnetic microspheres coated with myoglobin antibody according to the present invention.

[0069]

[0070] Note: Baseline, control 1, control 2, and control 3 were all tested in 10 parallel samples.

[0071] 4.3 Stability Test of Trifluoromethanesulfonyl Magnetic Microspheres

[0072] Using magnetic nanospheres stored at 4℃ as a control, p-toluenesulfonyl magnetic nanospheres and trifluoromethanesulfonyl magnetic nanospheres were placed in a 37℃ incubator for 3 days and 5 days respectively for accelerated treatment. Then, myoglobin antibodies were prepared according to the coating protocol in 3.1 and detected by chemiluminescence method.

[0073] Table 4. Test of the magnetic stability of the trifluoromethanesulfonyl group in this invention.

[0074]

[0075] As shown in Table 44, after 5 days of acceleration, the luminescence value of the p-toluenesulfonyl magnetic nanospheres coated with myoglobin antibody decreased by about 60% compared with the control; while after 5 days of acceleration, the luminescence value of the trifluoroformyl magnetic nanospheres coated with myoglobin antibody hardly decreased compared with the control, indicating that the thermal stability of the trifluoroformyl magnetic nanospheres of the present invention is better than that of the p-toluenesulfonyl magnetic nanospheres.

[0076] Table 5 compares the protein-coupled components of the trifluoroformyl magnetic nanospheres of this invention with those of conventional p-toluenesulfonyl magnetic nanospheres. Regarding thermal stability, the trifluoroformyl magnetic nanospheres prepared in this method exhibit better thermal stability than the p-toluenesulfonyl magnetic nanospheres. After 5 days of accelerated processing, the signal remained unchanged even after coating with myoglobin antibodies. Trifluoromethanesulfonyl chloride, used as a modifier, can improve the thermal stability of the magnetic beads. Furthermore, [the following text appears to be incomplete and requires further context: "from..."] Figure 1SEM images of trifluoromethanesulfonyl magnetic nanospheres show no aggregation between the magnetic beads. Compared with carboxyl magnetic nanospheres, the trifluoromethanesulfonyl magnetic nanospheres prepared by this method do not require pre-activation with activators (EDC, NHS, or glutaraldehyde) and can be directly coupled to proteins without cross-linking the proteins themselves.

[0077] Table 5. Comparison of the trifluoroformyl magnetic nanospheres of the present invention with conventional p-toluenesulfonyl magnetic nanospheres and their coupling proteins.

[0078]

[0079] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The application of trifluoromethanesulfonyl magnetic nanospheres in chemiluminescent immunoassay, characterized in that, The trifluoromethanesulfonyl magnetic nanospheres are used for direct covalent coupling with antigens or antibodies; The trifluoromethanesulfonyl magnetic nanospheres are obtained by reacting the modifier trifluoromethanesulfonyl chloride with epoxy-based magnetic nanospheres. The specific preparation process of the trifluoromethanesulfonyl magnetic nanospheres includes the following steps: 1) Formation of epoxy-based magnetic nanosphere micelles; 2) Trifluoromethanesulfonyl chloride, a modifier, was added dropwise to the epoxy-based magnetic nanosphere micelles prepared in step 1) at 0-25℃ to obtain crude trifluoromethanesulfonyl magnetic nanospheres. 3) The crude product of trifluoromethanesulfonyl magnetic nanospheres was subjected to particle size screening by column chromatography and centrifugation to obtain trifluoromethanesulfonyl magnetic nanospheres. The specific preparation process of epoxy-based magnetic nanosphere micelles is as follows: sodium dodecyl sulfate is added to an organic solvent, and after emulsification by stirring under alkaline conditions, epoxy-based magnetic nanospheres are added, and epoxy-based magnetic nanosphere micelles are formed under stirring conditions.

2. The application according to claim 1, characterized in that, The mass-to-volume ratio of the epoxy magnetic nanospheres to trifluoromethanesulfonyl chloride is 4 mg: 5-15 μL.

3. The application according to claim 1, characterized in that, In the specific preparation process of epoxy-based magnetic nanosphere micelles, the mass-to-volume ratio of sodium dodecyl sulfate to organic solvent is 5 mg: 4-5 mL.

4. The application according to claim 3, characterized in that, The organic solvent is dichloromethane or tetrahydrofuran.

5. The application according to claim 1, characterized in that, In the specific preparation process of epoxy-based magnetic nanosphere micelles, potassium hydroxide solution or triethylamine is added to form alkaline conditions.

6. The application according to claim 5, characterized in that, The mass ratio of sodium dodecyl sulfate to potassium hydroxide is 0.04-0.06:1; the dosage ratio of sodium dodecyl sulfate to triethylamine is 5-10 mg:1 mL.

7. The application according to claim 1, characterized in that, The trifluoromethanesulfonyl magnetic nanospheres have a diameter of 1-2 μm.

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