Self-antifouling super-hydrophilic immunomagnetic microspheres and application thereof

Abstract

The application provides a self-anti-fouling super-hydrophilic immunomagnetic microsphere and application thereof. Specifically, the application provides a super-hydrophilic immunomagnetic microsphere, comprising: a magnetic polymer-based sphere, the magnetic polymer-based sphere comprising a polymer inner core and a magnetic material coated on the outer surface of the polymer inner core; a super-hydrophilic layer coated on the outer surface of the magnetic polymer-based sphere, the super-hydrophilic layer comprising a polymer containing primary amine and / or secondary amine grafted with non-ionic water-based polyurethane; and an immunoligand layer coated on the outer surface of the super-hydrophilic coating layer; wherein the polymer containing primary amine and / or secondary amine is selected from polyethyleneimine and polyimide; and raw materials of the non-ionic water-based polyurethane comprise polyethylene glycol and an isocyanate-containing substance. The application also provides a method for preparing the super-hydrophilic immunomagnetic microsphere. And the application provides application of the super-hydrophilic immunomagnetic microsphere in chemiluminescence immunoassay.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of chemiluminescence immunoassay, in particular to a self-anti-fouling super-hydrophilic immunomagnetic microsphere and application thereof. BACKGROUND

[0002] Immunomagnetic beads are a kind of spherical microparticles with magnetism and surface modified by functional groups. The functional groups on the microparticles can fix the immunological ligands to the surface of the microparticles through covalent coupling or physical adsorption, and under the action of an external magnetic field, the microparticles can be directionally enriched to achieve the purposes of high-efficiency separation, purification, detection, etc. In recent years, they have been widely used in the fields of chemiluminescence immunoassay, cell sorting, nucleic acid purification, etc.

[0003] Chemiluminescence immunomagnetic beads are generally divided into carboxyl magnetic beads, tosyl magnetic beads, amino magnetic beads, etc. according to the types of modification groups. By coating antibodies, antigens, streptavidin, etc. on the surface of the magnetic beads, the immunomagnetic beads can specifically bind to the antigens or antibodies to be detected in the body fluids such as plasma and serum, so as to achieve the purpose of quantitative detection. However, the composition of serum is abnormally complex, and impurities other than the substances to be detected in the serum can easily interfere with the results and affect the accuracy. Therefore, the anti-fouling ability (i.e. the ability to resist non-specific adsorption) of the surface of the immunomagnetic beads greatly affects the sensitivity and accuracy of the test.

[0004] Super-hydrophilicity is a hot spot in the research of material surface modification in recent years, and is currently more applied in membrane separation materials, biomedical materials, etc. The construction of super-hydrophilic surface is mainly determined by the roughness and chemical composition of the material surface, and the water contact angle is less than 10°. According to different application scenarios, the existing methods for constructing super-hydrophilic surface mainly include vapor deposition, electrostatic spraying, sol-gel, layer-by-layer self-assembly, etc. These methods have certain advantages in their respective application scenarios. Among them, the electrostatic spraying method requires high equipment, is not suitable for large-scale production, and the designed super-hydrophilic coating cannot uniformly coat the microspheres; the layer-by-layer self-assembly method has low utilization rate of coating materials, and the adjustable range of surface hydrophilicity is limited. In addition, CN112871097A and CN112871098A disclose super-hydrophilic magnetic microspheres prepared by ultrasonic atomization and spray drying methods, but the above methods also have high preparation cost, high energy consumption, and high requirements for preparation instruments, and are also not suitable for large-scale production.

[0005] Therefore, it is necessary to provide a super-hydrophilic immunomagnetic bead which is simple to prepare and has highly adjustable surface properties. SUMMARY

[0006] In order to solve the problems of the prior art, the present application provides a self-anti-fouling super-hydrophilic immunomagnetic microsphere and application thereof, and the hydrophilic and hydrophobic properties of the surface of the magnetic microsphere are highly adjustable, the adjustment cost is low, and the method is suitable for large-scale production.

[0007] In particular, the present application includes, but is not limited to, the following:

[0008] In one aspect, the present application provides a super-hydrophilic immunomagnetic microsphere, comprising:

[0009] a magnetic polymer-based sphere, the magnetic polymer-based sphere comprising a polymer inner core and a magnetic material coated on an outer surface of the polymer inner core;

[0010] a super-hydrophilic layer coated on an outer surface of the magnetic polymer-based sphere, the super-hydrophilic layer comprising a primary amine and / or secondary amine containing compound grafted with a non-ionic waterborne polyurethane; and

[0011] an immunoligand layer coupled on an outer surface of the super-hydrophilic layer;

[0012] wherein the primary amine and / or secondary amine containing compound is selected from one or more of polyethyleneimine, polyimide, diethylene triamine, and ethylene diamine;

[0013] the raw material of the non-ionic waterborne polyurethane comprises polyethylene glycol and an isocyanate-containing substance.

[0014] In one aspect, the primary amine and / or secondary amine containing compound of the present application is selected from one or more of polyethyleneimine and diethylene triamine.

[0015] In one aspect, the primary amine and / or secondary amine containing compound of the present application is selected from polyethyleneimine. In one aspect, the primary amine and / or secondary amine containing compound of the present application is selected from diethylene triamine.

[0016] In one aspect, the polymer inner core of the present application is selected from one or more of polystyrene microspheres, polymethyl methacrylate microspheres, polyglycidyl methacrylate microspheres, polylactic acid microspheres, and polystyrene-divinylbenzene microspheres;

[0017] and / or, the raw material of the magnetic material is selected from one or both of a ferric salt, a ferrous salt, and a manganous salt;

[0018] and / or, the immunoligand is selected from one or more of an antibody, an antigen, and a protein.

[0019] In one aspect, the polymer inner core of the present application is selected from polystyrene microspheres.

[0020] In one aspect, the raw material of the magnetic material of the present application is a mixture of a ferrous salt and a manganous salt. In one aspect, the raw material of the magnetic material of the present application is FeSO4·7H2O and MnSO4·H2O.

[0021] In one aspect, the immunoligand of the present invention is streptavidin.

[0022] In one aspect, the molecular weight of the polyethylene glycol of the present invention is in the range of 2000-5000;

[0023] and / or the molecular weight of the polymer containing primary and / or secondary amines is in the range of 30,000-70,000;

[0024] And / or, the super hydrophilic layer is a multi-layer super hydrophilic layer, and the multi-layer super hydrophilic layer has 2-4 layers.

[0025] In one aspect, the molecular weight of the polyethylene glycol of the present invention is in the range of 2000-5000.

[0026] In one aspect, the molecular weight of the polyethyleneimine of the present invention is in the range of 30000 to 70000. Preferably, the molecular weight of the polyethyleneimine of the present invention is in the range of 50000 to 70000.

[0027] In one aspect, the molecular weight of the polyethylene glycol of the present invention is 2000.

[0028] In one aspect, the super-hydrophilic layer of the present invention is a multi-layer super-hydrophilic layer. Specifically, the outer surface of the magnetic polymer-based sphere is coated once with a compound containing primary and / or secondary amines to form a first super-hydrophilic layer, followed by grafting a non-ionic aqueous polyurethane onto the compound containing primary and / or secondary amines to form a second super-hydrophilic layer, and then the coating of the first and second super-hydrophilic layers is repeated. On this basis, the multi-layer super-hydrophilic layer of the present invention has 2-4 layers, and preferably, the multi-layer super-hydrophilic layer has 4 layers.

[0029] In one aspect, the present invention provides a method for preparing superhydrophilic immunomagnetic microspheres, comprising the following steps:

[0030] (1) mixing the magnetic polymer-based spheres dispersed in a first solvent with a compound containing primary amines and / or secondary amines to obtain magnetic polymer-based spheres having the compound containing primary amines and / or secondary amines attached to their surfaces after the reaction, and separating and washing the magnetic polymer-based spheres having the compound containing primary amines and / or secondary amines attached to their surfaces;

[0031] (2) dispersing the magnetic polymer-based balls obtained in step (1) with the compound containing primary amine and / or secondary amine attached to the surface in a second solvent, and then mixing with polyethylene glycol and an isocyanate-containing substance to graft nonionic waterborne polyurethane onto the compound containing primary amine and / or secondary amine to obtain magnetic polymer-based balls grafted with nonionic waterborne polyurethane, adding glycidol after the reaction, and then separating and washing the magnetic polymer-based balls grafted with nonionic waterborne polyurethane; and

[0032] (3) repeating the coating reaction in steps (1) and (2) on the magnetic polymer-based balls grafted with the non-ionic water-based polyurethane, and replacing the glycidol with hydroxyethyl methacrylate or hydroxypropyl methacrylate after the reaction is completed, to obtain magnetic polymer-based balls coated with a multi-layer super-hydrophilic layer on the surface,

[0033] wherein the magnetic polymer-based balls comprise a polymer core and a magnetic material coated on the outer surface of the polymer core; and the polymer containing primary and / or secondary amines is selected from one or more of polyethyleneimine, polyimide, diethylene triamine, and ethylenediamine.

[0034] In one aspect, the method of the present application further comprises step (4) comprising modifying the surface of the magnetic polymer-based balls coated with a multi-layer super-hydrophilic layer obtained in step (3) with a functional group to obtain modified coated balls.

[0035] Preferably, the functional group modification of the present application is selected from carboxyl modification, epoxy modification, amino modification, or tosyl modification.

[0036] More preferably, the functional group modification of the present application is carboxyl modification.

[0037] In one aspect, the carboxyl modification process of the present application comprises dispersing the magnetic polymer-based balls coated with a multi-layer super-hydrophilic layer obtained in step (3) in an organic solvent, mixing with azobisisobutyronitrile and an organic acid in a protective atmosphere to obtain carboxyl-modified coated balls, and separating and washing the carboxyl-modified coated balls, wherein the organic acid is selected from acrylic acid, methacrylic acid, and itaconic acid. Preferably, the organic acid of the present application is selected from acrylic acid.

[0038] In one aspect, the method of the present application further comprises step (5) of coating an immunoligand on the surface of the modified coated balls to obtain super-hydrophilic immunomagnetic microspheres coated with an immunoligand layer.

[0039] In one aspect, in step (1) of the method of the present application, the polymer core is selected from one or more of polystyrene microspheres, polymethyl methacrylate microspheres, polyglycidyl methacrylate microspheres, polylactic acid microspheres, and polystyrene-divinylbenzene microspheres.

[0040] And / or, in step (1), the raw material of the magnetic material is selected from one or both of a trivalent iron salt, a divalent iron salt, and a divalent manganese salt.

[0041] And / or, in step (1), the molecular weight of the compound containing primary and / or secondary amines is in the range of 30,000-70,000.

[0042] and / or, in step (1), the first solvent is selected from one or more of absolute ethanol, isopropyl alcohol, acetonitrile, N,N-dimethylformamide;

[0043] and / or, in step (1), the mass ratio of the compound containing primary amine and / or secondary amine to the magnetic polymer-based ball or the magnetic polymer-based ball grafted with non-ionic water-based polyurethane is (1-10): 1.

[0044] and / or, in step (1), the reaction temperature is 30-70°C.

[0045] and / or, in step (1), the reaction time is 8-24 hours.

[0046] In the present application, by implementing step (1), the compound containing primary amine and / or secondary amine can be adsorbed on the surface of the magnetic polymer-based ball through non-covalent force.

[0047] Preferably, in step (1) of the present application, the polymer core is a polystyrene microsphere.

[0048] Preferably, in step (1) of the present application, the raw material of the magnetic material is selected from a mixture of divalent iron salt and divalent manganese salt. In one aspect, the raw material of the magnetic material of the present application is FeSO4·7H2O and MnSO4·H2O.

[0049] Preferably, in step (1) of the present application, the compound containing primary amine and / or secondary amine is one or more of polyethyleneimine and diethylenetriamine.

[0050] In one aspect, in step (1) of the present application, the compound containing primary amine and / or secondary amine is polyethyleneimine.

[0051] In one aspect, in step (1) of the present application, the compound containing primary amine and / or secondary amine is diethylenetriamine.

[0052] More preferably, the molecular weight of the polyethyleneimine used in the present application is in the range of 30000-70000. Most preferably, the molecular weight of the polyethyleneimine used in the present application is in the range of 50000-70000.

[0053] Preferably, in step (1) of the present application, the first solvent is absolute ethanol.

[0054] Preferably, in step (1) of the present application, the mass ratio of the magnetic polymer-based ball or the magnetic polymer-based ball grafted with non-ionic water-based polyurethane to the compound containing primary amine and / or secondary amine is 1:5. More preferably, in step (1), the mass ratio of the magnetic polymer-based ball or the magnetic polymer-based ball grafted with non-ionic water-based polyurethane to polyethyleneimine or diethylenetriamine is 1:5.

[0055] In one aspect, the mass ratio of the magnetic polymer-based sphere or the magnetic polymer-based sphere grafted with non-ionic water-based polyurethane and polyethyleneimine in step (1) of the present application is 1:5.

[0056] In one aspect, the mass ratio of the magnetic polymer-based sphere or the magnetic polymer-based sphere grafted with non-ionic water-based polyurethane and diethylenetriamine in step (1) of the present application is 1:5.

[0057] Preferably, the reaction temperature in step (1) of the present application is 70°C.

[0058] Preferably, the reaction time in step (1) of the present application is 14 hours.

[0059] In one aspect, the molecular weight of the polyethylene glycol in step (2) of the present application is in the range of 2000-5000;

[0060] And / or, the second solvent in step (2) is selected from acetone;

[0061] And / or, the mass ratio of the magnetic polymer-based sphere or the magnetic polymer-based sphere grafted with non-ionic water-based polyurethane, polyethylene glycol and isocyanate-containing substance to which the compound containing primary amine and / or secondary amine is attached in step (2) is (1-10):1:0.1;

[0062] And / or, the addition amount of the magnetic polymer-based sphere grafted with non-ionic water-based polyurethane and glycidol in step (2) is (1-5):1;

[0063] And / or, the reaction temperature before adding glycidol in step (2) is 40-58°C;

[0064] And / or, the reaction time before adding glycidol in step (2) is 1-3 hours;

[0065] And / or, the reaction temperature after adding glycidol in step (2) is 40-58°C;

[0066] And / or, the reaction time after adding glycidol in step (2) is 8-24 hours.

[0067] In one aspect, the molecular weight of the polyethylene glycol in step (2) of the present application is 2000-5000. Preferably, the molecular weight of the polyethylene glycol in step (2) of the present application is 2000.

[0068] Preferably, the mass ratio of the magnetic polymer-based sphere or the magnetic polymer-based sphere grafted with non-ionic water-based polyurethane, polyethylene glycol and isocyanate-containing substance to which the compound containing primary amine and / or secondary amine is attached in step (2) of the present application is (1-10):1:0.1.

[0069] Preferably, in step (2) of the present application, the mass ratio of the non-ionic waterborne polyurethane magnetic polymer-based ball and glycidol is (1-5): 1.

[0070] Preferably, in step (2) of the present application, the reaction temperature before adding glycidol is 50°C.

[0071] Preferably, in step (2) of the present application, the reaction time before adding glycidol is 2 hours.

[0072] Preferably, in step (2) of the present application, the reaction time after adding glycidol is 14 hours.

[0073] In one aspect, in step (2) of the present application, a catalyst is contained in the process of grafting non-ionic waterborne polyurethane on the compound containing primary amine and / or secondary amine, preferably, the catalyst is dibutyltin dilaurate.

[0074] In one aspect, in step (3) of the present application, the coating reaction of one step (1) and one step (2) is completed in the form of grafting reaction to coat 2 layers of super-hydrophilic layer, the multi-layer super-hydrophilic layer is 2-4 layers of super-hydrophilic layer, preferably, the multi-layer super-hydrophilic layer is 4 layers of super-hydrophilic layer;

[0075] And / or, in step (3), the reaction temperature after adding hydroxyethyl methacrylate or hydroxypropyl methacrylate is 40-58°C;

[0076] And / or, in step (3), the reaction time after adding hydroxyethyl methacrylate or hydroxypropyl methacrylate is 8-24 hours.

[0077] Preferably, in step (3) of the present application, the reaction temperature after adding hydroxyethyl methacrylate or hydroxypropyl methacrylate is 50°C.

[0078] Preferably, in step (3) of the present application, the multi-layer super-hydrophilic layer is 4 layers of super-hydrophilic layer.

[0079] Preferably, in step (3) of the present application, the reaction time after adding hydroxyethyl methacrylate or hydroxypropyl methacrylate is 14 hours.

[0080] Preferably, in step (3) of the present application, glycidol is replaced by hydroxyethyl methacrylate after the reaction is completed.

[0081] In one aspect, in step (4) of the present application, the organic solvent is selected from one or more of anhydrous ethanol, isopropyl alcohol, acetonitrile and N, N-dimethylformamide;

[0082] And / or, in step (4), the protective atmosphere is selected from an inert gas protective atmosphere;

[0083] And / or, in step (4), the mass ratio of the magnetic polymer-based ball coated with the multi-layer super-hydrophilic layer, azobisisobutyronitrile and acrylic acid is: 1: (0.01-0.04): (1-3);

[0084] And / or, in step (4), the reaction temperature is 60-80°C;

[0085] And / or, in step (4), the reaction temperature time is 8-24 hours.

[0086] Preferably, in step (4) of the present application, the organic solvent is anhydrous ethanol.

[0087] Preferably, in step (4) of the present application, the protective atmosphere is a nitrogen protective atmosphere.

[0088] Preferably, in step (4) of the present application, the mass ratio of the magnetic polymer-based ball coated with the multi-layer super-hydrophilic layer, azobisisobutyronitrile and acrylic acid is: 25:1:50.

[0089] Preferably, in step (4) of the present application, the reaction temperature is 70°C.

[0090] Preferably, in step (4) of the present application, the reaction temperature time is 16 hours.

[0091] In one aspect, in step (5) of the present application, the immunoligand is selected from one or more of an antibody, an antigen and a protein;

[0092] And / or, in step (5), the mass ratio of the modified coated ball and the immunoligand is 1: (0.01-0.2);

[0093] And / or, in step (5), the immunoligand is dissolved in a MES (2-morpholinoethanesulfonic acid) solution with a pH of 5.0-6.5, the modified coated ball is added, and incubation is carried out at 10-30°C for 30 minutes, then a MES solution of EDC is added, and after reaction, the super-hydrophilic immunomagnetic microspheres coupled with the immunoligand layer are obtained.

[0094] Preferably, in step (5) of the present application, the immunoligand is biotin.

[0095] Preferably, in step (5) of the present application, the mass ratio of the modified coated ball and the immunoligand is 10:1.

[0096] Preferably, in step (5) of the present application, the immunoligand is dissolved in a MES solution with a pH of 6.0, the modified coated ball is added, and incubation is carried out at 10-30°C for 30 minutes, then a MES solution of EDC is added, and after reaction, the super-hydrophilic immunomagnetic microspheres coupled with the immunoligand layer are obtained.

[0097] In one aspect, the step (1) of the present application further comprises a step of preparing magnetic polymer-based spheres.

[0098] In one aspect, the magnetic polymer-based spheres of the present application have a particle size of 500 nm.

[0099] In one aspect, the modified coated spheres of the present application have a particle size of 0.8 µm to 5.5 µm. Preferably, the modified coated spheres have a particle size of 2.8 µm.

[0100] In one aspect, the isocyanate-containing substance of the present application is selected from hexamethylene diisocyanate, 4,4-diphenyl diisocyanate and isophorone diisocyanate.

[0101] In one aspect, the isocyanate-containing substance of the present application is hexamethylene diisocyanate.

[0102] In one aspect, the present application further provides the use of the super-hydrophilic immunomagnetic microspheres of the present application in chemiluminescence immunoassay.

[0103] The beneficial effects that can be achieved by the present application include, but are not limited to, the following:

[0104] (1) The immunomagnetic microspheres obtained by the present application can maintain a WCA of less than 10°, i.e., super-hydrophilicity, even after coupling with ligands such as antibodies, antigens, proteins, etc., although the surface hydrophilic carboxyl groups are consumed;

[0105] (2) The immunomagnetic microspheres obtained by the present application have excellent anti-non-specific adsorption properties and exhibit high coupling efficiency of ligands such as antibodies, proteins, etc.;

[0106] (3) The immunomagnetic microspheres obtained by the present application exhibit super-hydrophilicity (WCA < 10°) and have excellent hydrolysis resistance such as acid resistance and alkali resistance;

[0107] (4) Through the grafting modification means provided by the present application, magnetic microspheres with different degrees of super-hydrophilic / hydrophilic surfaces can be constructed according to the application requirements of different application scenarios. BRIEF DESCRIPTION OF DRAWINGS

[0108] Figure 1 . Static water contact angle photos of different grafted modified microspheres.

[0109] Figure 2 . Surface morphology of super-hydrophilic magnetic microspheres.

[0110] Figure 3 . Non-specific adsorption test standard curve and formula. DETAILED DESCRIPTION

[0111] In order to make the purposes, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely. Obviously, the described embodiments are some embodiments but not all embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present application.

[0112] Example 1 Preparation of super-hydrophilic immunomagnetic microspheres

[0113] 1. Preparation of a ball

[0114] Take 925 g of purified water into a three-necked flask with a mechanical stirrer, set the stirrer speed to 250 rpm, pass nitrogen for 30 minutes, add 20 g of styrene and 10 mL of methyl methacrylate (previously pass nitrogen for 30 minutes), set the temperature to 75℃, add 75 g of 0.6% (W / W) potassium persulfate aqueous solution (previously pass nitrogen for 30 minutes), and react for 16 hours.

[0115] After the reaction is completed, the reaction solution is filtered through a 400-mesh nylon screen to remove large agglomerated particles, the filtrate is transferred to a dialysis bag, and then placed in 50 kg of laboratory water for dialysis, with liquid exchange once a day, for a total of 7 times. After dialysis is completed, samples are taken for testing of solid content. The dispersion is placed in a plastic sample bottle and stored in a refrigerator at 2-8℃.

[0116] 2. Preparation of swollen polymer microspheres (PMS)

[0117] Solution A: Take 15 g of sodium dodecyl sulfate and add 6000 g of laboratory water. After complete dissolution, it is ready for use.

[0118] Dispersion B: Take 145 g of dibutyl phthalate and 1450 g of solution A, first use an ultrasonic cleaning machine for ultrasonic treatment for 10 minutes, and then use an ultrasonic cell crusher for ultrasonic treatment for 30 minutes (set parameters: 25# amplitude bar, 25-60% power, working time 2 seconds, interval time 4 seconds, total time 30 minutes).

[0119] Dispersion C: Add 230.4 g of styrene, 235.5 g of divinylbenzene, 811 g of toluene, 63.5 g of dibenzoyl peroxide and 20.5 g of A010010001 into a beaker, respectively, and stir until completely dissolved. Then add 3931 g of solution A and 784 g of laboratory water, first use a high-shear dispersion emulsifier for dispersion at 4000 RPM for 40 minutes, and then use an ultrasonic cell crusher for ultrasonic treatment for 30 minutes (set parameters: 25# amplitude bar, 25-60% power, working time 2 seconds, interval time 4 seconds, total time 30 minutes).

[0120] S1 : Weigh 1 g of the seed dispersion solution (containing solid 23.85 g), add it to the reaction vessel, and raise the temperature to 35 °C. Then add 1577.5 g of dispersion solution B, and stir at a speed of 100 rpm for 24 hours.

[0121] S2: Add 5493 g of dispersion solution C to the reaction vessel, and stir at a speed of 100 rpm for 24 hours at 35 °C.

[0122] S3: Pass nitrogen gas for 30 minutes, set the temperature to 80 °C, and stir the reaction for 24 hours.

[0123] S4: Filter the reaction solution through a 400-mesh nylon screen to remove large agglomerated particles, and use suction filtration. Wash the product with 5000 g of laboratory water and 2400 g of ethanol three times, and dry it to obtain a white solid powder (PMS) with a particle size of 500 nm.

[0124] 3. Nitration

[0125] Cool a mixture of 25 g of 95% sulfuric acid and 7.5 g of 65% nitric acid to 10 °C, and then add 1 g of PMS. Raise the temperature to 30 °C, and stir at a speed of 250 rpm for 2 hours. Then, add 150 g of ice water. Filter, wash with ethanol three times, and wash with pure water three times, and dry it to obtain a yellow solid powder (PMSN).

[0126] 4. Magnetization

[0127] Take 1 g of PMSN, disperse it with 10 g of pure water, and add it to a three-necked flask containing 10 g of FeS04·7H20 and 0.02 g of MnS04·H20. Start stirring at a speed of 250 rpm, and pass nitrogen gas for 30 minutes. Raise the temperature to 60 °C, add 10 mL of 25% ammonia water, and react at 60 °C for 4 hours. Magnetically separate it, and wash it with pure water to obtain magnetic microspheres.

[0128] 5. Hydrophilic coating

[0129] H1 : Take 1 g of the magnetic microspheres obtained in step 4, magnetically separate them, wash them with anhydrous ethanol three times, disperse them with 10 g of anhydrous ethanol, and add them to a three-necked flask containing 5 g of polyethyleneimine (PEI). Raise the temperature to 70 °C, and stir at a speed of 250 rpm for 14 hours.

[0130] H2: After step H1 above, magnetic separation, washed with 3 times of anhydrous ethanol, then washed with 3 times of acetone. Then dispersed with 10 g of acetone, added into a three-necked flask containing 2 g of PEG2000 and 0.01 g of catalyst dibutyltin dilaurate. The temperature was raised to 50℃, 0.2 g of hexamethylene diisocyanate (HDI) was added, reacted for 2 h, then 2 g of glycidol (GL) was added, reacted for 14 h.

[0131] H3: After step H2 above, magnetic separation, washed with 3 times of anhydrous ethanol, then dispersed with 10 g of anhydrous ethanol, added into a three-necked flask containing 5 g of polyethyleneimine (PEI). The temperature was raised to 70℃, stirred at a speed of 250 rpm for 14 h.

[0132] H4: After step H3 above, magnetic separation, washed with 3 times of anhydrous ethanol, then washed with 3 times of acetone. Then dispersed with 10 g of acetone, added into a three-necked flask containing 2 g of PEG2000 and 0.01 g of catalyst dibutyltin dilaurate. The temperature was raised to 50℃, 0.2 g of hexamethylene diisocyanate (HDI) was added, reacted for 2 h, then 2 g of hydroxyethyl methacrylate (HEMA) was added, reacted for 14 h. After completion, magnetic separation, washed with 3 times of acetone, then washed with 3 times of pure water, to obtain super-hydrophilic magnetic microspheres with suspended double bonds on the surface, and the solid content was determined.

[0133] 6. Carboxyl modification

[0134] Take 1 g of the coated spheres in step 5-H4, magnetic separation, washed with 3 times of ethanol, then dispersed with 10 g of ethanol, added into a three-necked flask containing 0.04 g of AIBN and 2 g of acrylic acid, set the speed of the stirrer to 250 rpm, and nitrogen was passed for 30 minutes. The temperature was raised to 70℃, and reacted at 70℃ for 16 h. After completion, washed with 3 times of ethanol, 3 times of pure water, dispersed with pure water, and the solid content was determined and stored for later use.

[0135] The solid content test procedure is as follows: ① A clean and dry glass weighing bottle was weighed, and the mass was recorded to 0.001 g, denoted as m1. The analytical balance was skinned. ② A certain mass of microsphere dispersion prepared in step 2 of Example 1-2 or Comparative Example 1 was weighed and placed in the glass weighing bottle, and the weight of the microsphere dispersion was recorded to 0.001 g, denoted as m2. ③ The glass bottle containing the microsphere dispersion was placed in a forced air drying oven with a temperature of 120℃, and heated for 4 h. ④ After the test time reached, the glass bottle was taken out and placed in a desiccator to cool to room temperature, and the mass was weighed to 0.001 g, denoted as m3. ⑤ The solid content of the WPU solution was calculated according to the following formula, accurate to 0.01%.

[0136] Microsphere dispersion liquid solid content = (m3-m1) / m2*100%.

[0137] 7. Immobilization of streptavidin

[0138] (1) Take 10 mg of carboxyl-modified magnetic beads in step 6, magnetically separate, remove the supernatant, and wash with pH 6.0 MES for 3 times. 1 mg of streptavidin dissolved in 900 μL of pH 6.0 MES is added to the magnetic beads.

[0139] (2) Incubate the mixed dispersion of magnetic beads and streptavidin at room temperature for 30 minutes with a mixing device.

[0140] (3) Weigh 1 mg of EDC, dissolve it in 100 μL of pH 6.0 MES, and add it to the mixed dispersion, and react at room temperature for 5 hours.

[0141] (4) Magnetically separate, remove the supernatant, wash with pH 7.4 phosphate buffer containing 0.01% (W / V) Tween-20 for 3 times to remove excess EDC and streptavidin, and finally disperse with 1 mL of pH 7.4 phosphate buffer containing 0.01% (W / V) Tween-20, store at 2-8°C, and use.

[0142] Example 2 Preparation of super-hydrophilic immunomagnetic microspheres

[0143] 1. Preparation of seed particles

[0144] Take 925 g of purified water and add it to a three-necked flask with a mechanical stirrer, set the stirrer speed to 250 rpm, and pass nitrogen gas for 30 minutes. Add 20 g of styrene and 10 mL of methyl methacrylate (previously pass nitrogen gas for 30 minutes), set the temperature to 75°C, and add 75 g of 0.6% (W / W) potassium persulfate aqueous solution (previously pass nitrogen gas for 30 minutes), and react for 16 hours.

[0145] After the reaction is completed, the reaction solution is filtered through a 400-mesh nylon screen to remove agglomerated large particles, and the filtrate is transferred to a dialysis bag, then placed in 50 kg of laboratory water for dialysis, and the liquid is changed once a day for a total of 7 times. After dialysis is completed, take samples to test the solid content. The dispersion is packed into a plastic sample bottle and stored in a 2-8°C refrigerator.

[0146] 2. Preparation of swollen polymer microspheres (PMS)

[0147] Solution A: Weigh 15 g of sodium dodecyl sulfate and add it to 6000 g of laboratory water. After complete dissolution, it is ready for use.

[0148] Dispersion B: 145 g of dibutyl phthalate and 1450 g of Solution A were measured, first ultrasonic cleaning machine for 10 minutes, then ultrasonic cell crusher for 30 minutes (set parameters: 25# amplitude bar, 25~60% power, working time 2 seconds, gap time 4 seconds, total time 30 minutes).

[0149] Dispersion C: 230.4 g of styrene, 235.5 g of divinylbenzene, 811 g of toluene, 63.5 g of dibenzoyl peroxide and 20.5 g of A010010001 were added to a beaker, respectively, and after complete dissolution by stirring, 3931 g of Solution A and 784 g of laboratory water were added, first dispersed by high shear dispersion emulsifier 4000 RPM for 40 minutes, and then ultrasonic cell crusher for 30 minutes (set parameters: 25# amplitude bar, 25~60% power, working time 2 seconds, gap time 4 seconds, total time 30 minutes).

[0150] S1: The seed dispersion solution (containing solid 23.85 g) was weighed into the reaction vessel, heated to 35 ℃, then 1577.5 g of dispersion B was added, and stirred at 100 rpm for 24 hours.

[0151] S2: 5493 g of dispersion C was added to the reaction vessel, and stirred at 100 rpm for 24 hours at 35 ℃.

[0152] S3: Nitrogen was passed for 30 minutes, the temperature was set to 80 ℃, and the stirring reaction was carried out for 24 hours.

[0153] S4: The reaction liquid was filtered through a 400 mesh nylon screen to remove agglomerated large particles, suction filtered, washed 3 times with 5000 g of laboratory water and 2400 g of ethanol, and dried to obtain a white solid powder (PMS) with a particle size of 500 nm.

[0154] 3. Nitration

[0155] A mixture of 25 g of 95% sulfuric acid and 7.5 g of 65% nitric acid was cooled to 10 ℃, then 1 g of PMS was added. The temperature was raised to 30 ℃, and stirred at 250 rpm for 2 hours. Then, 150 g of ice water was added. Filtered, washed 3 times with ethanol, and 3 times with pure water, and dried to obtain a yellow solid powder (PMSN).

[0156] 4. Magnetization

[0157] Take 1 g of PMSN, disperse with 10 g of pure water, add to a three-necked flask containing 10 g of FeS04-7H20 and 0.02 g of MnS04-H20, open the stirring, at a speed of 250 rpm, nitrogen for 30 minutes. The temperature is raised to 60°C, 10 mL of 25% ammonia is added, and the reaction is carried out at 60°C for 4 hours. Magnetic separation, washing with pure water, to obtain magnetic microspheres.

[0158] 5. Hydrophilic coating

[0159] H1: Take 1 g of the magnetic microspheres obtained in step 4, magnetically separate, wash with anhydrous ethanol 3 times, then disperse with 10 g of anhydrous ethanol, add to a three-necked flask containing 5 g of diethylenetriamine. The temperature is raised to 70°C, and stirring is carried out at a speed of 250 rpm for 14 hours.

[0160] H2: After the end of the above step H1, magnetically separate, wash with anhydrous ethanol 3 times, then wash with acetone 3 times. Then disperse with 10 g of acetone, add to a three-necked flask containing 2 g of PEG2000 and 0.01 g of catalyst dibutyltin dilaurate. The temperature is raised to 50°C, 0.2 g of hexamethylene diisocyanate (HDI) is added, and the reaction is carried out for 2 h, then 2 g of glycidol (GL) is added, and the reaction is carried out for 14 h.

[0161] H3: After the end of the above step H2, magnetically separate, wash with anhydrous ethanol 3 times, then disperse with 10 g of anhydrous ethanol, add to a three-necked flask containing 5 g of diethylenetriamine. The temperature is raised to 70°C, and stirring is carried out at a speed of 250 rpm for 14 hours.

[0162] H4: After the end of the above step H3, magnetically separate, wash with anhydrous ethanol 3 times, then wash with acetone 3 times. Then disperse with 10 g of acetone, add to a three-necked flask containing 2 g of PEG2000 and 0.01 g of catalyst dibutyltin dilaurate. The temperature is raised to 50°C, 0.2 g of hexamethylene diisocyanate (HDI) is added, and the reaction is carried out for 2 h, then 2 g of hydroxyethyl methacrylate (HEMA) is added, and the reaction is carried out for 14 h. After the end, magnetically separate, wash with acetone 3 times, then wash with pure water 3 times, to obtain super-hydrophilic magnetic microspheres with suspended double bonds on the surface, and measure the solid content.

[0163] 6. Carboxyl modification

[0164] Take 1 g of coated ball in step 5-H4, magnetic separation, wash with ethanol 3 times, then disperse with 10 g of ethanol, add to a three-necked flask containing 0.04 g of AIBN and 2 g of acrylic acid, set the stirrer speed to 250 rpm, pass nitrogen for 30 minutes, raise the temperature to 70℃, react at 70℃ for 16 hours, after completion, wash with ethanol 3 times, pure water 3 times, disperse with pure water, measure the solid content, and store for later use.

[0165] 7. Immobilization of streptavidin

[0166] (1) Take 10 mg of carboxyl-modified magnetic beads in step 6, magnetic separation, remove the supernatant, wash with pH 6.0 MES 3 times, add 1 mg of streptavidin dissolved in 900 μL of pH 6.0 MES to the magnetic beads.

[0167] (2) Incubate the mixed dispersion of magnetic beads and streptavidin at room temperature for 30 minutes with a mixing device.

[0168] (3) Weigh 1 mg of EDC, dissolve in 100 μL of pH 6.0 MES, add to the mixed dispersion, and react at room temperature for 5 hours.

[0169] (4) Magnetic separation, remove the supernatant, wash with pH 7.4 phosphate buffer containing 0.01% (W / V) Tween-20 3 times to remove excess EDC and streptavidin, finally disperse with 1 mL of pH 7.4 phosphate buffer containing 0.01% (W / V) Tween-20, store at 2-8℃, and reserve for later use.

[0170] Comparative Example 1

[0171] 1. Preparation of seed balls

[0172] Take 925 g of purified water and add it to a three-necked flask with a mechanical stirrer, set the stirrer speed to 250 rpm, pass nitrogen for 30 minutes, add 20 g of styrene and 10 mL of methyl methacrylate (previously pass nitrogen for 30 minutes), set the temperature to 75℃, add 75 g of 0.6% (W / W) potassium persulfate aqueous solution (previously pass nitrogen for 30 minutes), and react for 16 hours.

[0173] After the reaction is completed, the reaction solution is filtered through a 400-mesh nylon screen to remove agglomerated large particles, the filtrate is transferred to a dialysis bag, and then placed in 50 kg of laboratory water for dialysis, with the liquid changed once a day for a total of 7 times. After dialysis is completed, samples are taken to test the solid content. The dispersion is placed in a plastic sample bottle and stored in a 2-8℃ refrigerator.

[0174] 2. Preparation of swollen polymer microspheres (PMS)

[0175] Solution A: Weigh 15 g of sodium dodecyl sulfate, add 6000 g of laboratory water, and dissolve completely for later use.

[0176] Dispersion B: Measure 145 g of dibutyl phthalate and 1450 g of solution A, first use the ultrasonic cleaner for 10 minutes, then use the ultrasonic cell crusher for 30 minutes (set parameters: 25# amplitude rod, 25~60% power, working time 2 seconds, interval time 4 seconds, total time 30 minutes).

[0177] Dispersion C: Add 230.4 g of styrene, 235.5 g of divinylbenzene, 811 g of toluene, 63.5 g of dibenzoyl peroxide, and 20.5 g of A010010001 to the beaker, respectively, stir and dissolve completely, then add 3931 g of solution A and 784 g of laboratory water, first use the high shear dispersion emulsifier for 40 minutes at 4000 RPM, then use the ultrasonic cell crusher for 30 minutes (set parameters: 25# amplitude rod, 25~60% power, working time 2 seconds, interval time 4 seconds, total time 30 minutes).

[0178] S1: Weigh the seed dispersion (containing 23.85 g of solid), add it to the reaction container, heat to 35 ℃, then add 1577.5 g of dispersion B, stir at 100 rpm for 24 hours.

[0179] S2: Add 5493 g of dispersion C to the reaction container, stir at 100 rpm for 24 hours at 35 ℃.

[0180] S3: Pass nitrogen for 30 minutes, set the temperature to 80 ℃, and stir for 24 hours.

[0181] S4: Filter the reaction liquid through a 400-mesh nylon screen to remove agglomerated large particles, vacuum filter, wash 3 times with 5000 g of laboratory water and 2400 g of ethanol, and dry to obtain a white solid powder (PMS) with a particle size of 500 nm.

[0182] 3. Nitration

[0183] Cool a mixture of 25 g of 95% sulfuric acid and 7.5 g of 65% nitric acid to 10 ℃, then add 1 g of PMS. Raise the temperature to 30 ℃, stir at 250 rpm for 2 hours. Then, add 150 g of ice water. Filter, wash 3 times with ethanol, and then wash 3 times with pure water, and dry to obtain a yellow solid powder (PMSN).

[0184] 4. Magnetization

[0185] Take 1 g of PMSN, disperse with 10 g of pure water, add to a three-necked flask containing 10 g of FeSO4.7H2O and 0.02 g of MnSO4.H2O, open the stirrer at 250 rpm, and pass nitrogen gas for 30 minutes. Raise the temperature to 60°C, add 10 mL of 25% ammonia water, and react at 60°C for 4 hours. Magnetically separate, wash with pure water, and obtain magnetic microspheres.

[0186] 5. Hydrophilic coating

[0187] H1: Take 1 g of the magnetic microspheres obtained in step 4, magnetically separate, wash with anhydrous ethanol 3 times, disperse with 10 g of anhydrous ethanol, and add to a three-necked flask containing 5 g of polyethyleneimine (PEI). Raise the temperature to 70°C, stir at 250 rpm for 14 hours.

[0188] H2: After the end of step H1 above, magnetically separate, wash with anhydrous ethanol 3 times. Then disperse with 10 g of ethanol, and add to a three-necked flask containing 5 g of bisphenol A diglycidyl ether (ER4). Raise the temperature to 70°C, stir at 250 rpm for 14 hours.

[0189] H3: After the end of step H2 above, magnetically separate, wash with anhydrous ethanol 3 times, disperse with 10 g of anhydrous ethanol, and add to a three-necked flask containing 5 g of polyethyleneimine (PEI). Raise the temperature to 70°C, stir at 250 rpm for 14 hours.

[0190] H4: After the end of step H3 above, magnetically separate, wash with anhydrous ethanol 3 times. Then disperse with 10 g of ethanol, and add to a three-necked flask containing 5 g of glycidyl methacrylate (GMA). Raise the temperature to 70°C, stir at 250 rpm for 14 hours. After the end, magnetically separate, wash with ethanol 3 times, and then with pure water 3 times, to obtain hydrophilic magnetic microspheres with suspended double bonds on the surface, and measure the solid content.

[0191] 6. Carboxyl modification

[0192] Take 1 g of the coated spheres in step 5-H4, magnetically separate, wash with ethanol 3 times, then disperse with 10 g of ethanol, add to a three-necked flask containing 0.04 g of AIBN and 2 g of acrylic acid, set the stirrer speed to 250 rpm, pass nitrogen gas for 30 minutes, raise the temperature to 70°C, and react at 70°C for 16 hours. After the end, wash with ethanol 3 times, wash with pure water 3 times, disperse with pure water, measure the solid content, and store for use.

[0193] 7. Streptavidin immobilization

[0194] (1) Take 10 mg of the carboxyl-modified magnetic beads in step 6, magnetically separate, remove the supernatant, and wash with 3 times of pH 6.0 MES, and then add 1 mg of streptavidin dissolved in 900 μL of pH 6.0 MES to the magnetic beads.

[0195] (2) Incubate the mixed dispersion of the magnetic beads and streptavidin at room temperature for 30 minutes using a mixing device.

[0196] (3) Weigh 1 mg of EDC, dissolve it in 100 μL of pH 6.0 MES, and add it to the mixed dispersion, and react at room temperature for 5 hours.

[0197] (4) Magnetically separate, remove the supernatant, wash with 3 times of pH 7.4 phosphate buffer containing 0.01% (W / V) Tween-20 to remove excess EDC and streptavidin, and finally disperse with 1 mL of pH 7.4 phosphate buffer containing 0.01% (W / V) Tween-20, and store at 2-8°C for standby use.

[0198] Example 4 Performance detection of super-hydrophilic immunomagnetic microspheres

[0199] (1) Static water contact angle measurement

[0200] The microsphere dispersion was uniformly coated on a glass sheet, and after the microspheres were completely dried, the water contact angle was measured at room temperature using an XG-CAM contact angle tester. The volume of each droplet was about 3 μL, and the average value was obtained by parallel testing 5 times. The shortest distance between the measurement points of the same sample was 10 mm, and the results are shown in Figure 1 .

[0201] Wherein, a is Comparative Example 1, b is Example 2, and c is Example 1. As can be seen from the size of the static water contact angle, the use of PEI and diethylenetriamine as the spacer layer of the hydrophilic layer both achieved the hydrophilic coating of the magnetic bead surface. The static water contact angle decreased from 65.1° of the comparative example to 14.0° and 6.9°, respectively. Compared with diethylenetriamine, polyethyleneimine achieved true super-hydrophilicity (static water contact angle < 10°). The difference is mainly due to the fact that the molecular weight of polyethyleneimine is larger than that of diethylenetriamine, the molecular chain is composed of more secondary amines, secondary amines and tertiary amines, and the grafting site is more. The subsequent coating layer is more complete on the surface of the microspheres.

[0202] (2) Surface morphology test of microspheres

[0203] Then, the surface morphology of the super-hydrophilic magnetic beads prepared in Examples 1 and 2 was analyzed by a JSM-6380LV scanning electron microscope of Japan, and the results are shown in Figure 2 .

[0204] (3) Non-specific adsorption test

[0205] Adsorption capacity of super-hydrophilic magnetic microspheres to BSA:

[0206] 1) In this experiment, bovine serum albumin (BSA) was selected as the target protein for non-specific adsorption. The adsorption capacity of super-hydrophilic magnetic microspheres to BSA was determined by Lowry method using a multifunctional enzyme marker. Among them, reagent A refers to the standard configuration of Folin phenol reagent A: Folin phenol reagent B = 50: 1, and Folin phenol reagent is provided by the improved Lowry protein concentration determination kit of Biyun Tian; reagent B refers to Folin phenol reagent, which is provided by the improved Lowry protein concentration determination kit of Biyun Tian.

[0207] 2) Determination of BSA standard curve: Configure 0 mg / mL, 0.01 mg / mL, 0.02 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.15 mg / mL, 0.2 mg / mL, 0.25 mg / mL, 0.4 mg / mL concentration gradient of BSA protein solution, use enzyme marker 96 hole plate with pipette gun to add different concentrations of BSA protein solution, duplicate holes, pipette gun to add 200 μL of reagent A to each sample hole, mix and shake for 30 seconds, then stand for 10 minutes. Use a pipette to add 20 μL of reagent B to each well, mix and shake for 30 seconds, then incubate in a 37 ℃ constant temperature drying oven for 30 minutes.

[0208] 3) After incubation, the corresponding absorbance at A650 is obtained, the absorbance at 0 mg / mL (background value) is subtracted, the average value is taken, and the standard curve and formula are fitted.

[0209] 4) Determination of BSA adsorption capacity of super-hydrophilic magnetic microspheres: Take 10 mg of complex magnetic microspheres, disperse with 1 mL of pH=6.0 MES buffer solution, add 1 mg of BSA solution prepared in step 2) above, dissolve and shake evenly, then shake at room temperature for 16 hours, centrifuge, and take the supernatant for determination.

[0210] Use a pipette to add 20 μL of MES solution as a blank control and the supernatant to be tested, duplicate holes, and use a pipette to add 200 μL of reagent A to each sample hole, mix and shake for 30 seconds, then stand for 10 minutes. Use a pipette to add 20 μL of reagent B to each well, mix and shake for 30 seconds, then incubate in a 37 ℃ constant temperature drying oven for 30 minutes, and detect the absorbance at A650.

[0211] Table 1 Non-specific adsorption capacity of magnetic beads with different hydrophilic surfaces

[0212]

[0213] From Table 1, it can be seen that after the surface of the magnetic microspheres is modified by the super-hydrophilic material, the physical adsorption (non-specific adsorption) of the magnetic microspheres to BSA is significantly reduced, and the amount of physical adsorption decreases with the increase of the hydrophilicity of the surface of the microspheres, which is consistent with the results of the static water contact angle. Figure 1

[0214] (4) Indirect method for detecting thyroid peroxidase antibody by magnetic beads

[0215] 1) A series of different concentrations of self-prepared thyroid peroxidase antibody solutions are prepared, and the concentration range is 0-100 mIU / mL; 2) Different concentrations of biotin-thyroid peroxidase solution are mixed with pH=7.4 PBS buffer containing magnetic microspheres (SA) prepared in Examples 1 and 2 or Comparative Example 1, and then acridinester-goat anti-mouse secondary antibody is added for oscillation and incubation, followed by magnetic separation of the product. After the product is washed with pH=7.4 PBS buffer and then dispersed in pH=7.4 PBS buffer again, hydrogen peroxide with a final concentration of 0.4-1.0 mM and sodium hydroxide with a final concentration of 0.1-0.3 mM are added, and the chemiluminescence intensity value of the immune complex is measured by a chemiluminescence immunoassay instrument; the chemiluminescence intensity value of the SA magnetic beads after detection of different concentrations of thyroid peroxidase is recorded as Y, and the concentration of peroxidase antibody is recorded as X. The chemiluminescence intensity is taken as the ordinate, and calibration testing is performed.

[0216] In Table 2, TC1, TC2, TC3, TC4, TC5 and TC6 respectively represent different concentrations of thyroid peroxidase antibody, TC1 represents an antibody concentration of 0 mIU / L, TC2 represents an antibody concentration of 45 mIU / L, TC3 represents an antibody concentration of 90 mIU / L, TC4 represents an antibody concentration of 180 mIU / L, TC5 represents an antibody concentration of 360 mIU / L, and TC6 represents an antibody concentration of 720 mIU / L.

[0217] Table 2 Comparison of the performance of magnetic beads with different hydrophilic surfaces in the indirect method

[0218]

[0219] As can be seen from Table 2, in the chemiluminescence indirect method test, the background value (TC1) is greatly reduced with the increase of hydrophilicity, the signal-to-noise ratio TC2 / TC1 is also improved, and at the same time, the high value TC6 does not show attenuation or significant decrease, indicating that the magnetic microspheres modified by super-hydrophilicity reduce non-specific adsorption, improve test sensitivity, and at the same time, retain the immunological activity of the immunoligand on the surface of the magnetic beads, thereby retaining or even improving the linear range of the test. RLU1 and RLU2 refer to 2 repeated tests. ​

[0220] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, and are not intended to limit the present application; although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that the technical solutions recorded in the foregoing embodiments can still be modified, or some or all of the technical features can be replaced by equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A super-hydrophilic immunomagnetic microsphere, characterized in that: Include: A magnetic polymer-based ball comprising a polymer core and a magnetic material coated on the outer surface of the polymer core; A super-hydrophilic layer coated on the outer surface of the magnetic polymer-based ball, wherein the super-hydrophilic layer comprises a compound containing primary amine and / or secondary amine grafted with non-ionic waterborne polyurethane; as well as an immune ligand layer coupled to the outer surface of the super hydrophilic layer; Wherein, the compound containing primary amine and / or secondary amine is selected from polyethyleneimine; The raw materials of the non-ionic waterborne polyurethane include polyethylene glycol and an isocyanate-containing substance, and the isocyanate-containing substance is hexamethylene diisocyanate; The super hydrophilic layer is a multi-layer super hydrophilic layer, and the multi-layer super hydrophilic layer is a 4-layer super hydrophilic layer.

2. The superhydrophilic immunomagnetic microspheres according to claim 1, characterized in that The polymer core is selected from one or more of polystyrene microspheres, polymethyl methacrylate microspheres, polyglycidyl methacrylate microspheres, polylactic acid microspheres and polystyrene-divinylbenzene microspheres; And / or, the raw material of the magnetic material is selected from one or two of ferric salts, divalent iron salts and divalent manganese salts; And / or, the immune ligand is selected from one or more of antibodies, antigens and proteins.

3. The super-hydrophilic immunomagnetic microspheres according to claim 1, characterized in that The molecular weight of the polyethylene glycol is in the range of 2000-5000; And / or, the molecular weight of the compound containing primary amine and / or secondary amine is in the range of 30,000-70,000.

4. A method for preparing superhydrophilic immunomagnetic microspheres, characterized in that: The following steps are involved: (1) mixing the magnetic polymer-based spheres dispersed in a first solvent with a compound containing primary amines and / or secondary amines to obtain magnetic polymer-based spheres having the compound containing primary amines and / or secondary amines attached to their surfaces after the reaction, and separating and washing the magnetic polymer-based spheres having the compound containing primary amines and / or secondary amines attached to their surfaces; (2) dispersing the magnetic polymer-based balls obtained in step (1) with the compound containing primary amine and / or secondary amine attached to the surface in a second solvent, and then mixing with polyethylene glycol and an isocyanate-containing substance to graft nonionic waterborne polyurethane onto the compound containing primary amine and / or secondary amine to obtain magnetic polymer-based balls grafted with nonionic waterborne polyurethane, adding glycidol after the reaction, and then separating and washing the magnetic polymer-based balls grafted with nonionic waterborne polyurethane; and (3) Repeating the coating reaction in step (1) and / or step (2) on the magnetic polymer-based ball grafted with non-ionic waterborne polyurethane, and replacing glycidol with hydroxyethyl methacrylate or hydroxypropyl methacrylate after the reaction to obtain a magnetic polymer-based ball with a multi-layer super hydrophilic layer coated on the surface, Wherein, the magnetic polymer-based ball comprises a polymer core and a magnetic material coated on the outer surface of the polymer core; the compound containing primary amine and / or secondary amine is selected from polyethyleneimine; The isocyanate-containing substance is hexamethylene diisocyanate; In step (3), the coating reaction of step (1) and step (2) is completed once to coat two super-hydrophilic layers in the form of a grafting reaction, and the multi-layer super-hydrophilic layer is four super-hydrophilic layers.

5. The method according to claim 4, characterized in that The method further comprises step (4), which comprises performing functional group modification on the surface of the magnetic polymer-based spheres coated with multiple super-hydrophilic layers obtained in step (3) to obtain modified coated spheres.

6. The method according to claim 5, characterized in that The method further comprises step (5): coupling an immune ligand to the surface of the modified coated sphere to obtain super-hydrophilic immunomagnetic microspheres coupled with an immune ligand layer.

7. The method according to claim 4, characterized in that In step (1), the polymer core is selected from one or more of polystyrene microspheres, polymethyl methacrylate microspheres, polyglycidyl methacrylate microspheres, polylactic acid microspheres and polystyrene-divinylbenzene microspheres; And / or, in step (1), the raw material of the magnetic material is selected from one or two of trivalent iron salt, divalent iron salt and divalent manganese salt; and / or, in step (1), the molecular weight of the polymer containing primary amine and / or secondary amine is in the range of 30,000-70,000; And / or, in step (1), the first solvent is selected from one or more of anhydrous ethanol, isopropanol, acetonitrile, and N,N-dimethylformamide; And / or, in step (1), the mass ratio of the compound containing primary amine and / or secondary amine to the magnetic polymer-based ball or the magnetic polymer-based ball grafted with non-ionic waterborne polyurethane is (1-10):1; and / or, in step (1), the reaction temperature is 30-70°C; And / or, in step (1), the reaction time is 8 to 24 hours.

8. The method according to claim 4, characterized in that In step (2), the molecular weight of the polyethylene glycol is in the range of 2000-5000; and / or, in step (2), the second solvent is selected from acetone; And / or, in step (2), the mass ratio of the magnetic polymer-based spheres connected with a compound containing primary amine and / or secondary amine or the magnetic polymer-based spheres grafted with non-ionic waterborne polyurethane, polyethylene glycol and hexamethylene diisocyanate is (1-10):1:0.1; and / or, in step (2), the mass ratio of the magnetic polymer-based spheres to glycidol is (1-5):1; and / or, in step (2), the reaction temperature before adding glycidol is 40-58°C; and / or, in step (2), the reaction time before adding glycidol is 1 to 3 hours; and / or, in step (2), the reaction temperature after adding glycidol is 40-58°C; And / or, in step (2), the reaction time after adding glycidol is 8 to 24 hours.

9. The method according to claim 4, characterized in that In step (3), the reaction temperature after adding hydroxyethyl methacrylate or hydroxypropyl methacrylate is 40-58°C; And / or, in step (3), the reaction time after adding hydroxyethyl methacrylate or hydroxypropyl methacrylate is 8 to 24 hours.

10. Use of the superhydrophilic immunomagnetic microspheres according to any one of claims 1 to 3 in chemiluminescent immunoassay.

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