Preparation method of low non-specific streptavidin magnetic microspheres and chemiluminescence application

Abstract

The application relates to preparation of low non-specific streptavidin magnetic microspheres and chemical luminescence application, and belongs to the technical field of nanomaterials. The application provides a preparation method of magnetic microspheres, hydrophilic modification is carried out on magnetic nanoparticles, a layer-by-layer self-assembly method is used to adsorb the magnetic particles on the surface of polymer microspheres, then carboxyl modification and non-specificity reduction modification are carried out on the surface, and functionalized magnetic composite microspheres are obtained by coupling streptavidin. The target protein binding rate of the microspheres is improved, and the non-specificity is reduced. The magnetic microspheres prepared by the method have good linear correlation and detection sensitivity when applied to chemical luminescence detection.

Description

Technical Field

[0001] This invention relates to the field of nanomaterials technology, specifically to a method for preparing low-nonspecific streptavidin magnetic microspheres and their chemiluminescent application. Background Technology

[0002] Magnetic microspheres were first used in chemiluminescence immunoassay as early as the 1990s. Currently, the surface functional groups modified on magnetic microspheres used in chemiluminescence are mostly tosylate (Tosyl), carboxyl, amino, and streptavidin (SA), with particle sizes ranging from 1 μm to 3 μm. These magnetic microspheres mostly have hydrophilic surfaces, and antibodies are immobilized on their surfaces through covalent coupling to provide a homogeneous system for the immunoreaction.

[0003] Magnetic microspheres, as an important raw material for chemiluminescence detection, are one of the key factors determining the detection effect. Currently, the main methods for preparing magnetic microspheres include emulsion polymerization, dispersion polymerization, suspension polymerization, and seed polymerization. However, current magnetic bead products still have many shortcomings in application, such as: 1. Polydispersity: Inhomogeneity in particle size or ligand density leads to decreased detection accuracy and significant batch-to-batch variability. 2. High nonspecificity, resulting in inaccurate results, false positives, or false negatives. 3. Poor stability: Adhesion or sedimentation, short shelf life, and limited use. 4. Low production volume, preventing large-scale production and also causing batch-to-batch variability.

[0004] CN112007620A discloses a method for preparing streptavidin magnetic microspheres. However, this method involves complex and difficult-to-control polymer modification on the silica layer surface. Furthermore, the microsphere size is 300 nm, while commonly available microspheres are 1 μm, 1.5 μm, and 3 μm, requiring re-adjustment to replace commercially available microspheres. Additionally, this method uses aminohexanoic acid to introduce the carboxyl group; the six carbon chains of aminohexanoic acid have relatively short arms, resulting in steric hindrance that could affect subsequent streptavidin coupling and chemiluminescence applications. Therefore, there is an urgent need for a novel method for preparing functionalized magnetic microspheres to overcome the shortcomings of existing technologies. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a method for preparing low-nonspecificity streptavidin magnetic microspheres and their chemiluminescent application. This method solves the problems of uneven particle size, high nonspecificity, and poor stability of magnetic microspheres, making the magnetization process stable, the magnetic content controllable, and improving the binding capacity of the target protein. Subsequent chemiluminescent detection shows that the magnetic composite microspheres obtained by this method have high detection limits and high detection sensitivity.

[0006] The technical solution of the present invention to solve the above-mentioned technical problems is as follows:

[0007] A method for preparing low-nonspecific streptavidin magnetic microspheres includes the following steps:

[0008] (1) Preparation of amino magnetic microspheres: Using polymer microspheres as a substrate, several layers of positively charged polyelectrolytes and negatively charged iron oxide magnetic particles are deposited on the surface of the microspheres through layer-by-layer self-assembly to prepare amino-based magnetic microspheres.

[0009] (2) First step of surface modification: The amino magnetic microspheres obtained in step (1) are introduced with carboxyl groups by coupling.

[0010] (3) Second step: surface modification, further coupling hydrophilic compounds to the carboxyl group magnetic microspheres obtained in step (2);

[0011] (4) The magnetic microspheres obtained in step (3) are coupled with streptavidin to prepare streptavidin magnetic microspheres.

[0012] Preferably, in step (1), the polymer microspheres are selected from one or more of polystyrene microspheres, polymethyl methacrylate microspheres, silica microspheres, and glycidyl methacrylate microspheres, with a particle size of 500 nm to 3 μm.

[0013] Preferably, in step (1), the positively charged polyelectrolyte used in the layer-by-layer self-assembly method is one or more of the following: polydienedimethylammonium chloride (molecular weight 100,000-200,000), polydienedimethylammonium chloride (molecular weight 200,000-300,000), polyethyleneimine (molecular weight 10,000), polyethyleneimine (molecular weight 70,000), and polyurethane.

[0014] Preferably, in step (1), the selected magnetic particles are one or more of the following: citric acid modified magnetic particles, sodium oleate modified magnetic particles, polyvinylpyrrolidone modified magnetic particles, and dextran modified magnetic particles.

[0015] Preferably, in step (2), the carboxyl polymer is one or more of polyacrylic acid, polymethacrylic acid, polymaleic acid, polybenzenesulfonic acid-maleic acid copolymer, and dicarboxylated polyethylene glycol.

[0016] Preferably, in step (3), the second modification uses one or more of the following: aminocaproic acid, carboxyl-polyethylene glycol-amino, aspartic acid, cysteine, sodium polyacrylate, and polybenzenesulfonic acid-maleic acid copolymer. The carboxyl magnetic microspheres obtained in step (2) are coupled with hydrophilic compounds to reduce steric hindrance and decrease non-specific adsorption.

[0017] Preferably, in step (4), the activator used for coupling is one or more of EDC, EDC / NHS, SO-NHS, and DCC.

[0018] In one embodiment of the present invention, in step (1), the polymer microspheres are selected from one or more of polystyrene microspheres, polymethyl methacrylate microspheres, silica microspheres, and glycidyl methacrylate microspheres, with a particle size of 500 nm to 3 μm.

[0019] In one embodiment of the present invention, in step (1), the selected magnetic particles are one or more of the following: magnetic particles modified with sodium citrate, magnetic particles modified with sodium oleate, magnetic particles modified with polyvinylpyrrolidone, and magnetic particles modified with dextran.

[0020] In one embodiment of the present invention, in step (1), the sodium citrate-modified aqueous magnetic Fe3O4 nanoparticles have a particle size of 8 nm to 50 nm, and the amount of magnetic particles added during monolayer deposition accounts for 2% to 20% of the mass percentage of the base spheres, preferably 10% to 20%. The positive electrolyte polyethyleneimine has a molecular weight of 5000 to 100000, and the amount of polyethyleneimine added during monolayer adsorption accounts for 2% to 20% of the mass percentage of the base spheres.

[0021] In one embodiment of the present invention, in step (1), the magnetic particles are coated with 3 to 9 layers.

[0022] In one embodiment of the present invention, in step (2), the molecular weight of the carboxyl polymer polyacrylic acid is 1,000 to 100,000, and the amount of the feed is 5% to 50% of the mass of the magnetic balls.

[0023] In one embodiment of the present invention, in step (3), an amino-polyethylene glycol-carboxyl group with one end of carboxyl and one end of amino is used as a surface modifier, the molecular weight of the intermediate polyethylene glycol is 400 to 3000, and the EDC concentration in the coupling process is 0.1 mg / mL to 10 mg / mL.

[0024] In one embodiment of the present invention, the streptavidin in step (4) is produced by our company, and the coupling of streptavidin is carried out by a one-step or two-step method. The activator used for coupling is one or more of EDC, EDC / NHS, SO-NHS, and DCC.

[0025] A second objective of this invention is to provide magnetic microspheres obtained by the aforementioned preparation method.

[0026] The third objective of this invention is the application of the magnetic microspheres described herein in the field of chemiluminescence immunoassay.

[0027] The beneficial effects of this invention are as follows: This invention hydrophilically modifies magnetic nanoparticles, employs a layer-by-layer self-assembly method to magnetize the surface of monodisperse polymer-based microspheres, and obtains uniformly controllable magnetic microspheres with rapid magnetic response by adjusting the particle size of the base spheres. Surface carboxyl modification yields microspheres with high carboxyl content, and coupling with long-chain hydrophilic polymers such as polyethylene glycol further disperses the carboxyl functional groups away from the microspheres, reducing steric hindrance and non-specific adsorption. Finally, coupling with streptavidin yields functionalized magnetic composite microspheres. The magnetic microspheres prepared by this invention can be applied in the field of chemiluminescence immunoassay, exhibiting lower background values, higher signal-to-noise ratios, and higher detection sensitivity compared to imported magnetic microspheres. Attached Figure Description

[0028] Figure 1 These are photographs of the actual object and a diagram showing the magnetic separation effect in Example 1;

[0029] Figure 2 This is the TGA thermogravimetric analysis curve of Example 1;

[0030] Figure 3 This is the magnetometer detection curve of the VSM vibration sample in Example 1;

[0031] Figure 4 The curve for detecting carboxyl density by conductivity titration in Comparative Example 1 is shown.

[0032] Figure 5 The results of scanning electron microscopy for Example 1: 1μm magnetic microspheres;

[0033] Figure 6 Example 2: Scanning electron microscopy results of 3μm magnetic microspheres;

[0034] Figure 7 Example 3: Scanning electron microscopy results of 500nm magnetic microspheres. Detailed Implementation

[0035] The present invention will be further described in detail below with reference to embodiments, but is not limited thereto.

[0036] Example 1:

[0037] This embodiment provides a method for preparing polyethylene glycol-modified 1μm polystyrene magnetic microspheres, specifically including the following steps:

[0038] (1) Preparation of aqueous magnetic particles by co-precipitation method: 50 g of ferric chloride hexahydrate (FeCl3·6H2O) solid, 150 mL of ultrapure water, 30 g of ferrous sulfate heptahydrate (FeSO4·7H2O) solid, and 10 g of sodium citrate dihydrate (C6H5Na3O7·2H2O) solid were added sequentially to a 250 mL three-necked flask and ultrasonically stirred until completely dissolved. The mixture was heated in a water bath under nitrogen and condensation conditions to 72 °C. 80 mL of ammonia water was added by pouring while stirring at 500 rpm. The reaction was carried out at 72 °C for 1 hour, then heated to 85 °C and aged for 1.5 hours. One neck of the three-necked flask was opened to evaporate the ammonia water, allowing the iron oxide particles to settle. After cooling, the product was magnetically separated and repeatedly washed with ethanol and ultrapure water to obtain hydrophilic magnetic particles.

[0039] (2) Preparation of magnetic microspheres by layer-by-layer self-assembly:

[0040] 1) Add 300 mL of polyethyleneimine solution (5 mg / mL) and 25 mL of 3M NaCl solution to a 1 L three-necked flask, place it in an ultrasonic cleaner, and mechanically stir at 500 rpm. Add 100 mL of a solution of 10 g of 900 nm polystyrene microspheres (prepared by dispersion polymerization at Changzhou Boyi) dropwise to the PEI solution. Sonicate for 45 minutes. During sonication, use ice packs to control the water temperature to not exceed 30°C. Centrifuge the product at 10000 rpm / 8 minutes and wash it with water 4 times. Obtain positively electrolyte-coated magnetic microspheres.

[0041] 2) Add 200 mL of sodium citrate-modified Fe3O4 aqueous solution (10 mg / mL) and 25 mL of NaCl solution to a 1 L three-necked flask. While ultrasonically stirring, add 200 mL of the microspheres obtained in the previous centrifugation step to the Fe3O4 solution. Stir ultrasonically at 500 rpm for 45 minutes. Centrifuge and wash the product with water at 8000 rpm for 5 minutes until the supernatant is colorless and transparent. This yields microspheres coated with a single layer of Fe3O4.

[0042] Repeat steps 1) and 2) above until 5 layers of Fe3O4 are coated, and then another layer of PEI is coated to obtain magnetic microspheres on the PEI surface.

[0043] (3) Surface carboxyl modification: Polyacrylic acid was selected as the modifying material.

[0044] Dissolve 10g of the amino magnetic microspheres obtained in the previous step in 400mL of ultrapure water, add 10mL of 40% PAA5000 solution, 15mL of ultrapure water, and 30mL of NaCl solution, and sonicate at 500rpm for 60 minutes. Wash with water and perform magnetic separation.

[0045] The microspheres obtained in the previous step were magnetically separated and washed twice with MES (10 mM, MES pH = 5.0), resuspended in 400 mL of MES solution, and sonicated for 5 min. 2 g of EDC dissolved in 20 mL of MES was added to the microsphere solution, and the mixture was stirred at 37 °C for 2.5 h. 65 mL of 2 M ethanolamine (pH = 8.0) was added, and the mixture was blocked for 0.5 h. The microspheres were then washed with water, magnetically separated, and brought to a final volume of 1 L to obtain polyacrylic acid-modified carboxyl magnetic microspheres.

[0046] (4) Surface hydrophilicity modification: amino polyethylene glycol carboxyl groups were selected as the modifying material.

[0047] Polyethylene glycol (PEG) is a widely used hydrophilic substance in the biomedical field. Its biocompatibility and non-immunogenic properties make it suitable for use as a modifier in many areas, such as the modification of antibodies, proteins, nucleotides, liposomes, and the surface modification of polymers and polymer microspheres. In immunoassays, PEG on the surface of nanoparticles can effectively reduce nonspecific adsorption, decrease background levels, improve reaction sensitivity, and reduce false positive and false negative results.

[0048] 3g of polyacrylic acid-modified carboxyl microspheres were washed twice with MES, resuspended in 300mL MES, activated at 37℃ for 0.5h with 300mg NHS / EDC (1:1), magnetically separated and washed three times with MES, resuspended in 250mL MES, and sonicated for 3min. 300mg of amino-polyethylene glycol carboxyl groups were dissolved in 50mL MES solution and added to the microsphere solution. The reaction was carried out at 37℃ for 4h. The product was washed with water, magnetically separated, and diluted to 280mL to obtain low-nonspecificity carboxyl microspheres.

[0049] (5) Two-step streptavidin conjugation

[0050] The microspheres obtained in the previous step were magnetically separated and washed twice with MES, resuspended in 300 mL of MES, activated with 300 mg of NHS / EDC (1:1) at 37 °C for 0.5 h, magnetically separated and washed three times with MES, resuspended in 250 mL of MES, sonicated for 3 min, and then 1% (by weight of the microspheres) of streptavidin was added. The reaction was carried out at 37 °C for 4 h. The product was washed with water, magnetically separated, and brought to a final volume of 280 mL to obtain streptavidin magnetic microspheres.

[0051] Figure 1 The images show photographs of the actual product and the magnetic separation effect of Example 1. It can be seen that the product of Example 1 exhibits good suspension and stability in aqueous solution. Under an applied magnetic field, it can be completely separated, yielding a clear supernatant. This indicates that the microspheres possess good magnetic responsiveness.

[0052] Figure 2The results of thermogravimetric analysis for Example 1 are shown. The thermogravimetric curves indicate that the non-combustible inorganic component is 40%, suggesting a magnetic content mass fraction of 40%. Figure 3 The results from the vibrating sample magnetometer showed that the saturation magnetic intensity of the magnetic microspheres was 40 emu / g.

[0053] Figure 4 The results are from the conductivity titration of the carboxyl microspheres obtained in step 4 without streptavidin coupling. The calculated carboxyl content is 1360 nmol / g, indicating a high carboxyl density.

[0054] Figure 5 The image shown is a scanning electron microscope (SEM) image of Example 1. The microspheres exhibit good dispersibility. There is no aggregation or detachment of magnetic particles. The particle size is 1 μm.

[0055] Example 2:

[0056] This embodiment provides a method for preparing aspartic acid-modified 3μm polystyrene magnetic microspheres, specifically including the following steps:

[0057] (1) Preparation of aqueous magnetic particles by coprecipitation method:

[0058] In a 250 mL three-necked flask, 50 g of ferric chloride hexahydrate (FeCl3·6H2O) solid, 150 mL of ultrapure water, 30 g of ferrous sulfate heptahydrate (FeSO4·7H2O) solid, and 10 g of sodium citrate dihydrate (C6H5Na3O7·2H2O) solid were added sequentially, and the mixture was ultrasonically stirred until completely dissolved. The mixture was heated in a water bath under nitrogen purging and condensation to 72 °C. 80 mL of ammonia water was poured in while stirring at 500 rpm. The reaction was carried out at 72 °C for 1 hour, then the temperature was raised to 85 °C and allowed to mature for 1.5 hours. One neck of the flask was opened to evaporate the ammonia water, allowing the iron(III) oxide particles to settle. After cooling, the product was magnetically separated and repeatedly washed with ethanol and ultrapure water to obtain hydrophilic magnetic particles.

[0059] (2) Preparation of magnetic microspheres by layer-by-layer self-assembly:

[0060] 1) Add 300 mL of polyethyleneimine solution (5 mg / mL) and 25 mL of 3M NaCl solution to a 1 L three-necked flask, place in an ultrasonic cleaner, and mechanically stir at 500 rpm. Add 10 g of 3 μm polystyrene microspheres to 100 mL of the PEI solution dropwise. Sonicate for 45 minutes. During sonication, use ice packs to control the water temperature to not exceed 30°C. Centrifuge the product at 10000 rpm / 8 minutes and wash with water 4 times. Obtain positively electrolyte-coated magnetic microspheres.

[0061] 2) Add 200 mL of sodium citrate-modified Fe3O4 aqueous solution (10 mg / mL) and 25 mL of NaCl solution to a 1 L three-necked flask. While ultrasonically stirring, add 200 mL of the microspheres obtained in the previous centrifugation step to the Fe3O4 solution. Stir ultrasonically at 500 rpm for 45 minutes. Centrifuge and wash the product with water at 8000 rpm for 5 minutes until the supernatant is colorless and transparent. This yields microspheres coated with a single layer of Fe3O4.

[0062] Repeat steps 1) and 2) above until 5 layers of Fe3O4 are coated, and then another layer of PEI is coated to obtain magnetic microspheres on the PEI surface.

[0063] (3) Surface carboxyl modification: Polyacrylic acid was selected as the modifying material.

[0064] Dissolve 10g of the amino magnetic microspheres obtained in the previous step in 400mL of ultrapure water, add 10mL of 40% PAA5000 solution, 15mL of ultrapure water, and 30mL of NaCl solution, and sonicate at 500rpm for 60 minutes. Wash with water and perform magnetic separation.

[0065] The microspheres obtained in the previous step were magnetically separated and washed twice with MES (10 mM, MES pH = 5.0), resuspended in 400 mL of MES solution, and sonicated for 5 min. 2 g of EDC dissolved in 20 mL of MES was added to the microsphere solution, and the mixture was stirred at 37 °C for 2.5 h. 65 mL of 2 M ethanolamine (pH = 8.0) was added, and the mixture was blocked for 0.5 h. The microspheres were then washed with water, magnetically separated, and brought to a final volume of 1 L to obtain polyacrylic acid-modified carboxyl magnetic microspheres.

[0066] (4) Surface hydrophilicity modification: Aspartic acid was selected as the modifying material.

[0067] Aspartic acid, in its levorotatory form, is one of the 20 essential amino acids that make up proteins and has wide applications in biochemical reagents and clinical medicine. Its chemical name is aminosuccinic acid. In the coupling process, it increases the carboxyl group density. Coupled to the surface of microspheres, it reduces non-specific adsorption.

[0068] 3g of polyacrylic acid carboxyl microspheres were washed twice with MES, resuspended in 300mL of MES, activated at 37℃ for 0.5h with 300mg of NHS / EDC (1:1), magnetically separated and washed three times with MES, resuspended in 250mL of MES, and sonicated for 3min. 300mg of aspartic acid was dissolved in 50mL of MES solution and added to the microsphere solution. The reaction was carried out at 37℃ for 4h. The product was washed with water, magnetically separated, and diluted to 280mL to obtain low-nonspecificity carboxyl microspheres.

[0069] (5) Two-step streptavidin conjugation

[0070] The microspheres obtained in the previous step were magnetically separated and washed twice with MES, resuspended in 300 mL of MES, activated with 300 mg of NHS / EDC (1:1) at 37 °C for 0.5 h, magnetically separated and washed three times with MES, resuspended in 250 mL of MES, sonicated for 3 min, and then 1% (by weight of the microspheres) of streptavidin was added and reacted at 37 °C for 4 h. The product was washed with water, magnetically separated, and brought to a final volume of 280 mL to obtain streptavidin magnetic microspheres.

[0071] Figure 6 The image shown is a scanning electron microscope image from Example 2. The microspheres exhibit good dispersibility. There is no aggregation or detachment of magnetic particles. The particle size is 3.2 μm.

[0072] Example 3:

[0073] This embodiment provides a method for preparing polyethylene glycol-modified 500nm silica magnetic microspheres, specifically including the following steps:

[0074] (1) Preparation of aqueous magnetic particles by co-precipitation method: 50 g of ferric chloride hexahydrate (FeCl3·6H2O) solid, 150 mL of ultrapure water, 30 g of ferrous sulfate heptahydrate (FeSO4·7H2O) solid, and 10 g of sodium citrate dihydrate (C6H5Na3O7·2H2O) solid were added sequentially to a 250 mL three-necked flask and ultrasonically stirred until completely dissolved. The mixture was heated in a water bath under nitrogen and condensation conditions to 72 °C. 80 mL of ammonia water was added by pouring while stirring at 500 rpm. The reaction was carried out at 72 °C for 1 hour, then heated to 85 °C and aged for 1.5 hours. One neck of the three-necked flask was opened to evaporate the ammonia water, allowing the iron oxide particles to settle. After cooling, the product was magnetically separated and repeatedly washed with ethanol and ultrapure water to obtain hydrophilic magnetic particles.

[0075] (2) Preparation of magnetic microspheres by layer-by-layer self-assembly:

[0076] 1) Add 300 mL of polyethyleneimine solution (5 mg / mL) and 25 mL of 3M NaCl solution to a 1 L three-necked flask, place in an ultrasonic cleaner, and mechanically stir at 500 rpm. 10 g of 500 nm silica microspheres (Changzhou Boyi) 100 mL of a solution (prepared by [method name]) was added dropwise to a PEI solution. The mixture was sonicated for 45 minutes. During sonication, an ice pack was used to control the water temperature to not exceed 30°C. The product was centrifuged at 10000 rpm for 8 minutes and washed four times with water. Positive electrolyte-coated magnetic microspheres were obtained.

[0077] 2) Add 200 mL of sodium citrate-modified Fe3O4 aqueous solution (10 mg / mL) and 25 mL of NaCl solution to a 1 L three-necked flask. While ultrasonically stirring, add 200 mL of the microspheres obtained in the previous centrifugation step to the Fe3O4 solution. Stir ultrasonically at 500 rpm for 45 minutes. Centrifuge and wash the product with water at 8000 rpm for 5 minutes until the supernatant is colorless and transparent. This yields microspheres coated with a single layer of Fe3O4.

[0078] Repeat steps 1) and 2) above until 5 layers of Fe3O4 are coated, and then another layer of PEI is coated to obtain magnetic microspheres on the PEI surface.

[0079] 3) Surface silica encapsulation:

[0080] Add 20 mL of an aqueous solution of Fe3O4 microspheres obtained in the previous step to 200 mL of ethanol, stir at 300 rpm for 15 minutes under sonication, add 5 mL of ammonia, continue sonication for 15 minutes, add 5 mL of tetraethyl orthosilicate (TOES), react at 30 °C and 300 rpm for 2 hours under magnetic separation, wash 4 times with anhydrous ethanol and 4 times with water. The product is dispersed in anhydrous ethanol.

[0081] (3) Surface carboxyl modification and hydrophilic modification:

[0082] Silane-PEG-COOH was selected as the modifying material.

[0083] The microspheres obtained in the previous step were dispersed in 200 mL of anhydrous ethanol, and 5 mL of ammonia water were added. The mixture was stirred at 300 rpm for 15 minutes under sonication. Then, 2 mL of silane-PEG-COOH was added, and the reaction was carried out at 30°C and 300 rpm for 6 hours. The mixture was then magnetically separated, washed four times with anhydrous ethanol, and four times with water. The product was dispersed in pure water.

[0084] Carboxyl microspheres with low nonspecificity were obtained.

[0085] (4) Two-step streptavidin coupling

[0086] The microspheres obtained in the previous step were magnetically separated and washed twice with MES, resuspended in 300 mL of MES, activated with 300 mg of NHS / EDC (1:1) at 37 °C for 0.5 h, magnetically separated and washed three times with MES, resuspended in 250 mL of MES, sonicated for 3 min, and then 1% (by weight of the microspheres) of streptavidin was added and reacted at 37 °C for 4 h. The product was washed with water, magnetically separated, and brought to a final volume of 280 mL to obtain streptavidin magnetic microspheres.

[0087] Figure 7The image shown is a scanning electron microscope image from Example 3. The microspheres exhibit good dispersibility. There is no aggregation or detachment of magnetic particles. The particle size is 500 nm.

[0088] Test case

[0089] Evaluation Example 1 Application on CA242 Chemiluminescence Platform

[0090] The magnetic microspheres of Example 1 of this invention were applied to the field of chemiluminescent immunoassay. The application performance of the magnetic microspheres of this invention and commercially available imported magnetic microspheres was compared. Under the same coupling conditions, CA242 antibody was bound to both the magnetic microspheres of this invention and the commercially available magnetic microspheres. The instrument used was a Yingkai i2910. Reagent components: M, magnetic beads from Example 1; R1, biotin-labeled antibody CA242-SL1-Bio; R2, AE-labeled antibody: CA242-RM0799-AE calibrator (0, 5, 20, 50, 200, 500 IU / ml, 6-point calibrators (antigen from Kangnaige).

[0091] The detection procedure, using the delayed one-step sandwich method: Sample + R1 + R2 → Incubate for 15 min → + M → Incubate for 5 min → Wash 3 times → + Substrate → Detection. The detection results are shown in the table below:

[0092]

[0093] As can be seen from the detection results in the table above, the nonspecificity of Example 1 was one-fifth that of the control, the luminescence value at high concentrations was twice that of the control, and the overall signal-to-noise ratio was nearly ten times that of the control. This indicates that the streptavidin magnetic microspheres obtained in this invention have good sensitivity and linear range in chemiluminescence applications.

[0094] Example 1: Accelerated Stability Verification

[0095]

[0096] Under normal storage conditions (4°C), the signal value decreased to 9% after 30 days of accelerated storage at 37°C, while non-specific adsorption did not increase. The correlation of the detection was good, and the overall signal-to-noise ratio was also high. This indicates that Example 1 has good time stability and maintains high performance under these accelerated conditions.

[0097] To verify the effect of each modification step, two comparative examples were set up.

[0098] Comparative Example 1:

[0099] The preparation method of the magnetic microspheres in this comparative example is similar to that in Example 1, except that:

[0100] Without performing the modification step (4) to reduce nonspecificity and the coupling with streptavidin (5), polyacrylic acid-modified carboxyl magnetic microspheres were obtained. Chemiluminescence detection was performed according to the method in Example 1, and the results are shown in the table below:

[0101]

[0102] The test results show that, in chemiluminescence applications, the carboxyl magnetic beads of Comparative Example 1 have similar nonspecific adsorption and high adsorption values ​​to imported magnetic beads. Compared with Example 1, they have higher nonspecificity and lower high adsorption values. This indicates that, without subsequent hydrophilic coating and streptavidin coupling, Comparative Example 1 still suffers from the defects of commercially available imported magnetic beads.

[0103] Performance needs further improvement.

[0104] Comparative Example 2:

[0105] The preparation method of the magnetic microspheres in this comparative example is similar to that in Example 1, except that:

[0106] Without the (5) streptavidin coupling, low-nonspecificity carboxyl magnetic microspheres were obtained. Chemiluminescence detection was performed according to the method of Example 1. The chemiluminescence detection results showed that, compared to commercially available magnetic beads, the nonspecificity of Comparative Example 2 was significantly reduced, while the high value was close to the control, indicating that the additional hydrophilic coating step on the surface effectively reduced nonspecificity. Compared to Example 1, surface streptavidin coupling increased the high value, further improving sensitivity.

[0107]

[0108] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the inventive concept of the present invention, and these all fall within the protection scope of the present invention.

Claims

1. A method for preparing low-nonspecific streptavidin magnetic microspheres, characterized in that, Includes the following steps: (1) Preparation of amino magnetic microspheres: Using polymer microspheres as a substrate, several layers of positively charged polyelectrolytes and negatively charged iron oxide magnetic particles are deposited on the surface of the microspheres through layer-by-layer self-assembly to prepare surface amino magnetic microspheres; (2) First step surface modification: The amino magnetic microspheres obtained in step (1) are introduced with carboxyl groups by coupling to obtain carboxyl group magnetic microspheres; In step (2), the carboxyl groups are derived from carboxyl polymers, which are one or more of polyacrylic acid, polymethacrylic acid, polymaleic acid, polybenzenesulfonic acid-maleic acid copolymer, dicarboxyl polyethylene glycol, and silane polyethylene glycol carboxyl groups; (3) Second step surface modification: The magnetic microspheres with carboxyl groups obtained in step (2) are further coupled with hydrophilic compounds to obtain magnetic microspheres; In step (3), the hydrophilic compounds used in the second step surface modification are one or more of aminocaproic acid, amino polyethylene glycol carboxyl, aspartic acid, polyethylene glycol, silane polyethylene glycol carboxyl, cysteine, sodium polyacrylate, and polybenzenesulfonic acid maleic acid copolymer. (4) The magnetic microspheres obtained in step (3) are coupled with streptavidin to prepare streptavidin magnetic microspheres.

2. The preparation method according to claim 1, characterized in that, In step (1), the polymer microspheres are selected from one or more of polystyrene microspheres, polymethyl methacrylate microspheres, silica microspheres, and glycidyl methacrylate microspheres, with a particle size of 500 nm to 3 μm.

3. The preparation method according to claim 1, characterized in that, In step (1), the positively charged polyelectrolyte used in the layer-by-layer self-assembly method is one or more of polydienedimethylammonium chloride, polydienedimethylammonium chloride, polyethyleneimine, and polyurethane.

4. The preparation method according to claim 1, characterized in that, In step (1), the selected magnetite magnetic particles are one or more of the following: citric acid-modified magnetite magnetic particles, sodium oleate-modified magnetite magnetic particles, polyvinylpyrrolidone-modified magnetite magnetic particles, and dextran-modified magnetite magnetic particles.

5. The preparation method according to claim 1, characterized in that, In step (4), a coupling activator is also used, which is one or more of EDC, EDC / NHS, SO-NHS, and DCC.

6. The preparation method according to claim 1, characterized in that, During monolayer deposition, the amount of the magnetite magnetic particles is 2% to 20% of the mass of the substrate, the amount of the polyelectrolyte is 2% to 20% of the mass of the substrate, the amount of the hydrophilic polymer is 5% to 50% of the mass of the carboxyl group magnetic microspheres, and the amount of streptavidin is 1% to 5% of the mass of the magnetic microspheres obtained in step (3).

7. The preparation method according to claim 1, characterized in that, The amount of carboxyl polymer fed into the amino magnetic microspheres is 5% to 50% of the total mass.

8. The application of the magnetic microspheres obtained by the preparation method according to any one of claims 1 to 7 in the field of chemiluminescence immunoassay.

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