Chemiluminescence immunoassay immune magnetic beads, and preparation method and application thereof

Abstract

The application belongs to the technical field of biological detection, and particularly relates to an immunomagnetic bead for chemiluminescence immunoassay and a preparation method and application thereof. The immunomagnetic bead is of a core-shell structure, and comprises a magnetic core, an epoxy shell layer, a hydrophilic spacer wall, a boronic acid group coupling layer and an antibody. The magnetic core is prepared by using a solvothermal method, the epoxy polymer shell layer is prepared by using a backflow precipitation polymerization, the hydrophilic PEG long chain is modified by using click chemistry reaction of the double-end thiol-containing PEG molecule and the epoxy group, and finally the boronic acid group is fixed on the surface of the microsphere by thiol-double bond click chemistry reaction to obtain the magnetic composite microsphere. The antibody is coupled to the surface of the magnetic bead carrier by coordination bonding of the cis-diol group in the structure and the boronic acid group on the surface of the microsphere to obtain the chemiluminescence immunomagnetic bead. The immunomagnetic bead can be applied to detection of tumor markers, has the advantages of simple synthesis route, easy operation, high antibody coupling efficiency, high detection sensitivity and strong specificity, and can realize efficient immunoassay.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological detection, and relates to an immunomagnetic bead for chemiluminescence immunoassay and a preparation method and application thereof. BACKGROUND

[0002] Early screening of cancer can greatly reduce the risk of tumor-related diseases and improve the survival rate of patients. Therefore, developing tumor-related biomarker analysis techniques with high sensitivity and specificity is crucial for early diagnosis of tumor-related diseases. Currently, a variety of analysis methods are used to detect tumor markers in biological fluids, such as ELISA, chemiluminescence immunoassay (CLIA), electrochemiluminescence immunoassay (ECLIA), time-resolved fluorescence immunoassay, radioimmunoassay (RIA), gold-labeled immunofiltration assay and liquid chip technology. CLIA is considered as a fast and highly sensitive technique with the advantages of simple operation, high stability and complete automation. In a clinical environment, establishing different tumor marker detection thresholds can greatly affect the false positive or false negative rate in cancer screening. It is still a challenge to achieve high sensitivity and high selectivity detection of low-level tumor markers in serum.

[0003] CLIA based on magnetic beads is the preferred method for detecting cancer biomarkers in biological fluids. Magnetic beads are used as solid carriers in combination with CLIA, and under the influence of an external magnetic field, the magnetic beads can be quickly separated from the liquid matrix. These magnetic beads have high separation and enrichment capacity, which can significantly reduce the influence of antigens on fixed antibodies during the immunoassay process. As a carrier for the entire immune response and signal collection process, magnetic beads allow antigens or antibodies to be coupled and adsorbed on their chemically modified surfaces. The reactive groups on the surface of the magnetic beads, including carboxyl, amino, hydroxyl and sulfate groups, provide many sites for covalent attachment of antigens or antibodies. Among the commercially available magnetic beads used for CLIA detection, carboxyl and tosyl magnetic beads are the two commonly used commercial magnetic beads. Among them, carboxyl magnetic beads need to be activated before antibody coupling during use, while tosyl magnetic beads do not require activation, but have high coupling requirements, which can greatly affect the detection efficiency of CLIA. Therefore, developing a new type of immunomagnetic bead that makes antibody coupling simple and efficient is crucial for improving the detection efficiency of CLIA.

[0004] Boric acid affinity chromatography technology has attracted extensive research interest. Boric acid molecules have strong affinity for biological macromolecules containing cis-diol structures, and can form stable cyclic esters with cis-diol moieties in weakly basic or neutral aqueous solutions. Antibodies are a kind of glycoprotein containing cis-diol groups in the domain. These characteristics indicate that borate functionalized materials are expected to become immunomagnetic beads, effectively coupling antibodies from the system. SUMMARY

[0005] The primary object of the present application is to provide a novel immunomagnetic bead for immunoassay, which has excellent chemiluminescence performance.

[0006] Another object of the present application is to provide a preparation method of the above-mentioned immunomagnetic bead, which has simple route, easy operation, mild antibody coupling condition, short coupling time, and does not need any activation step.

[0007] The immunomagnetic bead for immunoassay provided by the present application has a core-shell structure, which comprises a magnetic inner core, an epoxy shell layer, a hydrophilic spacer arm, a boronic acid group coupling layer, and an antibody.

[0008] The magnetic inner core is a magnetic nanocluster with a surface modified with a stabilizing agent, and the magnetic nanocluster is one of γ-Fe2O3, m-γ-Fe2O3, Fe3O4 or a mixture of several thereof.

[0009] The stabilizing agent is selected from polyglutamic acid, sodium citrate, oleic acid, or 4-styrene sulfonic acid-co-maleic acid sodium salt; the precursor for synthesizing the magnetic nanocluster is ferric chloride, ferric chloride hexahydrate, ferric chloride hexahydrate, ferric sulfate hydrate, or ferric sulfate heptahydrate; and the nucleation auxiliary agent used for synthesis includes sodium acetate, sodium hydroxide, potassium hydroxide, and ammonium hydroxide.

[0010] The epoxy shell layer is a cross-linked functional layer with a large number of reactive epoxy functional groups.

[0011] The hydrophilic spacer arm is a single-arm or multi-arm long-chain PEG molecule containing a double-end thiol group in the structure.

[0012] The boronic acid group adsorption layer is a functional layer containing a large number of boronic acid coordination bonds, and the boronic acid group is selected from boronic acid molecules containing a hydrocarbon group structure.

[0013] The antibody is coupled to the surface of the magnetic bead carrier by coordination bonding of a cis-diol group in the structure with the boronic acid group on the surface of the microsphere; and the antibody is specifically a human monoclonal complete antibody.

[0014] In the present application, the total particle size of the immunomagnetic bead with a core-shell structure is 200-500 nm; the particle size of the magnetic inner core is 170-400 nm; and the thickness of the epoxy polymer shell layer is 30-50 nm.

[0015] The preparation method of the above-mentioned immunomagnetic bead provided by the present application has the following specific steps:

[0016] Step 1: preparing a surface stabilizer modified magnetic nanocluster in a reaction bottle by using a modified solvothermal method to obtain a magnetic inner core in the form of a microsphere (referred to as MSP);

[0017] Step 2, a layer of cross-linked epoxy polymer is coated on the obtained microspheres as a shell layer (referred to as MSP@PGMA) by using reflux precipitation polymerization;

[0018] Step 3, a thiol hydrophilic spacer with a long PEG chain is modified on the surface by using a double-end thiol PEG molecule to interact with the epoxy group (referred to as MSP@PGMA-SH);

[0019] Step 4, a boronic acid group is fixed on the surface of the microspheres by using a click chemistry reaction between the thiol group and the double bond (referred to as MSP@PGMA-PBA);

[0020] Step 5, coupling of the magnetic beads and the antibody; the boronic acid group modified magnetic composite microspheres are coupled with the antibody in a coupling solution at a certain temperature for a certain time, and after magnetic separation, the deionized water is repeatedly washed twice to obtain the antibody fixed immunomagnetic beads (referred to as MSP@PGMA-PBA-Ab).

[0021] Preferably, in step 1, the surface stabilizer is selected from polyglutamic acid, sodium citrate, oleic acid or 4-styrene sulfonic acid-copoly-maleic acid sodium salt; the reaction precursor is ferric chloride, ferric chloride hexahydrate, ferric chloride hexahydrate, ferric sulfate hydrate, ferric sulfate heptahydrate, and the nucleation auxiliary agent includes sodium acetate, sodium hydroxide, potassium hydroxide, ammonium hydroxide.

[0022] Preferably, in step 2, the cross-linking agent is one or more of N,N'-methylenebisacrylamide (MBA), divinylbenzene (DVB) or ethylene glycol dimethyl acrylate (EGDMA); and the preferred epoxy functional monomer is one or more of 2,3-epoxypropyl acrylate, (3,4-epoxycyclohexyl) methyl acrylate, 1,2-epoxy-5-hexene, 1,2-epoxy-7-octene, 3,4-epoxy-1-butene or glycidyl methacrylate (GMA).

[0023] Preferably, in step 3, the double-end thiol PEG molecule is one or more of SH-PEG-SH, 4Arm-PEG-SH or 8Arm-PEG-SH with different molecular weights.

[0024] Preferably, in step 4, the boronic acid group coupling layer is a boronic acid group functional layer containing a large number of coordination bonds, and the boronic acid molecules are selected from molecules containing a hydrocarbon group and a boronic acid structure.

[0025] Preferably, in step 5, the coupling solution is a Tris alkaline buffer solution with a pH of 7-9, the coupling temperature is 25-55°C, the coupling time is 2-24h; the concentration of the antibody solution is 4mg / mL, the volume ratio of the antibody to the coupling solution is 2-18:500, and the concentration of MSP@PGMA-PBA in the coupling solution is 0.5-1mg / mL.

[0026] Further, the specific process of step 1 is as follows: a certain amount of ethylene glycol is measured into a flask, and reaction precursors, surface stabilizers are added in turn, and they are fully dispersed into a uniform solution by ultrasonic, mechanical stirring and heating, then nucleation auxiliary agent is added, and continue to heat and stir at 50-60℃ to make it completely dissolved; the above solution is heated to 170-210℃ and kept constant temperature for 12-16h, after the reaction is completed, the product is separated by magnet and washed with ethanol and water for several times, finally the product is dispersed in deionized water, thus the magnetic inner core microparticles modified by stabilizer, MSPs, are prepared.

[0027] Further, the mass ratio of reaction precursors, surface stabilizers, nucleation auxiliary agent in step 1 is 1-2:8-9:9-12, and the amount of ethylene glycol is 250-300mL.

[0028] Further, the specific process of step 2 is as follows: a certain amount of crosslinking monomer, epoxy monomer and initiator are dissolved in acetonitrile, and MSPs are dispersed in a flask containing the above mixture; the flask is connected to a condenser tube, and reacted in an oil bath at 85-110℃ for 30-90min; after the reaction is completed, the product is collected by magnetic separation and washed with water and ethanol for several times to obtain MSP@PGMA magnetic composite microspheres.

[0029] Further, the molar ratio of crosslinking monomer and epoxy monomer in step 2 is 1:3, the amount of initiator is 2-3wt% of the total amount of monomers, and the amount ratio of acetonitrile and MSP is 40-50mL:50mg.

[0030] Further, the specific process of step 3 is as follows: MSP@PGMA magnetic composite microspheres are uniformly dispersed in DMF, and double-end mercapto PEG molecules are added, and ultrasonic stirring is mixed for 10min; DBU is placed on a microsyringe pump, and DBU is added dropwise while stirring at room temperature, and after the dropwise addition is completed, the reaction is continued for 6-24h; the product is collected by magnetic separation and washed with ethanol for several times to obtain MSP@PGMA-SH magnetic composite microspheres.

[0031] Further, the amount ratio of DMF, MSP@PGMA, double-end mercapto PEG molecules and DBU in step 3 is 20-50mL:150mg:100-200mg:20μL, and the dropwise speed of DBU is 100-200mm / h.

[0032] Further, the specific process of step 4 is as follows: the MSP@PGMA-SH magnetic composite microspheres are uniformly dispersed in DMF, the initiator and boric acid molecules are added thereto, and the mixture is stirred under ultrasonic for 10 min. Under the nitrogen atmosphere, the reaction is stirred at 60 DEG C for 12-48 h. The product is collected by magnetic separation and washed with ethanol for several times to obtain the MSP@PGMA-PBA magnetic composite microspheres.

[0033] Further, the amount ratio of DMF, MSP@PGMA-SH, boric acid molecules and initiator in step 4 is 20-50 mL:150 mg:100-200 mg:6-10 mg.

[0034] Further, the specific process of antibody coupling in step 5 is as follows: the immunomagnetic beads are placed in a 1.5 mL centrifuge tube, the coupling buffer is added, then the antibody solution is added thereto, and the mixture is uniformly vortexed for 3 min, and then the mixture is placed on a constant temperature mixing instrument at 25-55 DEG C for coupling reaction for 0.5-8 h. The product is collected by magnetic separation, and after the reaction is completed, the magnetic beads are washed with pure water for 2 times to obtain the MSP@PGMA-PBA-Ab immunomagnetic beads.

[0035] Further, the concentration of the antibody solution in step 5 is 4 mg / mL, the volume ratio of the antibody solution to the coupling solution is 2-18:500-1000, the concentration of MSP@PGMA-PBA in the coupling solution is 0.5-1 mg / mL, the coupling reaction temperature is 25-55 DEG C, and the coupling time is 2-24 h.

[0036] The immunocomposite microspheres prepared in the application have a shell-core structure, and the total particle size of the magnetic beads is 200-500 nm. In step 1, the particle size of the magnetic inner core is 170-400 nm; and in step 3, the thickness of the crosslinked polymer shell layer is 30-50 nm.

[0037] The magnetic beads prepared in the application can be used for detecting tumor markers in serum samples.

[0038] Compared with the prior art, the beneficial effects of the application are embodied in the following aspects:

[0039] (1) Based on the coordination bonding effect of boric acid group and cis-diol, the antibody can be rapidly and efficiently coupled by adjusting the pH value of the coupling solution under mild and simple conditions. Compared with other commercial magnetic beads, the application has significant advantages. For example, compared with the traditional carboxyl magnetic beads, the activation step is simplified, the operation is easy, and the process is more simple. Compared with the traditional tosyl magnetic beads, the antibody coupling time is significantly reduced, the detection time is greatly shortened, and the CLIA detection efficiency is improved.

[0040] (2) The magnetic beads introduce hydrophilic spacer PEG chains, which can effectively avoid non-specific adsorption due to hydrophobic interaction;

[0041] (3) The introduction of the long-chain spacer wall of the magnetic bead avoids the direct interaction between the borate group surface and the antibody to some extent, and is conducive to improving the coupling efficiency of the antibody;

[0042] (4) The epoxy polymer functional layer is modified on the surface of the magnetic core by backflow precipitation, so that the non-specific adsorption of the magnetic core itself is reduced, and the magnetic core is also protected, improving the anti-non-specific adsorption and acid and alkali resistance of the magnetic core;

[0043] (5) The high magnetic response makes the whole detection process simple and efficient, greatly shortens the detection time, and is conducive to improving the detection efficiency. BRIEF DESCRIPTION OF DRAWINGS

[0044] Figure 1 The preparation flowchart of the MSP@PGMA-PBA-Ab immunomagnetic beads of the present application.

[0045] Figure 2 The morphology and EDS element distribution spectrum of the MSP@PGMA-PBA composite microspheres prepared in Example 1 of the present application.

[0046] Figure 3 The linear fitting curve of the detection results of Examples 7-12 of the present application.

[0047] Figure 4 The comparison of the luminescence intensity of the detection results of Examples 13-18 of the present application and the commercial immunomagnetic beads on the detection of different concentrations of AFP in serum. DETAILED DESCRIPTION

[0048] In order to more clearly illustrate the present application, the present application will be further described below in conjunction with preferred embodiments. It should be clear that the described embodiments are only a part of the embodiments of the present application, not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application. The present application will be further introduced through specific embodiments.

[0049] Example 1: Preparation of chemiluminescence immunomagnetic beads for alpha-fetal protein (AFP) detection.

[0050] Preparation of magnetic core microspheres. 27.06 g of sodium acetate, 5.85 g of ferric chloride, 9 g of sodium citrate and 280 mL of ethylene glycol were measured into a flask, and were dispersed into a uniform solution by ultrasonic and heating. Then 5.4 g of sodium hydroxide was added, and the solution was heated at 70°C for 1 h to completely dissolve. The solution was quickly heated to 200°C and kept at this temperature for 9 h. After the reaction was completed, the product was separated by a magnet and washed with ethanol and water. Finally, the obtained product was dispersed in a certain amount of deionized water to obtain magnetic core microspheres modified by a surface stabilizer, denoted as MSPs. The results of particle size analysis showed that the average particle size of the microspheres was 300 nm.

[0051] Coating of a functional shell containing an epoxy group on the surface of the magnetic beads. 250 mg of MSP and 140 g of acetonitrile were added to a reaction flask, and ultrasonic dispersion was performed for 5 min to make the mixture uniform. Then 120 mg of MBA, 375 mg of GMA and 60 mg of initiator (AIBN) were sequentially added to the flask, and ultrasonic dispersion was continued for 10 min to dissolve the monomers and the initiator. The flask was placed in a 95°C oil bath for condensation reflux reaction for 30 min. After the reaction was completed, the product was collected by magnetic separation and washed with water and ethanol for several times to remove the excess reactants and a small amount of polymer microspheres formed by homogeneous nucleation, and finally MSP@PGMA magnetic composite microspheres were obtained. The thickness of the shell coating layer was about 30 nm.

[0052] Modification of PEG spacer shell on the surface of the magnetic beads. 50 mg of MSP@PGMA magnetic beads were dispersed in 50 mL of DMF, and 300 mg of single-arm double-end thiol-PEG-SH with a molecular weight of 2000 was added thereto. The mixture was stirred at 25°C, 20 μL of DBU was measured and placed on a microsyringe pump, and the dropwise addition speed was set to 100 mm / h. After the dropwise addition was completed, the stirring was continued for 24 h. The product was collected by magnetic separation and washed with ethanol and water for several times to remove the excess SH-PEG-SH and DBU, and finally MSP@PGMA-SH magnetic composite microspheres were obtained.

[0053] Modification of boronic acid groups on the surface of the magnetic beads. 25 mg of MSP@PGMA-SH magnetic beads were dispersed in 20 mL of DMF, and 150 mg of 4-vinylphenylboronic acid (PBA) and 3 mg of AIBN were added thereto. The mixture was placed in a 60°C oil bath and reacted under nitrogen protection for 48 h. After the reaction was completed, the product was collected by magnetic separation and washed with ethanol and water for several times, and finally magnetic beads MSP@PGMA-PBA were obtained. The morphology and element distribution of the magnetic beads are shown in Figure 2 (a) and (b), respectively.

[0054] Magnetic sphere coupling AFP antibody. 3.6 mg of prepared magnetic beads were dispersed in a 1.5 mL centrifuge tube, 1 mL of coupling buffer with pH = 8 was added, 18 μL of AFP antibody was added, vortexed for 3 min, placed in a constant temperature shaker at 37°C, and the coupling reaction was carried out for 2 h. The product was collected by magnetic separation and washed with deionized water twice to obtain MSP@PGMA-PBA-Ab.

[0055] Example 2: This example is basically the same as example 1, except that in the preparation process of modifying the thiol group on the surface of the magnetic sphere, the SH-PEG-SH molecule with a molecular weight of 2000 is replaced by an SH-PEG-SH molecule with a molecular weight of 200, and the concentration of the thiol group in the mixed solution remains unchanged.

[0056] Example 3: This example is basically the same as example 1, except that in the preparation process of modifying the thiol group on the surface of the magnetic sphere, the SH-PEG-SH molecule with a molecular weight of 2000 is replaced by an SH-PEG-SH molecule with a molecular weight of 8000, and the concentration of the thiol group in the mixed solution remains unchanged.

[0057] Example 4: This example is basically the same as example 1, except that in the preparation process of modifying the thiol group on the surface of the magnetic sphere, the SH-PEG-SH molecule with a molecular weight of 2000 is replaced by a four-arm SH-PEG-SH molecule with a molecular weight of 2000, and the concentration of the thiol group in the mixed solution remains unchanged.

[0058] Example 5: This example is basically the same as example 1, except that in the preparation process of modifying the thiol group on the surface of the magnetic sphere, the SH-PEG-SH molecule with a molecular weight of 2000 is replaced by an eight-arm SH-PEG-SH molecule with a molecular weight of 2000, and the concentration of the thiol group in the mixed solution remains unchanged.

[0059] Example 6: This example is basically the same as example 1, except that in the preparation process of modifying the thiol group on the surface of the magnetic sphere, the 4-vinylphenylboronic acid is replaced by 2-vinylphenylboronic acid, and the concentration of the boronic acid group in the mixed solution remains unchanged.

[0060] Example 7: The immunomagnetic beads prepared in example 1 were used for AFP sample detection.

[0061] The MSP@PGMA-PBA-Ab magnetic beads were placed in a 1.5 mL centrifuge tube, 1 mL of blocking buffer (1% BSA-PBS buffer) was added, vortexed for 3 min, then placed in a constant temperature shaker at 37°C, and the blocking reaction was carried out for 3 h. The product was collected by magnetic separation and washed with blocking buffer twice to obtain MSP@PGMA-PBA-Ab-BSA magnetic composite microspheres.

[0062] The MSP@PGMA-PBA-Ab-BSA magnetic composite microspheres were dispersed in 3 mL of blocking buffer. A mixed solution was prepared with a concentration of 1.2 mg / mL, 200 μL of the mixed solution was taken, 1.8 mL of blocking buffer was added to dilute the sample, and the diluted sample was added to sample slot 1 of the detection instrument. 3 mL of AFP-labeled monoclonal antibody (denoted as Ab-ALP) was added to sample slot 2. 1 mL of 10 ng / mL AFP standard sample was added to the sample slot of the instrument, and 20 mL of luminescent substrate solution (AMPPD solution) was added to the sample slot. The sample volume of AFP sample, enzyme-labeled antibody, and luminescent substrate was set to 100 μL, 50 μL, and 200 μL, respectively. In the instrument parameter interface, the detection time of the AFP sample was set to 30 min, the reaction time of the magnetic beads coupled with the AFP sample and the enzyme-labeled antibody was set to 30 min, and the incubation time with the reaction substrate was set to 30 min. Finally, the instrument was started for detection.

[0063] Example 8: This example is basically the same as Example 7, except that the concentration of the AFP-containing sample to be detected added to the sample slot was changed from 10 ng / mL AFP standard sample to 5 ng / mL AFP standard sample.

[0064] Example 9: This example is basically the same as Example 7, except that the concentration of the AFP-containing sample to be detected added to the sample slot was changed from 10 ng / mL AFP standard sample to 2.5 ng / mL AFP standard sample.

[0065] Example 10: This example is basically the same as Example 7, except that the concentration of the AFP-containing sample to be detected added to the sample slot was changed from 10 ng / mL AFP standard sample to 1.25 ng / mL AFP standard sample.

[0066] Example 11: This example is basically the same as Example 7, except that the concentration of the AFP-containing sample to be detected added to the sample slot was changed from 10 ng / mL AFP standard sample to 0.625 ng / mL AFP standard sample.

[0067] Example 12: This example is basically the same as Example 7, except that the concentration of the AFP-containing sample to be detected added to the sample slot was changed from 10 ng / mL AFP standard sample to a sample without AFP.

[0068] The results of Examples 7-12 were further analyzed, and the concentration and chemiluminescence intensity results were curve-fitted. The analysis results are shown in Table 1. Figure 3

[0069] ​From Figure 3 It can be seen that, at lower AFP detection concentrations, the AFP to be detected concentration (C) and the luminescence intensity (I) increase linearly, and the fitting curve satisfies I = 22728C + 678. The results of 20 repeated detections of Example 12 are shown in Table 1 below, the standard deviation (denoted as S) of the 20 test results is calculated, and the lowest detection limit is calculated according to the concentration of three times the signal-to-noise ratio. It is concluded that the lowest detection limit of the MSP@PGMA-PBA-Ab magnetic composite microspheres for AFP is 1.2 pg / mL.

[0070] Table 1 Chemiluminescence intensity results of 20 repeated detections of Example 12

[0071] Number of detections Relative luminescence intensity 1 686 2 674 3 672 4 679 5 667 6 698 7 699 8 672 9 676 10 680 11 682 12 684 13 686 14 688 15 690 16 692 17 694 18 696 19 698 20 672

[0072] Example 13: This example is basically the same as Example 7, except that the type of AFP-containing sample to be detected added to the sample groove is changed from a pure AFP sample with a concentration of 10 ng / mL to a serum sample containing 500 ng / mL of AFP.

[0073] Example 14: This example is basically the same as Example 7, except that the type of AFP-containing sample to be detected added to the sample groove is changed from a pure AFP sample with a concentration of 10 ng / mL to a serum sample containing 250 ng / mL of AFP.

[0074] Example 15: This example is basically the same as Example 7, except that the type of AFP-containing sample to be detected added to the sample groove is changed from a pure AFP sample with a concentration of 10 ng / mL to a serum sample containing 50 ng / mL of AFP.

[0075] Example 16: This example is basically the same as Example 7, except that the type of AFP-containing sample to be detected added to the sample groove is changed from a pure AFP sample with a concentration of 10 ng / mL to a serum sample containing 25 ng / mL of AFP.

[0076] Example 17: This example is basically the same as Example 7, except that the type of AFP-containing sample to be detected added to the sample groove is changed from a pure AFP sample with a concentration of 10 ng / mL to a serum sample containing 5 ng / mL of AFP.

[0077] Example 18: This example is basically the same as Example 7, except that the type of AFP-containing sample to be detected added to the sample groove is changed from a pure AFP sample with a concentration of 10 ng / mL to a serum sample containing no AFP.

[0078] The results of Examples 13-18 were further analyzed and compared with the detection effects of other commercial immunomagnetic beads (IMB). The comparison data results are shown in Table 2. Figure 4 As shown in Table 2.

[0079] As shown in Table 2. Figure 4 As shown in Table 2, with the increase of the AFP concentration in the sample to be detected, the chemiluminescence intensity gradually increased. This indicates the feasibility of detection in serum. Compared with other commercial IMB, it has stronger luminescence intensity at high AFP detection concentration, showing more excellent detection performance.

Claims

1. An immunomagnetic bead for chemiluminescent immunoassay, characterized in that, It has a core-shell structure, including a magnetic core, an epoxy shell, hydrophilic spacers, a borate coupling layer, and an antibody; wherein: The magnetic core is a magnetic nanocluster with a surface stabilizer modified on its surface. The magnetic nanocluster is one or a mixture of several of γ-Fe2O3, m-γ-Fe2O3, and Fe3O4. The surface stabilizer is selected from polyglutamic acid, sodium citrate, oleic acid, or 4-styrenesulfonic acid-copoly-sodium maleate; the precursor for the synthesis of magnetic nanoclusters is ferric chloride, ferric chloride hexahydrate, ferric chloride hexahydrate, ferric sulfate hydrate, or ferric sulfate heptahydrate; the nucleating aid used in the synthesis is selected from sodium acetate, sodium hydroxide, potassium hydroxide, or ammonium hydroxide. The epoxy shell is a cross-linked functional layer with a large number of reactive epoxy functional groups. The hydrophilic spacer arm is a single-armed or multi-armed long-chain PEG molecule containing double-terminal thiol groups in its structure. The boric acid coupling layer is a functional layer containing a large number of boric acid coordination bonds, and the boric acid groups are selected from boric acid molecules containing hydrocarbon structures. The antibody is coupled to the surface of the magnetic bead carrier by coordination bonding between the cis-diol group in its structure and the borate group on the surface of the microspheres; the antibody is specifically a human monoclonal complete antibody.

2. The immunomagnetic beads for chemiluminescent immunoassay according to claim 1, characterized in that, The core-shell structured immunomagnetic beads have a total particle size of 200-500 nm; a magnetic core particle size of 170-400 nm; and an epoxy polymer shell thickness of 30-50 nm.

3. The method for preparing immunomagnetic beads as described in claim 1 or 2, characterized in that, The specific steps are as follows: Step 1: Surface stabilizer-modified magnetic nanoclusters are prepared in a reaction flask using a modified solvothermal method to obtain magnetic cores in the form of microspheres, denoted as MSPs. The surface stabilizers are selected from polyglutamic acid, sodium citrate, oleic acid, or 4-styrenesulfonic acid-copoly-sodium maleate. The reaction precursors are ferric chloride, ferric chloride hexahydrate, ferric chloride hexahydrate, ferric sulfate hydrate, or ferric sulfate heptahydrate. The nucleation aids used in the reaction are selected from sodium acetate, sodium hydroxide, potassium hydroxide, and ammonium hydroxide. Step 2: Using reflux precipitation polymerization, a cross-linked epoxy polymer is coated onto the obtained microspheres as a shell, and the product is designated as MSP@PGMA; the cross-linking agent used is one or more of N,N'-methylenebisacrylamide (MBA), divinylbenzene (DVB) or ethylene glycol dimethacrylate (EGDMA); Step 3: Utilize the interaction between PEG molecules containing double-terminated thiol groups and epoxy groups to modify thiol hydrophilic spacers with long PEG chains. The product is denoted as MSP@PGMA-SH. The double-terminated thiol PEG molecules are one or more of SH-PEG-SH, 4Arm-PEG-SH, or 8Arm-PEG-SH with different molecular weights. Step 4: Using the click chemical reaction between the thiol group and the double bond, the boric acid group is fixed on the surface of the microsphere, and the product is denoted as MSP@PGMA-PBA; the boric acid molecule is selected from molecules containing hydrocarbon groups and boric acid structures. Step 5: Coupling of magnetic beads and antibodies; specifically, boric acid-modified magnetic composite microspheres and antibodies are coupled and incubated in a coupling solution at a certain temperature for a period of time. After magnetic separation, they are washed twice with deionized water to obtain antibody-fixed immunomagnetic beads, denoted as MSP@PGMA-PBA-Ab. The coupling solution is a Tris alkaline buffer solution with a pH of 7-9, the coupling temperature is 25-55℃, and the coupling time is 2-24 h. The concentration of the antibody solution is 4 mg / mL, the volume ratio of antibody to coupling solution is 2-18:500, and the concentration of MSP@PGMA-PBA in the coupling solution is 0.5-1 mg / mL.

4. The method for preparing immunomagnetic beads according to claim 3, characterized in that step 1 is as follows: ethylene glycol is placed in a flask, and a reaction precursor and a surface stabilizer are added sequentially. The mixture is then fully dispersed into a homogeneous solution by ultrasonication, mechanical stirring, and heating. A nucleation auxiliary agent is then added, and the mixture is heated and stirred at 50-60 °C until completely dissolved. The solution is heated to 170-210 °C and reacted at this temperature for 12-16 h. After the reaction, the product is separated by magnetic adsorption and washed multiple times with ethanol and water. Finally, the product is dispersed in deionized water, thereby obtaining surface stabilizer-modified magnetic core microparticles, denoted as MSPs. The mass ratio of the reaction precursor, surface stabilizer, and nucleating agent is (1~2): (8~9): (9~12), and the amount of ethylene glycol used is 250~300 mL.

5. The method for preparing immunomagnetic beads according to claim 3, characterized in that step 2 is as follows: the crosslinking monomer, epoxy monomer and initiator are dissolved in acetonitrile, and the MSPs are dispersed in a flask containing the above mixture; the flask is connected to a condenser and reacted in an oil bath at 85~110 ℃ for 30~90 min; after the reaction is completed, the product is collected by magnetic separation and washed multiple times with water and ethanol to obtain MSP@PGMA magnetic composite microspheres; The molar ratio of crosslinking monomer to epoxy monomer is 1:3, the amount of initiator is 2-3 wt% of the total monomers, and the ratio of acetonitrile to MSP is 40-50 mL:50 mg.

6. The method for preparing immunomagnetic beads according to claim 3, characterized in that step 3 is as follows: MSP@PGMA magnetic composite microspheres are uniformly dispersed in DMF, and double-terminated thiol-containing PEG molecules are added thereto and ultrasonically stirred for 10 min; DBU is placed on a microinjection pump and added dropwise while stirring at room temperature, and the reaction continues for 6-24 h after the addition is complete; the product is magnetically separated and collected, and washed multiple times with ethanol to obtain MSP@PGMA-SH magnetic composite microspheres; in, The ratio of DMF, MSP@PGMA, PEG molecules with 20~50 mL : 150 mg : (100~200 mg) : 20 μL is used, and the dropping rate of DBU is 100~200 mm / h.

7. The method for preparing immunomagnetic beads according to claim 3, characterized in that step 4 is as follows: MSP@PGMA-SH magnetic composite microspheres are uniformly dispersed in DMF, an initiator and boric acid molecules are added thereto, and the mixture is ultrasonically stirred for 10 min; under a nitrogen atmosphere, the mixture is stirred and reacted at 60 °C for 12~48 h; the product is magnetically separated and collected, and washed multiple times with ethanol to obtain MSP@PGMA-PBA magnetic composite microspheres; in, The ratio of DMF, MSP@PGMA-SH, boric acid molecules and initiator is (20~50 mL): 150 mg: (100~200 mg): (6~10 mg).

8. The method for preparing immunomagnetic beads according to claim 3, characterized in that the specific process of antibody conjugation in step 5 is as follows: place the immunomagnetic beads in a 1.5 mL centrifuge tube, add conjugation buffer, then add antibody solution, vortex mix for 3 min, place it on a constant temperature mixer at 25~55 ℃, and allow the conjugation reaction to proceed for 0.5~8 h. Collect the product by magnetic separation, and after the reaction is complete, magnetically separate and wash the magnetic beads twice with pure water to obtain MSP@PGMA-PBA-Ab immunomagnetic beads.

9. The method for preparing immunomagnetic beads according to claim 8, characterized in that, in step 5, the concentration of the antibody solution is 4 mg / mL, the volume ratio of the antibody solution to the coupling solution is (2~18): (500~1000), the concentration of MSP@PGMA-PBA in the coupling solution is 0.5~1 mg / mL, the coupling reaction temperature is 25~55 ℃, and the coupling time is 2~24 h.

10. The application of immunomagnetic beads as described in claim 1 or 2 in the detection of tumor markers.

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