A method for preparing magnetic beads with precisely controlled surface carboxyl content and application thereof

Abstract

The application provides a magnetic bead preparation method for precisely controlling surface carboxyl content and application thereof, and relates to the technical field of medical materials.The amino magnetic beads are reacted with a first compound and a second compound in the same reaction system, the molar ratio of the first compound and the second compound is adjusted to control the carboxyl content on the surface of the magnetic beads, and the magnetic beads with precisely controlled surface carboxyl content are obtained.The molar ratio of the first compound and the second compound is adjusted to accurately control the carboxyl content on the surface of the final magnetic beads;the reaction is completed in one step under the same system and conditions, and no intermediate separation and purification steps are needed;since the only control parameter is the molar ratio of the two compounds, batch differences caused by temperature and pH fluctuations are avoided, and the product consistency is good;the obtained carboxyl magnetic beads have low background adsorption, the coupling amount of biological ligands is controllable, and the activity is maintained, and the technical problems of difficult precise control of carboxyl content and poor activity after coupling in the prior art using one-step method or grafting method are solved.

Description

Technical Field

[0001] This invention relates to the field of medical materials technology, and in particular to a method for preparing magnetic beads with precise control of surface carboxyl content and its application. Background Technology

[0002] Magnetic microspheres (magnetic beads) have been widely used in bioseparation, immunoassay, and nucleic acid extraction due to their superparamagnetism, high specific surface area, and good biocompatibility. To immobilize biomolecules such as antibodies, antigens, and nucleic acids onto the surface of magnetic beads, functionalization modification is usually required, introducing active groups such as carboxyl groups (-COOH), amino groups (-NH2), and hydroxyl groups (-OH). Among these, surface-carboxylated magnetic beads are the most common, and they can covalently bind amino-containing biological ligands via EDC / NHS chemical coupling. However, existing methods for preparing carboxyl-containing magnetic beads have the following problems: Carboxyl content is difficult to control precisely: Traditional methods typically employ one-step methods (such as direct copolymerization of carboxyl-containing monomers) or grafting methods (such as encapsulation with polyacrylic acid). However, these methods often result in excessively high or low carboxyl density on the surface of magnetic beads, with large batch-to-batch variations, making continuous and precise control impossible.

[0003] Poor activity after coupling: Excessive carboxyl content leads to strong hydrophobicity on the surface of magnetic beads, resulting in increased non-specific adsorption; insufficient carboxyl content results in insufficient coupling of biological ligands, affecting detection sensitivity.

[0004] Existing control methods are complex: some methods control the reaction by changing the reaction time, temperature or the ratio of macromolecular monomers, but the process is cumbersome and has poor repeatability.

[0005] In view of this, the present invention is hereby proposed. Summary of the Invention

[0006] One of the objectives of this invention is to provide a method for preparing magnetic beads with precise control of surface carboxyl content, in order to solve the technical problems of difficulty in precisely controlling carboxyl content and poor activity after coupling in the one-step method or grafting method in the prior art.

[0007] The second objective of this invention is to provide a carboxyl magnetic bead.

[0008] A third objective of this invention is to provide the application of carboxyl magnetic beads prepared by the above-described magnetic bead preparation method in the preparation of products for immunodiagnostics.

[0009] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted: In a first aspect, the present invention provides a method for preparing magnetic beads with precise control of surface carboxyl content, comprising placing amino magnetic beads with a first compound and a second compound in the same reaction system for reaction, adjusting the molar ratio of the first compound and the second compound to control the carboxyl content on the surface of the magnetic beads, thereby obtaining magnetic beads with precise control of surface carboxyl content; The first compound is a reagent that can covalently bind to an amino group and introduce a free carboxyl group; the second compound is a reagent that can covalently bind to an amino group but does not introduce a free carboxyl group.

[0010] Furthermore, the carboxyl content is positively correlated with the molar ratio of the first compound and the second compound.

[0011] Furthermore, the first compound is a dicarboxylic anhydride compound.

[0012] Furthermore, the second compound is a monoacid anhydride compound; Preferably, the general formula of the monoacid anhydride compound is (CH2). x -CO-O-CO-(CH2) y , where x and y are independent integers from 1 to 10.

[0013] Furthermore, the second compound is a carboxylic acid ester compound; Preferably, the general formula of the active carboxylic acid ester compound is Sulfo-NHS-COO-(CH3). m Or NHS-COO-(CH3) m , where m is an integer from 1 to 10.

[0014] Furthermore, in the reaction system, the total amount of the first compound and the second compound added is 0.005 mol per 1g of amino magnetic beads.

[0015] Furthermore, the molar ratio of the first compound to the second compound is 1:(0.5~50).

[0016] Furthermore, the solvent in the reaction system is any one of DMF, DMSO, or DMAc.

[0017] Secondly, the present invention provides a carboxyl magnetic bead, which is prepared by the above-described magnetic bead preparation method.

[0018] Thirdly, the present invention provides carboxyl magnetic beads prepared by the above-described magnetic bead preparation method or the application of the above-described carboxyl magnetic beads in the preparation of products for immunodiagnosis.

[0019] This invention provides a method for precisely controlling the surface carboxyl content of magnetic beads. Amino magnetic beads are simultaneously reacted competitively with two compounds of different structures in the same system. The first compound introduces carboxyl functional groups onto the surface of the magnetic beads, while the second compound consumes unwanted amino groups, preventing further reaction and acting as a sealing agent. By adjusting the molar ratio of the two compounds, the final carboxyl content on the surface of the magnetic beads can be precisely controlled, much like "mixing reagents." The reaction is completed in one step under the same system and conditions, eliminating the need for intermediate separation and purification steps. This method is simple to operate and suitable for large-scale production. Since the control parameter is only the molar ratio of the two compounds, batch-to-batch differences caused by temperature and pH fluctuations are avoided, resulting in good product consistency. Data shows that the obtained carboxyl magnetic beads have low background adsorption, controllable bioligand coupling amount, and maintained activity. This solves the technical problems of difficult precise control of carboxyl content and poor activity after coupling in existing one-step or grafting methods. Attached Figure Description

[0020] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0021] Figure 1 The image shows a scanning electron microscope (SEM) image of the carboxyl magnetic beads provided in Example 1-1 of this invention. Figure 2 These are scanning electron microscope images of carboxyl magnetic beads provided in Examples 1-2 of this invention; Figure 3 These are scanning electron microscope images of carboxyl magnetic beads provided in Examples 1-3 of this invention; Figure 4 These are scanning electron microscope images of carboxyl magnetic beads provided in Examples 1-4 of this invention; Figure 5 These are scanning electron microscope images of carboxyl magnetic beads provided in Examples 1-5 of this invention; Figure 6 These are scanning electron microscope images of the carboxyl magnetic beads provided in Examples 1-6 of this invention; Figure 7 These are scanning electron microscope images of carboxyl magnetic beads provided in Examples 1-7 of this invention; Figure 8 These are scanning electron microscope images of carboxyl magnetic beads provided in Examples 1-8 of this invention; Figure 9 This is a scanning electron microscope image of carboxyl magnetic beads provided in Example 2 of the present invention; Figure 10This is a scanning electron microscope image of the carboxyl magnetic beads provided in Example 3 of the present invention; Figure 11 This is a scanning electron microscope image of the carboxyl magnetic beads provided in Example 4 of the present invention; Figure 12 This is a scanning electron microscope image of the carboxyl magnetic beads provided in Example 5 of the present invention; Figure 13 This is a scanning electron microscope image of the carboxyl magnetic beads provided in Comparative Example 1 of the present invention; Figure 14 This is a scanning electron microscope image of the carboxyl magnetic beads provided in Comparative Example 2 of the present invention. Detailed Implementation

[0022] Unless otherwise defined herein, the scientific and technical terms used in conjunction with this invention shall have the meanings commonly understood by one of ordinary skill in the art. The meaning and scope of terms shall be clear; however, in any case of potential ambiguity, the definitions provided herein shall prevail over any dictionary or foreign definitions. In this application, unless otherwise stated, the use of "or" means "and / or". Furthermore, the use of the term "comprising" and other forms is non-limiting.

[0023] Unless otherwise stated, the methods and techniques of the present invention are generally carried out according to conventional methods well known in the art and as described in various general and more specific references, which are cited and discussed throughout this specification.

[0024] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] The present invention provides a method for preparing magnetic beads with precise control of surface carboxyl content, comprising reacting amino magnetic beads with a first compound and a second compound in the same reaction system, adjusting the molar ratio of the first compound and the second compound to control the carboxyl content on the surface of the magnetic beads, thereby obtaining magnetic beads with precise control of surface carboxyl content; The first compound is a reagent that can covalently bind to an amino group and introduce a free carboxyl group; the second compound is a reagent that can covalently bind to an amino group but does not introduce a free carboxyl group.

[0026] This invention involves simultaneously reacting amino magnetic beads with two compounds of different structures in the same system in a competitive reaction. The first compound introduces carboxyl functional groups onto the surface of the magnetic beads, while the second compound consumes unwanted amino groups, preventing further reactions and acting as a blocking agent. By adjusting the molar ratio of the two compounds, the final carboxyl content on the surface of the magnetic beads can be precisely controlled, much like "mixing reagents." The reaction is completed in one step under the same system and conditions, eliminating the need for intermediate separation and purification steps, simplifying the operation and making it suitable for large-scale production. Since the control parameter is only the molar ratio of the two compounds, batch-to-batch differences caused by temperature and pH fluctuations are avoided, resulting in good product consistency. Data shows that the resulting carboxyl magnetic beads have low background adsorption, controllable bioligand coupling amount, and maintain activity, solving the technical problems of difficult precise control of carboxyl content and poor activity after coupling in existing one-step or grafting methods.

[0027] The amino loading of the amino magnetic beads is not limited and can be used for amino magnetic beads with any loading. In some specific embodiments, the amino magnetic beads are prepared by introducing amino groups through polymer coating copolymerization.

[0028] In some specific embodiments, the carboxyl content is positively correlated with the molar ratio of the first compound and the second compound; that is, the carboxyl content increases as the molar ratio of the first compound and the second compound increases. For example, increasing the proportion of the second compound decreases the final carboxyl content; increasing the proportion of the first compound increases the final carboxyl content. In some specific embodiments, the molar ratio of the first compound and the second compound is 1:(0.5~50).

[0029] In some specific embodiments, the first compound is a dicarboxylic anhydride compound.

[0030] In this process, the dicarboxylic anhydride reacts with the amino group to open the ring, forming an amide bond at one end and releasing a free carboxyl group at the other end. In some specific embodiments, the dicarboxylic anhydride compound includes any one of diethanolic anhydride, succinic anhydride, methylene succinic anhydride, maleic anhydride, glutaric anhydride, adipic anhydride, or phthalic anhydride.

[0031] In some specific embodiments, the second compound is a monoacid anhydride. In some specific embodiments, the general formula of the monoacid anhydride is (CH2). x -CO-O-CO-(CH2) y , where x and y are independent integers from 1 to 10.

[0032] In some specific embodiments, the second compound is a reactive carboxylic acid ester compound; in some specific embodiments, the general formula of the reactive carboxylic acid ester compound is Sulfo-NHS-COO-(CH3).m Or NHS-COO-(CH3) m , where m is an integer from 1 to 10. It can react with amino groups to form stable amide bonds without introducing additional carboxyl groups.

[0033] In some specific embodiments, the total amount of the first compound and the second compound added to the reaction system is 0.005 mol per 1g of amino magnetic beads.

[0034] In some specific embodiments, the solvent of the reaction system is any one of DMF, DMSO or DMAc.

[0035] According to another aspect of the present invention, a carboxyl magnetic bead is also provided, which is prepared by the above-described magnetic bead preparation method.

[0036] Carboxyl magnetic beads have low background adsorption and controllable biological ligand coupling, resulting in better signal-to-noise ratio and sensitivity in immunodiagnostics.

[0037] According to another aspect of the present invention, the application of carboxyl magnetic beads prepared by the above-described magnetic bead preparation method or the above-described carboxyl magnetic beads in the preparation of products for immunodiagnosis is also provided.

[0038] The present invention will be further illustrated by the following examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or purchased directly from the market.

[0039] The preparation of amino magnetic beads in the following examples: In this example, amino magnetic beads were prepared according to the preparation method described in Example 1 of Chinese Patent Application No. 202510816739.8. The specific operation steps are as follows: (1) Measure 100 mL of anhydrous ethanol, 50 mL of purified water and 8 g of PVP and add them to a 500 mL four-necked flask equipped with a mechanical stirrer. Stir the mixture to dissolve the PVP. Add 2 g of initiator AIBN and 10 mL of purified St monomer (molar ratio AIBN:St≈1:7). Stir at 300 rpm and purge with nitrogen for 30 min. Heat the oil bath to 70 °C and polymerize for 16 h. Centrifuge to obtain seed microspheres.

[0040] (2) Measure 15 mL of toluene, 15 mL of DVB and St monomer (St accounts for 10%, 1.5 mL), and about 0.2 g of initiator BPO, and add them to a 500 mL four-necked flask containing 200 mL of 0.2% SDS aqueous solution. Control the temperature at 30℃ and stir at 300 rpm for 16 h to emulsify. Then measure 2 g of the seed microspheres prepared in step (1), control the temperature at 30℃ and stir at 300 rpm for 16 h to swell. Then add 100 mL of 2% PVP solution and mix thoroughly. Raise the temperature to 70℃ and continue the reaction for 16 h.

[0041] (3) Centrifuge to collect the porous microspheres from step (2), wash them once with industrial alcohol, add an extractant (dichloromethane: toluene = 1:1) and extract overnight (12~16h), centrifuge to remove the extractant, wash them twice with ethanol, disperse the porous microspheres in water and determine the solid content.

[0042] (4) Measure 5g of porous microspheres and disperse them in a 500mL four-necked flask containing 200mL of anhydrous ethanol. Stir well and mix thoroughly. Then add 0.2g of initiator AIBN, 3mL of MAA monomer, and 0.5mL of DVB monomer. Stir at 300rpm and purge with nitrogen for 30min. Stir at room temperature for 2h to carry out adsorption. Gradually heat the water bath to 70℃ and continue the reaction for 24h. Centrifuge to collect the carboxylated porous microspheres and wash them alternately with water and ethanol to remove excess MAA. Determine the solid content.

[0043] (5) Take 5g of carboxylated porous spheres and disperse them in 150mL of water. Add 1mL of concentrated hydrochloric acid and purge with nitrogen gas for 30min while stirring at 200rpm. Dissolve 9.6g of FeCl3·6H2O(g) and 5.4g of FeSO4·7H2O in 40mL of deoxygenated purified water and add it to the component containing the carboxylated porous spheres. Continue to purge with nitrogen gas for 30min. After the purge is completed, adsorb at room temperature for 2h. Add 30mL of ammonia water and heat to 60℃. Quickly add 1mL of oleic acid and react at 250rpm for 5h. Cool to room temperature, enrich the magnetic microspheres with a magnet, wash with purified water to remove excess ammonia water, and dilute to ethanol solution for solid content determination.

[0044] (6) Measure 5g of magnetic microspheres and disperse them in a 500mL four-necked flask containing 200mL of ethanol solution. Stir and disperse evenly at 300rpm. Add 3mL of GMA monomer, 2mL of St monomer, 5mL of DVB monomer, and 1g of initiator AIBN. After purging at 300rpm for 30min, heat to 70℃ and react for 16h. Aminated magnetic microspheres (amino magnetic beads) are obtained by introducing amino groups through the ring-opening reaction of ethylenediamine on epoxy groups. The amino density is measured to be 810μm / g.

[0045] Example 1 In this embodiment, succinic anhydride is used as the first compound and acetic anhydride is used as the second compound to control the carboxyl content. The total molar amount of the first and second compounds remains unchanged at 0.005 mol / g. The amounts of the first and second compounds are adjusted according to the molar ratios of the first and second compounds in Tables 1-1 to 1-8. The specific preparation steps are as follows: 1 g of the prepared amino magnetic beads was dispersed in 10 mL of anhydrous DMF. The first compound (succinic anhydride, dissolved in DMF) and the second compound (acetic anhydride, dissolved in DMF) were added according to the proportions shown in Table 1. The reaction system was stirred at 25 °C for 2 hours. The microspheres were collected by magnetic separation and washed three times each with ethanol and pure water to obtain carboxyl magnetic beads. The prepared carboxyl magnetic beads were observed under a scanning electron microscope as shown below. Figures 1-8 As shown.

[0046] Example 2 Unlike Example 1, the reaction solvent was changed to DMAc, and maleic anhydride was used as the first compound, while octanoic anhydride (in the general formula x=6, y=6) was used as the second compound to control the carboxyl content. The specific preparation steps are as follows: Take 1 g of the prepared amino magnetic beads and disperse them in 10 mL of anhydrous DMF. Add the first compound (maleic anhydride, dissolved in DMF) and the second compound (octanoic anhydride, dissolved in DMF) at a molar ratio of 1:10, and other conditions are the same as in Example 1. The resulting carboxyl magnetic beads were observed under a scanning electron microscope as shown below. Figure 9 As shown.

[0047] Example 3 Unlike Example 1, glycolic anhydride was used as the first compound and acetic propionic anhydride (in the general formula x=1, y=2) was used as the second compound to regulate the carboxyl content. The specific preparation steps are as follows: Take 1 g of the prepared amino magnetic beads and disperse them in 10 mL of anhydrous DMF. Add the first compound (glycolic anhydride, dissolved in DMF) and the second compound (acetic acid propionic anhydride, dissolved in DMF) at a molar ratio of 1:10, and other conditions are the same as in Example 1. The resulting carboxyl magnetic beads were observed under a scanning electron microscope as shown below. Figure 10 As shown.

[0048] Example 4 Unlike Example 1, succinic anhydride was used as the first compound and Sulfo-NHS-acetic acid (m=1) was used as the second compound to regulate the carboxyl content. The specific preparation steps are as follows: Take 1 g of the prepared amino magnetic beads and disperse them in 10 mL of anhydrous DMF. Add the first compound (succinic anhydride, dissolved in DMF) and the second compound (Sulfo-NHS-acetic acid, dissolved in DMF) at a molar ratio of 1:10, and other conditions are the same as in Example 1. The resulting carboxyl magnetic beads were observed under a scanning electron microscope as shown below. Figure 11 As shown.

[0049] Example 5 Unlike Example 1, the reaction solvent was changed to DMSO, and succinic anhydride was used as the first compound and NHS-acetic acid (m=1) was used as the second compound to regulate the carboxyl content.

[0050] Take 1 g of the prepared amino magnetic beads and disperse them in 10 mL of anhydrous DMSO. Add the first compound (succinic anhydride, dissolved in DMF) and the second compound (NHS-acetic acid, dissolved in DMF) at a molar ratio of 1:20, and other conditions are the same as in Example 1. The resulting carboxyl magnetic beads were observed under a scanning electron microscope as shown below. Figure 12 As shown.

[0051] Comparative Example 1 Unlike Example 1, no second compound was added.

[0052] Take 1g of amino magnetic beads and add only the same total molar amount of succinic anhydride as in Examples 1-3 (i.e., use the first compound alone). The resulting carboxyl magnetic beads were observed under a scanning electron microscope as follows. Figure 13 As shown.

[0053] Comparative Example 2 Unlike Example 1, the first compound was not added.

[0054] Take 1g of amino magnetic beads and add only acetic anhydride (x=1, y=1) of the same total molar amount as in Examples 1-3 (i.e., use the second compound alone). The resulting carboxyl magnetic beads were observed under a scanning electron microscope as follows. Figure 14 As shown.

[0055] Experiment 1 The carboxyl density of the microspheres obtained in Examples 1-5 and Comparative Examples 1 and 2 was tested. The specific operating steps are as follows: (1) Preparation of titrant: 0.01M NaOH titrant: Take 2.50 mL of 1M NaOH standard solution and dilute to 250 mL in a volumetric flask with anhydrous ethanol.

[0056] (2) Sample preparation: Mix the sample to be tested evenly, calculate the required sample volume according to the solid content, and retain the precipitate by magnetic adsorption. Mix the precipitate with 50 mL of anhydrous ethanol, and then titrate it into the sample cup with 0.01 M NaOH-EtOH titrant.

[0057] (3) Instrument preparation: Select the corresponding pump head for the automatic potentiometric titrator, and install the ethanol electrode and pipeline after rinsing them.

[0058] The pipeline was flushed with pump air and then with 0.01M NaOH-EtOH solution.

[0059] Sample testing: Install the sample cup, select the corresponding test item, and start the test.

[0060] The equipment and reagents used in the experiment include: automatic potentiometric titrator, anhydrous ethanol, and 1M NaOH.

[0061] The results are shown in Table 1.

[0062] Table 1

[0063] The results in Examples 1-1 to 1-8 of Example 1 show that the content of carboxyl groups on the surface of immunomagnetic beads can be precisely controlled by controlling the ratio of the first compound and the second compound. Compared with Examples 1-4, Examples 2-4 changed the first compound and the second compound, or only changed the type of the second compound, but the ratio of the first compound and the second compound remained unchanged, and the carboxyl group content did not change significantly. Compared with Examples 1-5, Example 5 changed the type of the second compound, but the ratio of the first compound and the second compound remained unchanged, and the carboxyl group content did not change significantly. This verifies that the carboxyl group content on the surface of the magnetic beads is only related to the ratio of the first compound and the second compound, and changing the type of compound does not have a significant impact on the carboxyl group content.

[0064] Experiment 2 The microspheres obtained in Examples 1-5 and Comparative Examples 1 and 2 were subjected to immunochemiluminescence assays. The specific operating steps are as follows: 1. CA19-9 Packaging Process (150T): (1) Washing: Take 4.5 mg of carboxyl magnetic bead stock solution, add 200 μl of washing solution A, shake and wash for 3 min at 650 rpm, then magnetically adsorb for 3 min and discard the supernatant, leaving the magnetic bead precipitate. Wash a total of 5 times.

[0065] (2) Activation: Weigh out EDC and NHS and dissolve them in solution B to prepare solutions of 15 mg / ml. Note: Prepare solutions fresh for each use. Add 50 μl of each of the prepared EDC and NHS solutions to the magnetic beads and activate by shaking at 650 rpm for 1 h at room temperature. After the activation reaction is complete, perform magnetic adsorption for 4 min, discard the supernatant and keep the magnetic bead precipitate.

[0066] (3) Washing: Add 200 μl of washing solution B, shake and wash for 6 min at 650 rpm. After magnetic adsorption for 4 min, discard the supernatant and keep the magnetic bead precipitate. Wash twice in total.

[0067] (4) Coating: Add 88.2 μl of solution B and 11.8 μl of antibody solution, shake at 650 rpm for 2 h at room temperature. Discard the supernatant after magnetic adsorption for 5 min. Live material batch number: 2024061401, 5 mg / ml.

[0068] (5) Termination: Add 400 μl of ethanolamine A solution to terminate the reaction for 40 min. Magnetic adsorption for 5 min, discard the supernatant and keep the magnetic beads as precipitate.

[0069] (6) Blocking: Add 400 μl of sealing solution, shake and wash at 700 rpm for 10 min, then magnetically adsorb for 5 min and discard the supernatant, keeping the magnetic beads as precipitate. Wash a total of 3 times. Heat block at 37℃ for 3 days and then wash once.

[0070] (7) Volume adjustment: Use sealing solution to adjust the volume of magnetic beads to 3 ml, and label and store.

[0071] After preparing and correctly placing the test calibrators, click the start button to begin the calibration or sample testing procedure. The instrument will then perform the following operations: (1) Transfer the sample rack to the sample aspiration position and load the reaction vessel to the sample loading position.

[0072] (2) When performing the calibration procedure, dispense 25 μL of calibrator, 20 μL of magnetic microparticle suspension, and 50 μL of sample diluent.

[0073] (3) Mix the reaction solution and incubate it at 37°C for 15 minutes.

[0074] (4) After the incubation is completed, the reaction solution is cleaned and separated using a cleaning solution.

[0075] (5) Complete the dispensing of 100 μL of enzyme conjugate.

[0076] (6) Mix the reaction solution and incubate it at 37°C for 17 minutes.

[0077] (7) After the incubation is completed, the reaction solution is cleaned and separated using a cleaning solution.

[0078] (8) Complete the dispensing of 50 μL of substrate A solution and 50 μL of substrate B solution.

[0079] (9) Mix the reaction solution and test the luminescence intensity.

[0080] The equipment and reagents used in the experiment include: purified water, reaction vessel, sample container, sample rack, chemiluminescence detector (suitable for Autolumo A2000, Autolumo A2000 Plus, and Autolumo A2000 Plus B fully automated chemiluminescence analyzers), one set of calibrators (containing CA19-9) (concentrations of approximately 0 IU / mL, 2 IU / mL, 10 IU / mL, 20 IU / mL, 100 IU / mL, and 220 IU / mL), substrate solution for the fully automated immunoassay system (registration number: Yu Zheng Xie Bei 2014002), cleaning solution (registration number: Yu Zheng Xie Bei 20140021), and sample diluent (registration number: Yu Zheng Xie Bei 20140022).

[0081] 2. Result Calculation: The instrument automatically calculates the sample test results based on the luminescence values ​​obtained from the calibration curve and sample testing. The test results are shown in Table 2.

[0082] Table 2. Signal value data of enzyme-labeled antibodies for project verification.

[0083] The evaluation results of the CA19-9 project indicate a certain relationship between the luminescence value and the carboxyl group content. The luminescence value initially increases and then decreases with increasing carboxyl group content, with the optimal range being 110–380 µmol / g. Furthermore, with a fixed ratio of different types of the first and second compounds, as long as the carboxyl group content remains within this range, the luminescence value of this project can be maintained at a high level. The results of Comparative Examples 1 and 2 show that the magnetic beads have no functional groups on their surface. Due to hydrophobic interactions, the background of the evaluated project is high, and the luminescence value is extremely low, primarily due to passive adsorption.

[0084] Therefore, it can be seen that the present invention achieves continuous, precise and flexible control of the final effective carboxyl density on the surface of microspheres by adjusting the molar ratio of the first and second compounds through the competitive reaction mechanism of the first and second compounds. The process is simple and reliable, and achieves precise control of carboxyl density.

[0085] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing magnetic beads with precise control of surface carboxyl group content, characterized in that, This includes reacting amino magnetic beads with a first compound and a second compound in the same reaction system, adjusting the molar ratio of the first compound and the second compound to control the carboxyl content on the surface of the magnetic beads, and obtaining magnetic beads with precise control of the surface carboxyl content; The first compound is a reagent that can covalently bind to an amino group and introduce a free carboxyl group; the second compound is a reagent that can covalently bind to an amino group but does not introduce a free carboxyl group.

2. The method for preparing magnetic beads according to claim 1, characterized in that, The carboxyl content is positively correlated with the molar ratio of the first compound and the second compound.

3. The method for preparing magnetic beads according to claim 1, characterized in that, The first compound is a dicarboxylic anhydride compound.

4. The method for preparing magnetic beads according to claim 1, characterized in that, The second compound is a monoacid anhydride; Preferably, the general formula of the monoacid anhydride compound is (CH2). x -CO-O-CO-(CH2) y , where x and y are independent integers from 1 to 10.

5. The method for preparing magnetic beads according to claim 1, characterized in that, The second compound is a carboxylic acid ester compound; Preferably, the general formula of the active carboxylic acid ester compound is Sulfo-NHS-COO-(CH3). m Or NHS-COO-(CH3) m , where m is an integer from 1 to 10.

6. The method for preparing magnetic beads according to any one of claims 1 to 5, characterized in that, In the reaction system, the total amount of the first compound and the second compound added is 0.005 mol per 1g of amino magnetic beads.

7. The method for preparing magnetic beads according to claim 6, characterized in that, The molar ratio of the first compound to the second compound is 1:(0.5~50).

8. The method for preparing magnetic beads according to claim 6, characterized in that, The solvent for the reaction system is any one of DMF, DMSO or DMAC.

9. A carboxyl magnetic bead, characterized in that, The magnetic beads were prepared using the method described in any one of claims 1 to 8.

10. The use of carboxyl magnetic beads prepared by the method of any one of claims 1 to 8 or the carboxyl magnetic beads of claim 9 in the preparation of products for immunodiagnostics.

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