Microsphere as well as preparation method and application thereof

Abstract

The invention relates to the field of biological detection, in particular to microspheres and a preparation method and application thereof. The invention provides a preparation method of magnetic polymer microspheres. The preparation method comprises the following steps: S1, obtaining magnetic microspheres with carboxyl groups; s2, activating the magnetic microspheres with carboxyl in the step S1 to obtain activated magnetic microspheres; s3, mixing the activated magnetic microspheres obtained in S2 with a first compound and a second compound, reacting, and collecting the microspheres to obtain the magnetic polymer microspheres, the first compound and the second compound competitively react with the carboxyl of the activated magnetic microspheres. The preparation method provided by the invention is simple and convenient to operate, mild in condition and good in repeatability, and the prepared microspheres are accurate and controllable in carboxyl density and uniform in distribution, have the advantages of high magnetic responsiveness, good dispersity, dispersion stability, good biocompatibility and the like, and can be widely applied to the fields of biological separation, diagnosis and the like.

Description

Technical Field

[0001] This invention relates to the field of biological detection, and more particularly to microspheres, their preparation methods, and applications. Background Technology

[0002] Magnetic polymer microspheres, combining the excellent dispersibility and ease of modification of polymer materials with the magnetic responsiveness of magnetic particles, have wide applications in biomedical separation, immunoassay, targeted drug delivery, and cell sorting. Among them, magnetic microspheres with carboxyl groups (-COOH) on their surface are particularly important, as they can form stable amide bonds with primary amino groups (-NH2) on biomolecules such as proteins and nucleic acids through mature carbodiimide chemistry, thereby achieving the immobilization of biomolecules.

[0003] Currently, the traditional method for preparing carboxylated magnetic microspheres mainly involves copolymerizing carboxyl-containing monomers (such as acrylic acid and methacrylic acid) during the polymerization stage. However, these methods have significant limitations: First, the amount and distribution of carboxyl groups introduced are determined by the copolymerization reaction kinetics, which is difficult to control precisely, leading to performance fluctuations between different batches of products; second, the carboxyl groups formed by polymerization may be embedded inside the polymer network, resulting in poor accessibility and low reaction efficiency; third, the microsphere surface contains a large number of unreacted carboxyl groups and hydrophobic regions, which easily and non-specifically adsorb biomolecules, causing problems such as high detection background and poor specificity; finally, biomolecules are usually fixed on the microsphere surface in a random orientation, which may lead to the masking and inactivation of their active sites.

[0004] Therefore, developing a new method that can precisely control the surface carboxyl group density and improve the orientation of biomolecule immobilization is of great significance to the technological advancement in this field. Summary of the Invention

[0005] In view of this, the present invention provides microspheres, their preparation method, and applications. The preparation method provided by the present invention is simple to operate, has mild conditions, and good reproducibility. The resulting microspheres have precisely controllable carboxyl density and uniform distribution, and possess advantages such as high magnetic responsiveness, good dispersibility, stable dispersion, and excellent biocompatibility. They can be widely used in fields such as bioseparation and diagnostics.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides a method for preparing magnetic polymer microspheres, comprising the following steps:

[0008] S1: Obtain magnetic microspheres with carboxyl groups;

[0009] S2: Activate the carboxyl-containing magnetic microspheres in S1 to obtain activated magnetic microspheres;

[0010] S3: The activated magnetic microspheres obtained in S2 are mixed with the first compound and the second compound, reacted, and the microspheres are collected to obtain the magnetic polymer microspheres;

[0011] The first compound and the second compound competitively react with the carboxyl groups of the activated magnetic microspheres.

[0012] In some embodiments of the present invention, in the above preparation method, the general structural formula of the first compound is: H2N-R-COOH, wherein: R is a polyethylene glycol chain or an alkylene chain.

[0013] In some embodiments of the present invention, in the above preparation method, the first compound has the general structural formula: H2N-(PEG). x -COOH or H2N-(CH2) y -COOH, where: x is an integer from 2 to 24, and y is an integer from 2 to 24.

[0014] In some embodiments of the present invention, in the above preparation method, the second compound comprises: ethanolamine, tris(hydroxymethyl)aminomethane, 2-aminoethanesulfonic acid, and CH3-O-PEG. n -NH2 (where n is an integer from 2 to 12) or one or more of the following.

[0015] In some embodiments of the present invention, in the above preparation method, the molar ratio of the first compound and the second compound is 1:(0.5~50).

[0016] In some embodiments of the present invention, in the above preparation method, the molar ratio of the first compound and the second compound is: 1:0.5, 1:1, 1:2, 1:5, 1:10, 1:20, 1:30 or 1:50.

[0017] In some embodiments of the present invention, in the above preparation method, the carboxyl groups in the magnetic microspheres with carboxyl groups are introduced by copolymerization of carboxyl functional monomers.

[0018] In some embodiments of the present invention, in the above preparation method, the carboxyl functional monomer includes one or more of the following: acrylic acid, methacrylic acid, mono-2-(methacryloyloxy)ethyl maleate, β-(acryloyloxy)propionic acid, 6-acryloylaminohexanoic acid, mono-2-(methacryloyloxy)ethyl succinate, 3-butenoic acid, 4-pentenoic acid, 5-hexenoic acid, 6-heptenoic acid, 7-octenic acid, 8-nonenoic acid, 9-decenoic acid, 10-undecenoic acid, and 11-dodecenoic acid.

[0019] In some embodiments of the present invention, in the above preparation method, the mass ratio of the carboxyl functional monomer is 10%.

[0020] In some embodiments of the present invention, the activation in the above preparation method is performed using an activator; the activator includes one or more of EDC, NHS, DCC, CDI, HATU, HBTU and HCTU.

[0021] In some embodiments of the present invention, the activation in the above preparation method uses an activating agent; the activating agent is EDC and NHS.

[0022] The present invention also provides magnetic polymer microspheres obtained by the above preparation method.

[0023] The present invention also provides the application of the above-described magnetic polymer microspheres in any of the following:

[0024] (a) Immunological testing; and / or

[0025] (b) Preparation of products for immunoassay.

[0026] The present invention also provides a detection product comprising: the above-described magnetic polymer microspheres and acceptable additives or devices.

[0027] The beneficial effects of this invention include:

[0028] (1) Precise and controllable density: By adjusting the molar ratio of the first compound and the second compound through the competitive reaction mechanism, the final effective carboxyl density on the surface of the microspheres can be continuously, precisely and flexibly controlled. The process is simple and reliable.

[0029] (2) Low non-specific adsorption: The second compound, as a blocking agent, can react efficiently with the activation sites not occupied by the first compound, completely blocking them and fundamentally eliminating the main source of non-specific adsorption, thus significantly improving the signal-to-noise ratio and specificity of subsequent biological applications.

[0030] (3) Excellent biocompatibility: It is preferable to use PEG chains as spacer arms of the first compound or as the second compound itself, which can give the surface of microspheres excellent hydrophilicity and anti-fouling ability, forming a "inert" background with better biocompatibility, which is conducive to maintaining the activity of immobilized biomolecules.

[0031] (4) Orientation optimization: The carboxyl groups introduced by the first compound are all located at the end of the spacer arm, with consistent spatial orientation, which provides a more uniform and ideal immobilization environment for biomolecules, helps to reduce the masking of active sites and improve detection sensitivity.

[0032] (5) Flexible application: By adjusting the density parameters, a series of products suitable for different scenarios (such as conjugation of macromolecular antibodies or small molecule antigens) can be prepared, greatly enhancing the flexibility of application. Attached Figure Description

[0033] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0034] Figure 1 This shows a scanning electron microscope image of the acrylic substrate microspheres in Example 1;

[0035] Figure 2 This shows a scanning electron microscope image of the maleic acid mono-2-(methacryloyloxy)ethyl ester substrate microspheres in Example 2;

[0036] Figure 3 This shows a scanning electron microscope image of the β-(acryloyloxy)propionic acid-based microspheres in Example 3;

[0037] Figure 4 The scanning electron microscope image of the succinic acid mono-2-(methacryloyloxy)ethyl ester substrate microspheres in Example 4 shows that the first compound is H2N-(PEG). 24 -COOH (compound 1:compound 2 = 1:50);

[0038] Figure 5 The scanning electron microscope image of the succinic acid mono-2-(methacryloyloxy)ethyl ester substrate microspheres in Example 5 shows that the first compound is H2N-(CH2). 24 -COOH (compound 1:compound 2 = 1:50);

[0039] Figure 6 This shows a scanning electron microscope image of the succinic acid mono-2-(methacryloyloxy)ethyl ester substrate microspheres in Example 6;

[0040] Figure 7 This shows a scanning electron microscope image of the 4-pentenoic acid-based microspheres in Example 7;

[0041] Figure 8 This shows a scanning electron microscope image of the 11-dodecanoic acid-based microspheres in Example 8;

[0042] Figure 9 This shows a scanning electron microscope image of the 6-acryloylaminohexanoic acid substrate microspheres in Example 9;

[0043] Figure 10 This shows a scanning electron microscope image of the methacrylic acid substrate microspheres in Example 10;

[0044] Figure 11 This shows a scanning electron microscope image of the methacrylic acid substrate microspheres in Example 11;

[0045] Figure 12 This shows a scanning electron microscope image of the methacrylic acid substrate microspheres in Example 12;

[0046] Figure 13 The image shows a scanning electron microscope (SEM) image of the acrylic substrate microspheres in Comparative Example 1. Detailed Implementation

[0047] This invention discloses microspheres, their preparation methods, and applications.

[0048] It should be understood that the expression “one or more of…” individually includes each of the objects described after the expression, as well as various different combinations of two or more of the described objects, unless otherwise understood from the context and usage. The expression “and / or” combined with three or more described objects should be understood to have the same meaning, unless otherwise understood from the context.

[0049] The terms “including,” “having,” or “containing,” including the use of their grammatical synonyms, should generally be understood as open-ended and non-restrictive, for example, not excluding other unstated elements or steps, unless otherwise specifically stated or understood from the context.

[0050] It should be understood that the order of the steps or the order in which certain actions are performed is not important as long as the invention remains operational. Furthermore, two or more steps or actions can be performed simultaneously.

[0051] The use of any and all instances or exemplary language such as “e.g.” or “including” in this document is merely intended to better illustrate the invention and is not intended to limit the scope of the invention unless the claims are made. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0052] Furthermore, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any value inevitably contains standard deviations due to individual test methods. Therefore, unless explicitly stated otherwise, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified with the word "approximately". Here, "approximately" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specific value or range.

[0053] This invention provides a simple preparation process for magnetic polymer microspheres with flexibly adjustable carboxyl group density, and a method for preparing the same. Specifically, it includes:

[0054] In a first aspect, the present invention provides a method for preparing magnetic polymer microspheres with controllable surface carboxyl group density, comprising the following steps:

[0055] (1) Provide magnetic polymer microspheres with carboxyl groups on their surface;

[0056] (2) Activate the carboxyl groups on the surface of the magnetic microspheres obtained in step (1) with an activator to obtain magnetic microspheres with activated carboxyl groups on the surface;

[0057] (3) The magnetic microspheres obtained in step (2) are brought into contact with the first compound and the second compound in the same reaction system for reaction;

[0058] The first compound is a bifunctional compound having one primary amino group and one carboxyl group, with the general formula H2N-R-COOH, where R is a spacer group; the second compound is a monoamino compound having only one primary amino group; the first compound and the second compound competitively react with the activated carboxyl group, and the carboxyl group density finally grafted onto the surface of the microspheres is controlled by adjusting the molar ratio of the two compounds.

[0059] Preferably, the magnetic polymer microspheres with carboxyl groups on their surface described in step (1) have carboxyl groups introduced by a copolymerization reaction of a carboxyl-containing functional monomer; the carboxyl-containing functional monomer is a compound containing a carboxyl group at one end and a polymerizable double bond at the other end.

[0060] More preferably, the carboxyl-containing functional monomer is selected from at least one of the following: acrylic acid, methacrylic acid, mono-2-(methacryloyloxy)ethyl maleate, β-(acryloyloxy)propionic acid, 6-acryloylaminohexanoic acid, mono-2-(methacryloyloxy)ethyl succinate, 3-butenoic acid, 4-pentenoic acid, 5-hexenoic acid, 6-heptenoic acid, 7-octenic acid, 8-nonenoic acid, 9-decenoic acid, 10-undecenoic acid, and 11-dodecenoic acid.

[0061] Preferably, the activator in step (2) is selected from at least one of the following: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), dicyclohexylcarbodiimide (DCC), N,N'-carbonyldiimidazole (CDI), O-(7-azabenzotriazol-1-yl)-di(dimethylamino)carbodiium hexafluorophosphate (HATU), O-(benzotriazol-1-yl)-di(dimethylamino)carbodiium hexafluorophosphate (HBTU), and O-(5-chlorobenzotriazol-1-yl)-di(dimethylamino)carbodiium hexafluorophosphate (HCTU). More preferably, an activation system using EDC and NHS in combination is employed.

[0062] Preferably, in step (3), the spacer group R in the first compound is a polyethylene glycol chain or an alkylene chain; its general formula is H2N-(PEG). x -COOH or H2N-(CH2) y -COOH, where x is an integer between 2 and 24, and y is an integer between 2 and 24.

[0063] Preferably, in step (3), the second compound is selected from: ethanolamine, tris(hydroxymethyl)aminomethane (Tris), 2-aminoethanesulfonic acid, and methoxy-polyethylene glycol-amine (CH3-O-PEG). n At least one of the following: -NH2, where n is an integer from 2 to 12.

[0064] Preferably, in step (3), the molar ratio of the first compound to the second compound is 1:0.5 to 50. By adjusting the molar ratio within this range, the effective carboxyl group density on the surface of the microspheres that can be used for subsequent bioconjugation can be continuously and precisely controlled, much like using a "dimming switch".

[0065] Secondly, the present invention provides magnetic polymer microspheres with controllable surface carboxyl group density prepared by any of the above methods.

[0066] Thirdly, the present invention also provides the application of the above-mentioned magnetic polymer microspheres in immunoassay.

[0067] In Examples 1 to 12 and Comparative Example 1 of this invention, all raw materials and reagents used can be purchased from the market.

[0068] The present invention will be further illustrated below with reference to the embodiments:

[0069] Example 1: Acrylic acid was used as the functional monomer, H2N-PEG2-COOH was the first compound, and ethanolamine was the second compound.

[0070] (1) Preparation of substrate microspheres: Magnetic microspheres with polystyrene as matrix and embedded iron oxide nanoparticles (average particle size 2.0 μm) were prepared by seed swelling polymerization. During the polymerization process, acrylic acid, accounting for 10% of the total monomer mass, was introduced as a carboxyl-containing functional monomer to obtain magnetic polymer microspheres with carboxyl groups on the surface.

[0071] (2) Carboxyl activation: Take 10 mg of the above microspheres and wash them three times with 0.1 M MES buffer (pH 4.7). Redisperse the microspheres in 1 mL of MES buffer, and add EDC∙HCl and NHS sequentially to make their final concentrations 10 mM and 5 mM, respectively. Shake and react at room temperature for 30 minutes.

[0072] (3) Competitive grafting and blocking: The supernatant was discarded by magnetic separation, and the microspheres were washed once with pre-cooled PBS (pH 7.4). The activated microspheres were resuspended in 1 mL of PBS. The first compound H2N-PEG2-COOH (0.1 mmol / L) and the second compound ethanolamine (1.0 mmol / L) were added simultaneously to make a molar ratio of 1:10. The reaction was carried out at room temperature with shaking for 2 hours.

[0073] (4) Post-processing: After the reaction is completed, the microspheres are collected by magnetic separation, washed three times with PBS buffer containing 0.05% Tween-20, and finally dispersed in storage buffer (PBS containing 0.1% BSA) and stored at 4°C.

[0074] Example 2 uses mono-2-(methacryloyloxy)ethyl maleate as the functional monomer, H2N-PEG8-COOH as the first compound, and tris(hydroxymethyl)aminomethane (Tris) as the second compound.

[0075] (1) The substrate microspheres were prepared in the same way as in Example 1, but the carboxyl functional monomer was replaced with ethyl maleate mono-2-(methacryloyloxy) ester, which accounted for 10% of the total mass of the monomer.

[0076] (2) The carboxyl activation steps are the same as in Example 1.

[0077] (3) Competitive grafting and blocking: After resuspending the activated microspheres in PBS, the first compound H2N-PEG8-COOH (0.2 mmol / L) and the second compound tris(hydroxymethyl)aminomethane (Tris) (4.0 mmol / L) were added to make the molar ratio of the two compounds 1:20. The reaction conditions were the same as in Example 1.

[0078] (4) Post-processing is the same as in Example 1.

[0079] Example 3: H2N-PEG using β-(acryloyloxy)propionic acid as the functional monomer 12 -COOH is the first compound, and 2-aminoethanesulfonic acid is the second compound.

[0080] (1) The substrate microspheres were prepared in the same way as in Example 1, but the carboxyl functional monomer was replaced with β-(acryloyloxy)propionic acid, which accounted for 10% of the total mass of the monomer.

[0081] (2) The carboxyl activation steps are the same as in Example 1.

[0082] (3) Competitive grafting and blocking: After resuspending the activated microspheres in PBS, the first compound H2N-PEG was added. 12 -COOH (0.05 mmol / L) and the second compound 2-aminoethanesulfonic acid (1.5 mmol / L) were prepared at a molar ratio of 1:30. The reaction conditions were the same as in Example 1.

[0083] (4) Post-processing is the same as in Example 1.

[0084] Example 4: Using ethyl succinate mono-2-(methacryloyloxy)ethyl ester as the functional monomer, H2N-PEG 24 -COOH is the first compound, and CH3-O-PEG8-NH2 is the second compound.

[0085] (1) The substrate microspheres were prepared in the same way as in Example 1, but the carboxyl functional monomer was replaced with 10% of the total mass of the monomer, which was succinate mono-2-(methacryloyloxy)ethyl ester.

[0086] (2) The carboxyl activation steps are the same as in Example 1.

[0087] (3) Competitive grafting and blocking: After resuspending the activated microspheres in PBS, the first compound H2N-PEG was added. 24 -COOH (0.05 mmol / L) and the second compound CH3-O-PEG8-NH2 (2.5 mmol / L) were prepared in a molar ratio of 1:50. This ratio was intended to obtain a lower surface carboxyl group density. The reaction conditions were the same as in Example 1.

[0088] (4) Post-processing is the same as in Example 1.

[0089] Example 5

[0090] The steps are the same as in Example 4, except that the first compound is replaced with H2N-(CH2). 24 -COOH, so that the molar ratio of the first compound and the second compound is 1:50.

[0091] Example 6

[0092] The steps are the same as in Example 4, except that the first compound NH2-PEG is used. 24 The molar ratio of -COOH (0.05 mmol / L) to the second compound CH3-O-PEG8-NH2 (0.025 mmol / L) becomes 1:0.5.

[0093] Example 7 uses 4-pentenoic acid as the functional monomer, H2N-(CH2)2-COOH as the first compound, and CH3-O-PEG2-NH2 as the second compound.

[0094] (1) The substrate microspheres were prepared in the same way as in Example 1, but the carboxyl functional monomer was replaced with 4-pentenoic acid, which accounted for 10% of the total mass of the monomer.

[0095] (2) The carboxyl activation steps are the same as in Example 1.

[0096] (3) Competitive grafting and blocking: After resuspending the activated microspheres in PBS, add the first compound H2N-(CH2)2-COOH (1.0 mmol / L) and the second compound CH3-O-PEG2-NH2 (1.0 mmol / L) to make the molar ratio of the two 1:1.

[0097] (4) Post-processing is the same as in Example 1.

[0098] Example 8 uses 11-dodecenoic acid as the functional monomer, H2N-(CH2)6-COOH as the first compound, and CH3-O-PEG4-NH2 as the second compound.

[0099] (1) The substrate microspheres were prepared in the same way as in Example 1, but the carboxyl functional monomer was replaced with 11-dodecenoic acid, which accounted for 10% of the total mass of the monomer.

[0100] (2) The carboxyl activation steps are the same as in Example 1.

[0101] (3) Competitive grafting and blocking: After resuspending the activated microspheres in PBS, add the first compound H2N-(CH2)6-COOH (0.1 mmol / L) and the second compound CH3-O-PEG4-NH2 (0.2 mmol / L) to make the molar ratio of the two compounds 1:2.

[0102] (4) Post-processing is the same as in Example 1.

[0103] Example 9 uses 6-acryloylaminohexanoic acid as a functional monomer, H2N-(CH2). 12 -COOH is the first compound, and CH3-O-PEG8-NH2 is the second compound.

[0104] (1) The substrate microspheres were prepared in the same way as in Example 1, but the carboxyl functional monomer was replaced with 6-acryloylamino, which accounted for 10% of the total monomer mass.

[0105] (2) The carboxyl activation steps are the same as in Example 1.

[0106] (3) Competitive grafting and blocking: After resuspending the activated microspheres in PBS, the first compound H2N-(CH2) was added. 12 -COOH (0.1 mmol / L). The second compound is CH3-O-PEG8-NH2 (0.5 mmol / L), and its molar ratio with the first compound remains 1:5.

[0107] (4) Post-processing is the same as in Example 1.

[0108] Example 10 uses methacrylic acid as a functional monomer, H2N-(CH2) 24 -COOH is the first compound, CH3-O-PEG 12 -NH2 is the second compound.

[0109] (1) The substrate microspheres were prepared in the same way as in Example 1, but the carboxyl functional monomer was replaced with methacrylic acid, which accounted for 10% of the total mass of the monomer.

[0110] (2) The carboxyl activation steps are the same as in Example 1.

[0111] (3) Competitive grafting and blocking: After resuspending the activated microspheres in PBS, the first compound H2N-(CH2) was added. 24 -COOH (0.1 mmol / L). The second compound is CH3-O-PEG. 12 -NH2 (1 mmol / L), with the same molar ratio to the first compound, remains 1:10.

[0112] (4) Post-processing is the same as in Example 1.

[0113] Example 11 Using CDI activator

[0114] (1) The substrate microspheres were prepared in the same way as in Example 9 (using 10% methacrylic acid).

[0115] (2) Carboxyl activation: Take 10 mg of microspheres and wash twice with anhydrous DMF. Redisperse the microspheres in 1 mL of anhydrous DMF and add N,N'-carbonyldiimidazole (CDI) to make a final concentration of 20 mM. Shake and react at room temperature for 1 hour.

[0116] (3) Competitive grafting and blocking: Magnetic separation followed by rapid washing once with anhydrous DMF. The activated microspheres were resuspended in 1 mL of anhydrous DMF. The first compound, H2N-(CH2), was added. 24 -COOH (0.1 mmol / L) and the second compound CH3-O-PEG 12 A DMF solution containing -NH2 (1.0 mmol / L) (molar ratio 1:10) was prepared. The reaction was carried out at room temperature with shaking for 2 hours.

[0117] (4) Post-processing: magnetic separation, washing with ethanol 3 times, and finally dispersing in PBS storage buffer.

[0118] Example 12 Using HATU activator

[0119] (1) The substrate microspheres were prepared in the same way as in Example 9 (using 10% methacrylic acid).

[0120] (2) Carboxyl activation: Take 10 mg of microspheres and wash them twice with anhydrous DMF. Redisperse the microspheres in 1 mL of anhydrous DMF and add O-(7-azabenzotriazol-1-yl)-bis(dimethylamino)carbomony hexafluorophosphate (HATU) to a final concentration of 20 mM. Shake and react at room temperature for 1 hour.

[0121] (3) Competitive grafting and blocking: Magnetic separation followed by rapid washing once with anhydrous DMF. The activated microspheres were resuspended in 1 mL of anhydrous DMF. The first compound, H2N-(CH2), was added. 24 -COOH (0.1 mmol / L) and the second compound CH3-O-PEG12 A DMF solution containing -NH2 (1.0 mmol / L) (molar ratio 1:10) was prepared. The reaction was carried out at room temperature with shaking for 2 hours.

[0122] (4) Post-processing: magnetic separation, washing with ethanol 3 times, and finally dispersing in PBS storage buffer.

[0123] Comparative Example 1: Traditional carboxyl microspheres (without competitive grafting blocking treatment)

[0124] Prepare magnetic polymer microspheres with carboxyl groups on their surface that are exactly the same as those in Example 1, that is, only provide magnetic polymer microspheres with carboxyl groups on their surface, without performing the carboxyl activation and competitive grafting blocking treatment in steps (2) and (3).

[0125] The microspheres were washed directly with PBS, dispersed in the same storage buffer, and stored at 4°C for comparison with the embodiments of the present invention.

[0126] Effect verification experiment

[0127] 1. The carboxyl density of the microspheres obtained in Examples 1-12 and Comparative Example 1 was tested. The specific operating steps are as follows:

[0128] (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.

[0129] (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.

[0130] (3) Instrument preparation: Select the corresponding pump head for the automatic potentiometric titrator, and install the ethanol electrode and pipeline after rinsing them.

[0131] The pipeline was flushed with pump air and then with 0.01M NaOH-EtOH solution.

[0132] Sample testing: Install the sample cup, select the corresponding test item, and start the test.

[0133] The equipment and reagents used in the experiment include: automatic potentiometric titrator, anhydrous ethanol, and 1M NaOH.

[0134] 2. The microspheres obtained in Examples 1-12 and Comparative Example 1 were subjected to immunochemiluminescence assays. The specific operating steps are as follows:

[0135] 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:

[0136] (1) Transfer the sample rack to the sample aspiration position and load the reaction vessel to the sample loading position.

[0137] (2) When performing the calibration procedure, dispense 75 μL of calibrator, 20 μL of magnetic microparticle suspension, and 30 μL of sample diluent.

[0138] (3) Mix the reaction solution and incubate it at 37°C for 15 minutes.

[0139] (4) After the incubation is completed, the reaction solution is cleaned and separated using a cleaning solution.

[0140] (5) Complete the dispensing of 50 μL of enzyme conjugate.

[0141] (6) Mix the reaction solution and incubate it at 37°C for 17 minutes.

[0142] (7) After the incubation is completed, the reaction solution is cleaned and separated using a cleaning solution.

[0143] (8) Complete the dispensing of 50 μL of substrate A solution and 50 μL of substrate B solution.

[0144] (9) Mix the reaction solution and test the luminescence intensity.

[0145] 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 HBsAg) (concentrations of approximately 0, 0.05, 0.4, 4, 40, and 250 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).

[0146] Result calculation:

[0147] The instrument automatically calculates the sample test results based on the calibration curve and the luminescence values ​​obtained from the sample test. The test results are shown in Table 1.

[0148] Table 1. Signal value data of enzyme-labeled antibodies validated in the Cyfra211 project.

[0149]

[0150] 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.

[0151] Secondly, by preferably using PEG chains as spacer arms of the first compound or as the second compound itself, the surface of the microspheres can be endowed with excellent hydrophilicity and antifouling ability, forming a more biocompatible "inert" background, which is more conducive to maintaining the activity of immobilized biomolecules.

[0152] Furthermore, the carboxyl groups introduced by the first compound are all located at the ends of the spacer arms and have a consistent spatial orientation, providing a more uniform and ideal immobilization environment for biomolecules, which helps to reduce the masking of active sites and improve detection sensitivity.

[0153] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing microspheres, characterized in that, Includes the following steps: S1: Obtain magnetic microspheres with carboxyl groups; S2: Activate the carboxyl-containing magnetic microspheres in S1 to obtain activated magnetic microspheres; S3: The activated magnetic microspheres obtained in S2 are mixed with the first compound and the second compound, reacted, and the microspheres are collected to obtain the microspheres; The first compound and the second compound competitively react with the carboxyl groups of the activated magnetic microspheres.

2. The preparation method according to claim 1, characterized in that, The first compound has the general structural formula: H2N-R-COOH, where R is a polyethylene glycol chain or an alkylene chain.

3. The preparation method according to claim 2, characterized in that, The general structural formula of the first compound is: H2N-(PEG). x -COOH or H2N-(CH2) y -COOH, where: x is an integer from 2 to 24, and y is an integer from 2 to 24.

4. The preparation method according to any one of claims 1 to 3, characterized in that, The second compound comprises: ethanolamine, tris(hydroxymethyl)aminomethane, 2-aminoethanesulfonic acid, and CH3-O-PEG. n -NH2 (where n is an integer from 2 to 12) or one or more of the following.

5. The preparation method according to any one of claims 1 to 4, characterized in that, The molar ratio of the first compound to the second compound is 1:(0.5~50).

6. The preparation method according to any one of claims 1 to 5, characterized in that, In the magnetic microspheres with carboxyl groups, the carboxyl groups are introduced by copolymerization of carboxyl functional monomers.

7. The preparation method according to claim 6, characterized in that, The carboxyl-containing functional monomers include one or more of the following: acrylic acid, methacrylic acid, mono-2-(methacryloyloxy)ethyl maleate, β-(acryloyloxy)propionic acid, 6-acryloylaminohexanoic acid, mono-2-(methacryloyloxy)ethyl succinate, 3-butenoic acid, 4-pentenoic acid, 5-hexenoic acid, 6-heptenoic acid, 7-octenenoic acid, 8-nonenoic acid, 9-decenoic acid, 10-undecenoic acid, and 11-dodecenoic acid.

8. The preparation method according to any one of claims 1 to 7, characterized in that, The activation is performed using an activator; the activator includes one or more of EDC, NHS, DCC, CDI, HATU, HBTU and HCTU.

9. Microspheres obtained by the preparation method according to any one of claims 1 to 8.

10. The application of the microspheres as described in claim 9 in any of the following: (a) Immunological testing; and / or (b) Preparation of products for immunoassay.

11. A product for testing, characterized in that, include: The microspheres as described in claim 9, and acceptable additives or devices.

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