Carboxyl magnetic beads and their preparation method and application

Abstract

The present invention relates to the field of functional polymer modification, and in particular to carboxyl magnetic beads and their preparation method and application. The present invention provides carboxyl magnetic beads modified with N-(3-aminopropyl) methacrylamide. By reacting N-(3-aminopropyl) methacrylamide with acrylic acid, the carboxyl magnetic beads are modified into chain-extended carboxyl magnetic beads, thereby increasing the steric hindrance between the carboxyl magnetic beads. For the carboxyl magnetic beads modified using the modification method of the present invention, the dispersibility and stability of the magnetic beads after being coated with living materials are improved, thereby improving the quality of in vitro diagnostic kits and improving user experience.

Description

Technical Field

[0001] The present invention relates to the field of functional polymer modification, and in particular to carboxyl magnetic beads and a preparation method and application thereof. Background Art

[0002] Magnetic beads refer to colloidal composite materials that are uniformly dispersed in a certain base liquid. They have properties such as superparamagnetism, a high specific surface area, and modifiable functional groups. Therefore, antigens / antibodies, enzymes, nucleic acids / oligonucleotides, small molecule drugs, etc. can be fixed on their surface, making them widely used in the biomedical field. Based on the modifiability of the surface groups of magnetic beads, some magnetic beads containing amino, carboxyl, tosyl, hydroxyl and other groups on the surface have been widely used in the field of in vitro diagnosis, among which the application of carboxyl magnetic beads is more common. When using carboxyl magnetic beads for antigen or antibody coating, the coated magnetic beads usually have problems such as magnetic carrier aggregation and reduced thermal acceleration signal value. Through comparative studies, it was found that after the carboxyl magnetic beads are activated, the coating process of antigens or antibodies is prone to multi-layer coating and excessive adsorption of active materials themselves. The antigens or antibodies on these surfaces have poor dispersion and stability due to their own hydrophobic adsorption or small steric hindrance between the magnetic beads.

[0003] In existing patents or literature reports, polymer compounds are usually used to modify carboxyl magnetic beads. The synthesis of these polymer compounds usually requires iterative synthesis, which is complex and costly. Summary of the Invention

[0004] In view of this, the present invention provides carboxyl magnetic beads, their preparation methods, and applications. The present invention also provides a method for modifying carboxyl magnetic beads and their applications. The carboxyl magnetic beads modified using the present invention improve the dispersibility and stability of the beads after sample collection.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides application of N-(3-aminopropyl) methacrylamide in modifying carboxyl magnetic beads.

[0007] The present invention also provides modified carboxyl magnetic beads, which are modified with N-(3-aminopropyl) methacrylamide.

[0008] The present invention also provides a method for preparing the modified carboxyl magnetic beads: taking carboxyl magnetic beads, coupling them with an N-(3-aminopropyl) methacrylamide solution in the presence of a coupling agent, and polymerizing the coupling product with a cross-linking agent, acrylic acid and an initiator in the presence of nitrogen to obtain the modified carboxyl magnetic beads.

[0009] In some specific embodiments of the present invention, the carboxyl content of the carboxyl magnetic beads is 30 to 63 μmol / g; and / or

[0010] The particle size of the carboxyl magnetic beads ranges from 0.98 to 1.45 μm.

[0011] In some specific embodiments of the present invention, the concentration of the N-(3-aminopropyl)methacrylamide solution is 2.5 to 10 mg / mL; and / or

[0012] The coupling agent includes an EDC solution; the concentration of the EDC solution includes 10 to 30 mg / mL; and / or

[0013] The coupling time of the coupling comprises 1 to 3 hours.

[0014] In some specific embodiments of the present invention, the concentration of acrylic acid is 6.2 to 24.8 mg / mL; and / or

[0015] The cross-linking agent includes N,N-methylenebisacrylamide; the concentration of the N,N-methylenebisacrylamide is 1 to 5 mg / mL; and / or

[0016] The initiator includes ammonium persulfate or azobisisobutyronitrile.

[0017] In some specific embodiments of the present invention, the polymerization temperature is 40 to 60° C.; and / or

[0018] The polymerization time ranges from 8 to 12 hours.

[0019] In some specific embodiments of the present invention, the preparation method specifically comprises:

[0020] After washing the carboxyl magnetic beads, the supernatant is removed, and an EDC solution is added. After oscillation for 1 to 3 hours, the supernatant is removed by magnetic suction, and an N-(3-aminopropyl) methacrylamide solution is added and continued to oscillate and couple to obtain N-(3-aminopropyl) methacrylamide-modified carboxyl magnetic beads. The purpose of this step is to pre-modify the surface of the carboxyl magnetic beads with N-(3-aminopropyl) methacrylamide, preparing for the next step of further attaching acrylic acid to the surface of the carboxyl magnetic beads using free radical polymerization. This is also one of the key steps in the present invention to obtain carboxyl magnetic beads with greater steric hindrance by chain extension of ordinary carboxyl magnetic beads.

[0021] The preparation method further comprises:

[0022] The resulting N-(3-aminopropyl)methacrylamide-modified carboxyl magnetic beads were magnetically aspirated and the supernatant removed. N,N-methylenebisacrylamide, acrylic acid, and an initiator were added, and polymerization was carried out under a nitrogen atmosphere at 40-60°C for 8-12 hours to obtain modified chain-extended carboxyl magnetic beads. The purpose of this step is to use an initiator and a crosslinker to connect acrylic acid and the N-(3-aminopropyl)methacrylamide-modified carboxyl magnetic beads obtained in the first step (N-(3-aminopropyl)methacrylamide is now extended on the surface of the beads). N-(3-aminopropyl)methacrylamide and acrylic acid are further connected by a crosslinker to obtain the final modified carboxyl magnetic beads. After two-step modification, the surface carboxyl groups of the modified carboxyl magnetic beads have a certain degree of steric hindrance. This is the main difference between the modified carboxyl magnetic beads and the unmodified carboxyl magnetic beads, and it is also one of the key steps in modifying ordinary carboxyl magnetic beads into chain-extended carboxyl magnetic beads with greater steric hindrance.

[0023] The present invention also provides a reagent comprising any of the following items and acceptable adjuvants:

[0024] (I), the modified carboxyl magnetic beads; and / or

[0025] (II) Modified carboxyl magnetic beads prepared by the preparation method.

[0026] The present invention also provides the use of any of the following in the preparation of an in vitro diagnostic test kit:

[0027] (I), the modified carboxyl magnetic beads; and / or

[0028] (II), modified carboxyl magnetic beads obtained by the preparation method; and / or

[0029] (III) the reagent.

[0030] The present invention also provides a kit comprising any of the following items, and an acceptable adjuvant or carrier:

[0031] (I), the modified carboxyl magnetic beads; and / or

[0032] (II), modified carboxyl magnetic beads obtained by the preparation method; and / or

[0033] (III) the reagent.

[0034] The present invention has achieved the following beneficial effects including but not limited to:

[0035] The use of the carboxyl magnetic beads modified with N-(3-aminopropyl) methacrylamide and acrylic acid can improve the dispersibility and stability of the magnetic beads after coating with antigens / antibodies, thereby improving the quality of in vitro diagnostic kits and enhancing the customer's usage experience. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for describing the embodiments or the prior art.

[0037] Figure 1 Schematic diagram showing N-(3-aminopropyl) methacrylamide, cross-linker (N,N-methylenebisacrylamide), acrylic acid, and carboxyl magnetic beads;

[0038] Figure 2 Schematic diagram showing carboxyl magnetic beads modified with N-(3-aminopropyl) methacrylamide;

[0039] Figure 3 Schematic diagram showing the modification of magnetic beads by free radical polymerization of N-(3-aminopropyl)methacrylamide and acrylic acid using N,N-methylenebisacrylamide as a crosslinker;

[0040] Figure 4 The figure shows the comparison of the dispersibility of Merck carboxyl magnetic beads and the modified carboxyl magnetic beads of the present invention in different projects;

[0041] Figure 5 The figure shows the comparison of the dispersibility of Merck carboxyl magnetic beads, acrylamide + acrylic acid modified carboxyl magnetic beads and the modified carboxyl magnetic beads of the present invention in the PⅢNP project. DETAILED DESCRIPTION

[0042] The present invention discloses a carboxyl magnetic bead and its preparation method and application. Those skilled in the art can refer to the contents of this article and appropriately improve the process parameters to achieve. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art and they are all considered to be included in the present invention. The method and application of the present invention have been described by preferred embodiments, and relevant personnel can obviously change or appropriately change and combine the method and application described herein without departing from the content, spirit and scope of the present invention to realize and apply the technology of the present invention.

[0043] The present invention aims to provide a method for modifying carboxyl magnetic beads. By using free radical polymerization, N-(3-aminopropyl) methacrylamide and acrylic acid are polymerized together via the crosslinker N,N-methylenebisacrylamide. This chemical modification extends the carboxyl groups on the surface of conventional carboxyl magnetic beads, giving them a specific spatial structure. During antigen / antibody coating, the long-chain structure between the modified magnetic beads and the carboxyl groups acts as a "bridge" between the antigen / antibody and the magnetic beads. The carboxyl magnetic beads modified by this method exhibit greater stability during antigen / antibody coating. Furthermore, by improving the steric hindrance between the magnetic beads after antigen / antibody coating, the coated magnetic beads exhibit improved dispersibility.

[0044] The present invention also aims to provide an in vitro diagnostic kit that uses carboxyl magnetic beads modified with N-(3-aminopropyl) methacrylamide and acrylic acid to improve the dispersibility and stability of the magnetic beads after coating with antigens / antibodies, thereby improving the quality of the in vitro diagnostic kit and enhancing the customer's user experience.

[0045] To achieve the above-mentioned purpose, the present invention provides the following technical solution: after washing the carboxyl magnetic beads, the supernatant is removed, an EDC solution is added, and after shaking for a period of time, the supernatant is removed by magnetic attraction, and an N-(3-aminopropyl) methacrylamide solution is added and continued to shake and couple to obtain N-(3-aminopropyl) methacrylamide-modified carboxyl magnetic beads. After magnetic attraction of the obtained N-(3-aminopropyl) methacrylamide-modified carboxyl magnetic beads, the supernatant is removed, and N,N-methylenebisacrylamide, acrylic acid, and an initiator are added, and the beads are shaken and polymerized for a period of time under nitrogen atmosphere and a certain temperature to obtain modified chain-extended carboxyl magnetic beads.

[0046] The positive effects produced by the present invention are as follows:

[0047] The carboxyl magnetic beads modified by the present invention have a certain spatial structure in the carboxyl groups on the surface of the magnetic beads. When coated with antigens / antibodies, the long chain structure between the modified magnetic beads and the carboxyl groups acts as a "bridge" between the antigens / antibodies and the magnetic beads. The carboxyl magnetic beads modified by this method can significantly reduce nonspecific adsorption during the antigen / antibody coating process, increase the chemical connection ratio and improve stability. By improving the steric hindrance between the magnetic beads after antigen / antibody coating, the magnetic beads after coating have better dispersibility. This method can significantly improve the spatial structure of the groups on the surface of the magnetic beads, improving the dispersibility and stability during and after the coating process.

[0048] Unless otherwise specified, the carboxyl magnetic beads provided by the present invention and the raw materials and reagents used in their preparation and application can all be purchased from the market.

[0049] The present invention will be further described below in conjunction with the embodiments:

[0050] Preparation Example 1 Preparation of modified carboxyl magnetic beads of the present invention

[0051] (1) Merck carboxyl magnetic beads (batch number: M8998, carboxyl content: 30 μmol / g, particle size: 0.98 μm, concentration: 100 mg / mL, 30 μL) were taken and washed three times (0.1 mol / L PBS, 300 μL / time). The supernatant was removed and EDC solution (10 mg / mL, 100 μL) was added. The beads were shaken evenly in a rapid mixer and then placed on an oscillator for 1 h.

[0052] (2) After magnetic attraction, the supernatant was removed and N-(3-aminopropyl) methacrylamide (2.5 mg / mL, 60 μL) and 40 μL of buffer (0.1 mol / L PBS buffer, pH 7.4) were added. The mixture was shaken evenly with a rapid mixer and then placed on a shaker for 1 h.

[0053] (3) After magnetic attraction using a magnet, the supernatant was removed, and N,N-methylenebisacrylamide (1 mg / mL, 20 μL), acrylic acid (6.2 mg / mL, 60 μL), ammonium persulfate (10 mg / mL, 20 μL) and 100 μL of buffer (0.1 mol / L PBS buffer, pH 7.4) were added respectively. The mixture was sealed after being filled with nitrogen and continuously shaken at 40°C for 8 h to obtain the modified carboxyl magnetic beads of the present invention.

[0054] Preparation Example 2 Preparation of modified carboxyl magnetic beads of the present invention

[0055] (1) Merck carboxyl magnetic beads (batch number: M9163, carboxyl content: 47 μmol / g, particle size: 1.25 μm, concentration: 100 mg / mL, 30 μL) were taken and washed three times (0.1 mol / L PBS, 300 μL / time). The supernatant was removed and EDC solution (20 mg / mL, 100 μL) was added. The beads were shaken evenly in a rapid mixer and then placed on an oscillator for 1 h.

[0056] (2) After magnetic attraction, the supernatant was removed and N-(3-aminopropyl) methacrylamide (6.5 mg / mL, 60 μL) and 40 μL of buffer (0.1 mol / L PBS buffer, pH 7.4) were added. The mixture was shaken evenly with a rapid mixer and then placed on a shaker for 2 h.

[0057] (3) After magnetic attraction using a magnet, the supernatant was removed, and N,N-methylenebisacrylamide (3 mg / mL, 20 μL), acrylic acid (15.5 mg / mL, 60 μL), ammonium persulfate (10 mg / mL, 20 μL) and 100 μL buffer (0.1 mol / L PBS buffer, pH 7.4) were added respectively. The mixture was sealed after being filled with nitrogen and continuously shaken at 50°C for 10 h to obtain the modified carboxyl magnetic beads of the present invention.

[0058] Preparation Example 3 Preparation of modified carboxyl magnetic beads of the present invention

[0059] (1) Merck carboxyl magnetic beads (batch number: M8727, carboxyl content: 63 μmol / g, particle size: 1.45 μm, concentration: 100 mg / mL, 30 μL) were taken and washed three times (0.1 mol / L PBS, 300 μL / time). The supernatant was removed and EDC solution (30 mg / mL, 100 μL) was added. The beads were shaken evenly in a rapid mixer and then placed on an oscillator for 1 h.

[0060] (2) After magnetic attraction using a magnet, the supernatant was removed, and N-(3-aminopropyl) methacrylamide (10 mg / mL, 60 μL) and 40 μL of buffer (0.1 mol / L PBS buffer, pH 7.4) were added, respectively. The mixture was shaken evenly in a rapid mixer and then placed on an oscillator for 3 h to obtain N-(3-aminopropyl) methacrylamide-modified carboxyl magnetic beads.

[0061] (3) After magnetic attraction using a magnet, the supernatant was removed, and N,N-methylenebisacrylamide (5 mg / mL, 20 μL), acrylic acid (24.8 mg / mL, 60 μL), ammonium persulfate (10 mg / mL, 20 μL) and 100 μL of buffer (0.1 mol / L PBS buffer, pH 7.4) were added to the carboxyl magnetic beads modified with N-(3-aminopropyl)methacrylamide, respectively. The mixture was sealed after being filled with nitrogen and continuously shaken at 60°C for 12 h to obtain the modified carboxyl magnetic beads of the present invention.

[0062] Preparation Example 4 Preparation of acrylamide + acrylic acid modified carboxyl magnetic beads

[0063] (1) Merck carboxyl magnetic beads (batch number: M8727, carboxyl content: 63 μmol / g, particle size: 1.45 μm, concentration: 100 mg / mL, 30 μL) were taken and washed three times (0.1 mol / L PBS, 300 μL / time). The supernatant was removed and EDC solution (30 mg / mL, 100 μL) was added. The beads were shaken evenly in a rapid mixer and then placed on an oscillator for 1 h.

[0064] (2) After magnetic attraction using a magnet, the supernatant was removed, and acrylamide (10 mg / mL, 60 μL) and 40 μL buffer (0.1 mol / L PBS buffer, pH 7.4) were added respectively. The mixture was shaken evenly in a rapid mixer and then placed on an oscillator for 3 h to obtain acrylamide-modified carboxyl magnetic beads.

[0065] (3) After magnetic attraction using a magnet, the supernatant was removed and N,N-methylenebisacrylamide (5 mg / mL, 20 μL), acrylic acid (24.8 mg / mL, 60 μL), ammonium persulfate (10 mg / mL, 20 μL) and 100 μL buffer (0.1 mol / L PBS buffer, pH 7.4) were added to the acrylamide-modified carboxyl magnetic beads. The mixture was sealed after nitrogen filling and continuously shaken at 60°C for 12 h to obtain acrylamide + acrylic acid-modified carboxyl magnetic beads.

[0066] Example 1 Stability and dispersibility of carboxyl magnetic beads coated with Anti-SSA antigen

[0067] Unmodified carboxyl magnetic beads (i.e., Merck carboxyl magnetic beads (batch number: M8998, carboxyl content: 30 μmol / g, particle size: 0.98 μm, concentration: 100 mg / mL, 30 μL)) and the modified carboxyl magnetic beads of the present invention prepared in Preparation Example 1 were taken separately, and the supernatant was removed after magnetic attraction using a magnet, and the supernatant was removed. The mixture was washed 3 times (0.1 mol / L PBS, 300 μL / time) and the supernatant was removed. EDC solution (20 mg / mL, 100 μL) was added and shaken evenly on a rapid mixer and then placed on a shaker for 1 hour; the supernatant was removed after magnetic attraction using a magnet, and Anti-SSA antigen (3 mg / mL, 15 μL) and 85 μL buffer (0.1 mol / L PBS buffer, pH 7.4) were added and shaken evenly on a rapid mixer and then placed on a shaker for 2 hours. The supernatant was removed after continued magnetic attraction using a magnet, and the volume was fixed to 3 mL after blocking 3 times with a sealing solution.

[0068] The anti-SSA project used Merck carboxyl magnetic beads (batch number: M8998) and the modified carboxyl magnetic beads prepared in Preparation Example 1 to coat the beads. The dispersion was compared after magnetic acceleration for 3 days and loading for 30 minutes. Figure 4 As shown, it can be seen that the dispersion of the finished product coated with the carboxyl magnetic beads modified by the method of the present invention is significantly better than that of the conventional unmodified carboxyl magnetic beads. The excellent dispersion is due to the relatively large steric hindrance between the carboxyl magnetic beads modified by the method of the present invention, which makes the magnetic beads more dispersed.

[0069] The Anti-SSA project used Merck carboxyl magnetic beads (Batch No.: M8998) and the modified carboxyl magnetic beads of the present invention prepared in Preparation Example 1 for coating. After being placed in parallel at 4°C and 37°C for 10 days, the stability of the carboxyl magnetic beads coated with the Anti-SSA antigen was detected using an Antu Biotechnology A2000Plus instrument.

[0070] The stability results are shown in Table 1:

[0071] Table 1 Thermal Accelerated Stability of Anti-SSA Magnetic Particles (R10 is thermal acceleration for 10 days)

[0072]

[0073]

[0074] As can be seen from Table 1, the luminescence value of the unmodified carboxyl magnetic beads coated in the Anti-SSA project is compared with the luminescence value of the carboxyl magnetic beads coated with the carboxyl magnetic beads modified by the carboxyl magnetic beads modification method provided by the present invention. The coating titer of the modified carboxyl magnetic beads of the present invention is slightly lower than that of the unmodified carboxyl magnetic beads, but the thermal acceleration stability of the modified carboxyl magnetic beads of the present invention after coating is significantly better than that of the unmodified carboxyl magnetic beads. We believe that the spatial structure of the surface groups of the modified carboxyl magnetic beads of the present invention inhibits part of the adsorption effect, increases the chemical connection rate, and thus improves the thermal acceleration stability.

[0075] Example 2 Stability and dispersibility of carboxyl magnetic beads coated with β2-MG antibody

[0076] Unmodified carboxyl magnetic beads (i.e., Merck carboxyl magnetic beads (batch number: M9163, carboxyl content: 47 μmol / g, particle size: 1.25 μm, concentration: 100 mg / mL, 30 μL)) and the modified carboxyl magnetic beads of the present invention prepared in Preparation Example 2 were taken separately, and the supernatant was removed after magnetic attraction using a magnet, and the supernatant was removed. The mixture was washed 3 times (0.1 mol / L PBS, 300 μL / time) and EDC solution (20 mg / mL, 100 μL) was added and shaken evenly on a rapid mixer and then placed on a shaker for 1 h; the supernatant was removed after magnetic attraction using a magnet, β2-MG antibody (5 mg / mL, 15 μL) and 85 μL of buffer (0.1 mol / L PBS buffer, pH 7.4) were added and shaken evenly on a rapid mixer and then placed on a shaker for 2 h. The supernatant was removed after continued magnetic attraction using a magnet, and the volume was fixed to 3 mL after blocking 3 times with a sealing solution.

[0077] The β2-MG project used Merck carboxyl magnetic beads (batch number: M9163) and the modified carboxyl magnetic beads prepared in Preparation Example 2. The dispersion was compared after magnetic acceleration for 3 days and loading for 30 minutes. Figure 4 As shown, it can be seen that the dispersion of the finished product coated with the carboxyl magnetic beads modified by the method of the present invention is significantly better than that of the conventional unmodified carboxyl magnetic beads. The excellent dispersion is due to the relatively large steric hindrance between the carboxyl magnetic beads modified by the method of the present invention, which makes the magnetic beads more dispersed.

[0078] β2-MG antibody project Merck carboxyl magnetic beads (Batch No.: M9163) and the modified carboxyl magnetic beads of the present invention prepared in Preparation Example 2 were coated and placed in parallel at 4°C and 37°C for 10 days. The stability of the carboxyl magnetic beads after β2-MG antibody coating was detected using an Antu Bio A2000Plus instrument.

[0079] The stability results are shown in Table 2:

[0080] Table 2 Thermal Acceleration Stability of β2-MG Magnetic Particles (R10 is thermal acceleration for 10 days)

[0081]

[0082] As can be seen from Table 2: the luminescence value of the β2-MG project after coating with unmodified carboxyl magnetic beads is compared with the luminescence value of the carboxyl magnetic beads obtained by the carboxyl magnetic bead modification method provided by the present invention. The coating titer of the modified carboxyl magnetic beads of the present invention is slightly lower than the coating titer of the unmodified carboxyl magnetic beads, but the thermal acceleration stability of the modified carboxyl magnetic beads of the present invention after coating is significantly better than that of the unmodified carboxyl magnetic beads. We believe that the spatial structure of the surface group of the modified carboxyl magnetic beads of the present invention inhibits part of the adsorption effect, increases the chemical connection rate, and thus improves the thermal acceleration stability.

[0083] Example 3 Stability and dispersibility of carboxyl magnetic beads coated with PIIINP antigen

[0084] Merck carboxyl magnetic beads (batch number: M8727, carboxyl content: 63 μmol / g, particle size: 1.45 μm, concentration: 100 mg / mL, 30 μL), the modified carboxyl magnetic beads prepared in Preparation Example 3, and the acrylamide + acrylic acid modified carboxyl magnetic beads prepared in Preparation Example 4 were taken respectively. After magnetic attraction, the supernatant was removed. The supernatant was washed 3 times (0.1 mol / L PBS, 300 μL / time) and the supernatant was removed. EDC solution (20 mg / mL, 100 μL) was added and the mixture was shaken evenly on a rapid mixer and then placed on a shaker for 1 hour. After magnetic attraction, the supernatant was removed. PⅢNP antigen (5 mg / mL, 5 μL) and 95 μL buffer (0.1 mol / L PBS buffer, pH 7.4) were added and shaken evenly on a rapid mixer and then placed on a shaker for 2 hours. The supernatant was removed after continued magnetic attraction. The mixture was blocked 3 times with sealing solution and then fixed to 3 mL.

[0085] The PⅢNP project used Merck carboxyl magnetic beads (batch number: M8727), acrylamide + acrylic acid modified carboxyl magnetic beads prepared in Preparation Example 4, and modified carboxyl magnetic beads prepared in Preparation Example 3. The comparison chart after magnetic acceleration for 3 days and loading on the machine for 30 minutes is shown in the figure below. Figure 5 As shown, it can be seen that the N-(3-aminopropyl) methacrylamide used in the modification of the present invention benefits from its relatively extended spatial structure. Ultimately, the dispersibility of the modified carboxyl magnetic beads of the present invention is significantly better than that of the carboxyl magnetic beads modified with acrylamide + acrylic acid and Merck carboxyl magnetic beads (Batch No.: M8727). This is an advantage brought about by the greater steric hindrance between the modified carboxyl magnetic beads of the present invention.

[0086] The PⅢNP project was coated with Merck carboxyl magnetic beads (Batch No.: M8727), acrylamide + acrylic acid modified carboxyl magnetic beads prepared in Preparation Example 4, and the modified carboxyl magnetic beads of the present invention prepared in Preparation Example 3. After being placed in parallel at 4°C and 37°C for 10 days, the stability of the carboxyl magnetic beads after PⅢNP antigen coating was detected using an Antu Bio A2000Plus instrument.

[0087] The stability results are shown in Table 3:

[0088] Table 3 Thermal acceleration stability of PⅢNP magnetic particles (R10 is thermal acceleration for 10 days)

[0089]

[0090] As can be seen from Table 3: the thermal acceleration stability of the carboxyl magnetic beads (batch number: M8727) used in the PIIINP project, the carboxyl magnetic beads modified with acrylamide + acrylic acid prepared in Preparation Example 4, and the carboxyl magnetic beads modified by the present invention prepared in Preparation Example 3 are improved in sequence, among which the thermal acceleration stability of the carboxyl magnetic beads modified with acrylamide + acrylic acid after coating with antigen is still lower than that of the carboxyl magnetic beads modified by the present invention. After the N-(3-aminopropyl) methacrylamide used in the present invention is modified on the surface of the magnetic beads, since its carbon chain length is longer than that of acrylamide, the steric hindrance between the carboxyl groups on the surface of the carboxyl magnetic beads modified by the two is inconsistent. The chemical connection rate of the carboxyl magnetic beads modified by the present invention is higher and therefore more stable.

[0091] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for preparing modified carboxyl magnetic beads, characterized in that: The carboxyl magnetic beads are coupled with the N-(3-aminopropyl) methacrylamide solution in the presence of a coupling agent, and the coupling product is polymerized with a crosslinker, acrylic acid and an initiator in the presence of nitrogen to obtain modified carboxyl magnetic beads.

2. The preparation method according to claim 1, wherein The carboxyl content of the carboxyl magnetic beads is 30-63 µmol / g; and / or The particle size of the carboxyl magnetic beads is 0.98~1.45μm.

3. The preparation method according to claim 1, wherein The concentration of the N-(3-aminopropyl)methacrylamide solution is 2.5-10 mg / mL; and / or The coupling agent is an EDC solution; the concentration of the EDC solution is 10-30 mg / mL; and / or The coupling time of the coupling is 1 to 3 hours.

4. The preparation method according to claim 3, wherein The concentration of the acrylic acid is 6.2 to 24.8 mg / mL; and / or The cross-linking agent includes N,N-methylenebisacrylamide; the concentration of the N,N-methylenebisacrylamide is 1 to 5 mg / mL; and / or The initiator is ammonium persulfate or azobisisobutyronitrile.

5. The preparation method according to claim 1, wherein The polymerization temperature is 40-60°C; and / or The polymerization time is 8 to 12 hours.

6. Reagent, characterized in that The method comprises modified carboxyl magnetic beads prepared by the preparation method according to any one of claims 1 to 5 and an acceptable auxiliary agent.

7. Use of any of the following in the preparation of in vitro diagnostic test kits: (I) Modified carboxyl magnetic beads prepared by the preparation method according to any one of claims 1 to 5; or (II) The reagent according to claim 6.

8. A kit, characterized in that It includes any of the following, and acceptable adjuvants or carriers: (I) Modified carboxyl magnetic beads prepared by the preparation method according to any one of claims 1 to 5; or (II) The reagent according to claim 6.

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