HLB / WAX mixed magnetic bead and preparation method thereof

Abstract

The invention provides an HLB / WAX mixed magnetic bead and a preparation method thereof, and particularly relates to the technical field of solid-phase extraction magnetic beads. The HLB / WAX mixed magnetic bead comprises a magnetic core, a silicon dioxide coating layer, a polymer coating layer and an amino modification layer, the silicon dioxide coating layer comprises a first coating layer and a second coating layer. According to the HLB / WAX mixed magnetic bead, by combining two characteristics of HLB and WAX, the potential application field of the HLB / WAX mixed magnetic bead is remarkably broadened, the magnetic responsiveness of the magnetic bead is enhanced, the magnetic bead is more efficient in the separation and extraction process, additional functional groups are introduced through the amino modification layer, the selective adsorption capacity of ionic compounds with different polarities is improved, and the magnetic bead has a good application prospect. The HLB / WAX mixed magnetic beads have higher extraction efficiency and selectivity in the fields of drug monitoring, environmental analysis, biological sample treatment and the like, and a more efficient tool is provided for research and application in the related fields.

Description

Technical Field

[0001] The present invention relates to the technical field of solid-phase extraction magnetic beads, and particularly to an HLB / WAX hybrid magnetic bead and a preparation method thereof. Background Art

[0002] Liquid chromatography-tandem mass spectrometry (LC-MS / MS) is a powerful tool for analyzing small molecules, especially suitable for the clinical monitoring of drugs and their metabolites. In the analysis of biological samples, sample pretreatment before LC-MS / MS is a key step, and solid-phase extraction (SPE) is one of the commonly used pretreatment methods. Solid-phase extraction involves passing a sample through an extraction column filled with a solid adsorbent. Specific components in the sample are retained on the solid phase, and then appropriate solvents are used to elute impurities first and then the target substance, thereby achieving the purification and concentration of the target substance. However, the SPE process has many steps and is mostly manual operation, resulting in large deviations in parameters and affecting repeatability. In addition, the use of a large amount of elution solvents also makes the cost of SPE relatively high.

[0003] To overcome the limitations of traditional SPE, solid-phase extraction magnetic beads (SPE magnetic beads) have been developed. SPE magnetic beads consist of superparamagnetic nanomagnetic cores and functionalized polymer shells. They specifically bind to target substances through binding sites on the polymer shells and are enriched in a magnetic field. After removing the magnetic field, the extracted substances are released to achieve the purification and concentration of the target substance. The magnetic responsiveness of the magnetic beads makes them have the potential for high automation. Combined with LC-MS / MS, it can be developed into a fully automatic analyzer for pretreatment and detection, thereby improving the accuracy and efficiency of analysis.

[0004] According to functionality, SPE magnetic beads can be divided into hydrophilic-lipophilic balance type (HLB) and ion exchange type, etc. HLB magnetic beads are usually copolymerized from styrene-based monomers and hydrophilic monomers. This structure endows them with the selective adsorption ability for different polar non-ionic compounds, making them play an important role in many fields such as drug monitoring, environmental analysis, and food safety detection. However, since HLB magnetic beads do not carry charges, their ability to extract ionic compounds is limited, which restricts their application scope in some specific analysis fields.

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

[0006] One of the purposes of the present invention is to provide an HLB / WAX hybrid magnetic bead, aiming to solve at least one of the above technical problems in the prior art.

[0007] Another purpose of the present invention is to provide a preparation method of the HLB / WAX hybrid magnetic bead.

[0008] To solve the above technical problems, the present invention specifically adopts the following technical solutions:

[0009] The first aspect of the present invention provides an HLB / WAX hybrid magnetic bead, which includes a magnetic core, a silica coating layer, a polymer coating layer, and an amine-modified layer;

[0010] The silica coating layer includes a first coating layer and a second coating layer.

[0011] Further, the amine-modified layer includes amine functional groups.

[0012] Based on the mass of the HLB / WAX hybrid magnetic bead, the density of the amine functional groups is 0.01 - 3 mmol / g.

[0013] Further, the polymer in the polymer coating layer contains reactive functional groups.

[0014] Preferably, the reactive functional groups include at least one of epoxy group, carboxyl group, carbonyl group, and halogenated hydrocarbon group.

[0015] Further, the material of the magnetic core is magnetite.

[0016] Preferably, the particle size of the magnetite is 100 - 700 nm.

[0017] The second aspect of the present invention provides a method for preparing the HLB / WAX hybrid magnetic bead, including the following steps:

[0018] A. In an ethanol aqueous solution of magnetite, add a first silane coupling agent to form a first coating layer, and then add a second silane coupling agent to form a second coating layer, obtaining Fe 3 O 4 @SiO 2 ;

[0019] B. After making the Fe 3 O 4 @SiO 2 into a suspension, add a monomer solution and an initiator to obtain a mixture; add the mixture to a stabilizer solution for a first reaction to obtain an HLB magnetic bead;

[0020] C. Add the HLB magnetic bead to an amine compound solution for a second reaction to obtain an HLB / WAX hybrid magnetic bead.

[0021] Further, the structural formula of the amine compound in the amine compound solution is R 1 -NH-R 2 .

[0022] Wherein, R 1 is a substituted or unsubstituted hydrocarbon group, R2 is a hydrogen atom, a substituted or unsubstituted hydrocarbon group.

[0023] Further, the hydrocarbon group is a C1-C10 hydrocarbon group.

[0024] Preferably, the hydrocarbon group includes an alkyl group, an alkenyl group or an alkynyl group.

[0025] Preferably, the substituted hydrocarbon group includes at least one of a halogenated hydrocarbon group, a hydroxy hydrocarbon group, a carboxylic acid hydrocarbon group, an ester group hydrocarbon group, a keto hydrocarbon group, an ether group hydrocarbon group, a nitrile group hydrocarbon group, a sulfonic acid hydrocarbon group and a phosphoric acid hydrocarbon group.

[0026] Further, the first silane coupling agent includes at least one of acetyltrimethoxysilane, tetraethoxysilane, γ-aminopropyltriethoxysilane, 3-glycidoxypropyltrimethoxysilane, 3-aminopropyltriethoxysilane.

[0027] Preferably, the mass of the first silane coupling agent is 0.5-2.5 times the mass of iron trioxide.

[0028] Preferably, the temperature for forming the first coating layer is 20-40°C and the time is 4-6 h.

[0029] Preferably, the second silane coupling agent includes at least one of 3-(trimethoxysilyl)propyl methacrylate, isobutyl(trimethoxy)silane, octyltrimethoxysilane, octadecyltrimethoxysilane.

[0030] Preferably, the mass of the second silane coupling agent is 1.5-4 times the mass of iron trioxide.

[0031] Preferably, the temperature for forming the second coating layer is 70-90°C and the time is 8-10 h.

[0032] Further, in step B, the mass concentration of the suspension is 0.5-1.5%.

[0033] Preferably, the dosage of the initiator is 2-5% of the mass of the monomer.

[0034] Preferably, the concentration of the stabilizer solution is 2-4%.

[0035] Preferably, the stabilizer in the stabilizer solution includes at least one of sodium dodecyl sulfate, sodium dodecylbenzenesulfonate, gelatin, carboxymethyl cellulose, chitosan, polyvinyl alcohol, polyvinylpyrrolidone, alumina, magnesium hydroxide.

[0036] Preferably, the time of the first reaction is 6-10 h.

[0037] Further, in step C, the dosage of the amine compound is 0.5 to 10 times the mass of the HLB magnetic beads.

[0038] Preferably, in the reaction system for the second reaction, the concentration of the HLB magnetic beads is 3 to 6 wt%.

[0039] Preferably, the temperature of the second reaction is 70 to 90 °C, and the time is 16 to 48 h.

[0040] Compared with the prior art, the present invention has at least the following beneficial effects:

[0041] The HLB / WAX hybrid magnetic beads provided by the present invention significantly broaden their potential application fields by combining two characteristics of HLB (hydrophilic-lipophilic balance) and weak anion exchange (WAX). The structure of the magnetic beads includes a magnetic core, a silica coating layer, a polymer coating layer, and an amine-modified layer, wherein the silica coating layer is composed of a first coating layer and a second coating layer. This design not only enhances the magnetic responsiveness of the magnetic beads, making them more efficient in the separation and extraction processes, but also introduces additional functional functional groups through the amine-modified layer, improving the selective adsorption ability for different polar and ionic compounds.

[0042] The preparation method provided by the present invention first enhances the stability and chemical resistance of the magnetic beads by preparing a silica coating layer (Fe 3 O 4 @SiO 2 ) with a double-layer structure, and provides a good foundation for subsequent functionalization. Then, a polymer is coated on the surface of Fe 3 O 4 @SiO 2 to obtain HLB magnetic beads, ensuring that the surface of the magnetic beads has good hydrophilicity and lipophilicity and can effectively and selectively adsorb target substances with different polarities. Finally, by performing a second reaction on the HLB magnetic beads with an amine compound solution, the weak anion exchange property is introduced, enabling the magnetic beads to electrostatically adsorb ionic compounds with opposite charges, thereby broadening their application potential in the extraction and separation of ionic compounds. This preparation method ensures that the specific surface area and magnetic responsiveness of the magnetic beads reach the optimal state by precisely controlling the reaction conditions and material composition, making the HLB / WAX hybrid magnetic beads have higher extraction efficiency and selectivity in the fields of drug monitoring, environmental analysis, and biological sample processing, providing a more efficient tool for research and application in related fields. Description of the Drawings

[0043] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0044] Figure 1 The hysteresis loop obtained in Test Example 1;

[0045] Figure 2 The hysteresis loop obtained in Test Example 1;

[0046] Figure 3 The infrared spectrum of the HLB magnetic beads. Specific embodiments

[0047] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. Usually, the components of the embodiments of the present invention described and illustrated here can be arranged and designed in various different configurations.

[0048] The first aspect of the present invention provides an HLB / WAX hybrid magnetic bead, comprising a magnetic core, a silica coating layer, a polymer coating layer, and an amine-modified layer;

[0049] The silica coating layer includes a first coating layer and a second coating layer.

[0050] The HLB / WAX hybrid magnetic bead provided by the present invention significantly broadens its potential application fields by combining two characteristics of HLB (hydrophilic-lipophilic balance) and weak anion exchange (WAX). The structure of the magnetic bead includes a magnetic core, a silica coating layer, a polymer coating layer, and an amine-modified layer, wherein the silica coating layer consists of a first coating layer and a second coating layer. This design not only enhances the magnetic responsiveness of the magnetic bead, making it more efficient in the separation and extraction process, but also introduces additional functional functional groups through the amine-modified layer, improving the selective adsorption ability for different polar and ionic compounds.

[0051] Further, the amine-modified layer includes amine functional groups.

[0052] Based on the mass of the HLB / WAX hybrid magnetic beads, the density of the amino functional groups is 0.01 - 3 mmol / g. Within this density range, the adsorption efficiency of the magnetic beads with the target biomolecules is enhanced, while good dispersibility and resuspension are ensured, and agglomeration is avoided, thus showing excellent performance in the extraction, purification, and analysis of biomolecules, improving the experimental efficiency and the reliability of the results.

[0053] Typically but not restrictively, the density of the amino functional groups can be, for example, 0.01 mmol / g, 0.1 mmol / g, 0.2 mmol / g, 0.3 mmol / g, 0.5 mmol / g, 1 mmol / g, 2 mmol / g, or 3 mmol / g, or can also be any value within the range of 0.01 mmol / g to 3 mmol / g.

[0054] Furthermore, the polymer in the polymer coating layer contains reactive functional groups.

[0055] Preferably, the reactive functional groups include at least one of epoxy groups, carboxyl groups, carbonyl groups, and halogenated hydrocarbon groups. These reactive functional groups can react with amine compounds to make the magnetic beads carry amino functional groups.

[0056] Furthermore, the material of the magnetic core is magnetite.

[0057] Preferably, the particle size of the magnetite is 100 - 700 nm.

[0058] The second aspect of the present invention provides a preparation method of the HLB / WAX hybrid magnetic beads as described above, including the following steps:

[0059] A. In an aqueous ethanol solution of magnetite, add a first silane coupling agent to form a first coating layer, and then add a second silane coupling agent to form a second coating layer to obtain Fe 3 O 4 @SiO 2 ;

[0060] B. After making the Fe 3 O 4 @SiO 2 into a suspension, add a monomer solution and an initiator to obtain a mixed solution; add the mixed solution into a stabilizer solution for a first reaction to obtain HLB magnetic beads;

[0061] C. Add the HLB magnetic beads into an amine compound solution for a second reaction to obtain HLB / WAX hybrid magnetic beads.

[0062] The preparation method provided by the present invention, first, by preparing a silica coating layer with a double-layer structure (Fe 3 O 4 @SiO2 ), enhancing the stability and chemical resistance of the magnetic beads and providing a good foundation for subsequent functionalization. Then, on the surface of Fe 3 O 4 @SiO 2 a polymer was coated to obtain HLB magnetic beads, ensuring that the surface of the magnetic beads has good hydrophilicity and lipophilicity, and can effectively and selectively adsorb target substances with different polarities. Finally, by carrying out a second reaction between the HLB magnetic beads and an amine compound solution, weak anion exchange properties were introduced, enabling the magnetic beads to electrostatically adsorb ionic compounds with opposite charges, thereby broadening their application potential in the extraction and separation of ionic compounds. This preparation method ensures that the specific surface area and magnetic responsiveness of the magnetic beads reach the optimal state by precisely controlling the reaction conditions and material composition, making the HLB / WAX hybrid magnetic beads have higher extraction efficiency and selectivity in the fields of drug monitoring, environmental analysis, and biological sample processing, providing a more efficient tool for research and applications in related fields.

[0063] Furthermore, the structural formula of the amine compound in the amine compound solution is R 1 -NH-R 2 .

[0064] Among them, R 1 is a substituted or unsubstituted hydrocarbon group, and R 2 is a hydrogen atom, a substituted or unsubstituted hydrocarbon group.

[0065] It should be noted that the amine compound is a primary amine compound or a secondary amine compound.

[0066] In amine compounds, due to different steric hindrance effects, their abilities to participate in reactions also vary. Tertiary amine compounds usually have difficulty participating in the modification reaction of the magnetic bead polymer shell due to their large steric hindrance effect. In contrast, primary amine compounds have a smaller steric hindrance effect, which enables them to more effectively penetrate and enter the polymer shell of the magnetic beads and thus participate in the modification reaction. Therefore, when using primary amine compounds to modify magnetic beads, a higher amine group density can be formed on the surface of the magnetic beads. This high-density amine functional group provides more active sites for the magnetic beads, enhancing their functionality and efficiency in various applications. Especially compared with magnetic beads modified with secondary amine compounds, the latter have a relatively low amine group density due to their large steric hindrance effect, which affects their participation and effect in the reaction.

[0067] Furthermore, the hydrocarbon group is a C1-C10 hydrocarbon group.

[0068] The C1 to C10 hydrocarbon group includes starting from methyl (-CH 3 ), all the way to decyl (-C 10 H 21) A series of alkyl groups, which consist of 1 to 10 carbon atoms. As the number of carbon atoms increases, the number of branched isomers of these hydrocarbon groups also gradually increases. For example, ethyl (-C 2 H 5 ), propyl (-C 3 H 7 ), and its isopropyl (-CH(CH 3 )) 2 ), butyl (-C 4 H 9 ), and its isobutyl (-CH 2 CH(CH 3 )) 2 ), tert-butyl (-C(CH 3 )) 3 ), etc. As the number of carbon atoms increases, such as pentyl (-C 5 H 11 ), hexyl (-C 6 H 13 ), heptyl (-C 7 H 15 ), octyl (-C 8 H 17 ), nonyl (-C 9 H 19 ), and decyl (-C 10 H 21 ), their structures become more complex, including more branched isomers, such as isopentyl (-CH 2 CH 2 CH(CH 3 )) 2 ), neopentyl (-CH(CH 3 ))CH 2 CH 3 ), tert-pentyl (-C(CH 3 )) 3 ), isopentyl (-CH 2 CH 2 CH 2 CH(CH 3 )) 2 ), tert-hexyl (-CH 2 CH(CH 3 ))CH 2 CH 2 CH 3 ), etc.

[0069] Preferably, the hydrocarbon group includes an alkyl group, an alkenyl group, or an alkynyl group.

[0070] Preferably, the substituted hydrocarbon group includes at least one of a halogenated hydrocarbon group, a hydroxy hydrocarbon group, a carboxylic acid hydrocarbon group, an ester group hydrocarbon group, a ketone group hydrocarbon group, an ether group hydrocarbon group, a nitrile group hydrocarbon group, a sulfonic acid hydrocarbon group, and a phosphoric acid hydrocarbon group.

[0071] Further, the first silane coupling agent includes at least one of acetyltrimethoxysilane, tetraethoxysilane, γ-aminopropyltriethoxysilane, 3-glycidoxypropyltrimethoxysilane, and 3-aminopropyltriethoxysilane.

[0072] The first coating layer formed by the first silane coupling agent has a dense structure and can well coat the magnetic beads, reducing the direct contact between the magnetic core and the external environment, thereby avoiding oxidation or wear of the magnetic core under chemical reactions or physical actions and ensuring the stable magnetic properties of the magnetic beads.

[0073] Preferably, the mass of the first silane coupling agent is 0.5 to 2.5 times the mass of the iron oxide.

[0074] Typically but not restrictively, the mass of the first silane coupling agent can be 0.5 times, 1 time, 1.5 times, 2 times the mass of the iron oxide, or any value within the range of 0.5 to 2.5 times.

[0075] Preferably, the temperature for forming the first coating layer is 20 to 40 °C, and the time is 4 to 6 h.

[0076] Typically but not restrictively, the temperature for forming the first coating layer can be 20 °C, 25 °C, 30 °C, 35 °C, or 40 °C, or any value within the range of 20 °C to 40 °C; the time for forming the first coating layer can be 4 hours, 4.5 hours, 5 hours, 5.5 hours, or 6 hours, or any value within the range of 4 hours to 6 hours.

[0077] Preferably, the second silane coupling agent includes at least one of 3-(trimethoxysilyl)propyl methacrylate, isobutyl(trimethoxy)silane, octyltrimethoxysilane, and octadecyltrimethoxysilane. The main function of the second coating layer formed by the second silane coupling agent is to perform surface modification so that the magnetic beads can better bind to the polymer.

[0078] Preferably, the mass of the second silane coupling agent is 1.5 to 4 times the mass of the iron oxide.

[0079] Typically but not restrictively, the mass of the second silane coupling agent can be 1.5 times, 2 times, 2.5 times, 3 times, 3.5 times, or 4 times the mass of the iron oxide, or any value within the range of 1.5 to 4 times.

[0080] Preferably, the temperature for forming the second coating layer is 70 to 90 °C, and the time is 8 to 10 h.

[0081] Typically but not restrictively, the temperature for forming the second coating layer can be 70°C, 75°C, 80°C, 85°C or 90°C, or any value within the range of 70°C to 90°C; the time for forming the second coating layer can be 8 hours, 9 hours or 10 hours, or any value within the range of 8 hours to 10 hours.

[0082] Further, in step B, the mass concentration of the suspension is 0.5 - 1.5%.

[0083] Typically but not restrictively, the mass concentration of the suspension can be 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4% or 1.5%, or any value within the range of 0.5% to 1.5%.

[0084] Preferably, the dosage of the initiator is 2 - 5% of the mass of the monomer.

[0085] Typically but not restrictively, the dosage of the initiator can be 2%, 2.5%, 3%, 3.5%, 4%, 4.5% or 5% of the mass of the monomer, or any value within the range of 2% to 5%.

[0086] Preferably, the concentration of the stabilizer solution is 2 - 4%.

[0087] Typically but not restrictively, the concentration of the stabilizer solution can be 2%, 2.5%, 3%, 3.5% or 4%, or any value within the range of 2% to 4%.

[0088] Preferably, the stabilizer in the stabilizer solution includes at least one of sodium dodecyl sulfate, sodium dodecylbenzenesulfonate, gelatin, carboxymethyl cellulose, chitosan, polyvinyl alcohol, polyvinylpyrrolidone, alumina, magnesium hydroxide. The main function of these stabilizers is to provide a stable reaction environment. When the magnetic core suspension containing monomers is mixed in the stabilizer solution to form droplets, the stabilizer forms a physical barrier outside the droplets, preventing the adhesion or fusion between the droplets, thereby ensuring the sphericity and particle size of the magnetic beads and maintaining the integrity and functionality of the magnetic beads. Through this first reaction, the stabilizer helps to form magnetic beads with hydrophilic-lipophilic balance characteristics, providing a basis for subsequent surface modification and functionalization. Preferably, the time of the first reaction is 6 - 10h.

[0089] Typically but not restrictively, the time of the first reaction can be 6 hours, 7 hours, 8 hours, 9 hours or 10 hours, or any value within the range of 6 hours to 10 hours.

[0090] Further, in step C, the dosage of the amine compound is 0.5 - 10 times the mass of the HLB magnetic beads.

[0091] Typically but not limited thereto, the dosage of the amine compound can be 0.5 times, 1 time, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times or 10 times the mass of the HLB magnetic beads, or can be any value within the range of 0.5 to 10 times.

[0092] Preferably, in the reaction system for the second reaction, the concentration of the HLB magnetic beads is 3-6 wt%.

[0093] Typically but not limited thereto, the concentration of the HLB magnetic beads can be 3 wt%, 4 wt%, 5 wt% or 6 wt%, or can be any value within the range of 3 wt% to 6 wt%.

[0094] Preferably, the temperature of the second reaction is 70-90 °C and the time is 16-48 h.

[0095] Typically but not limited thereto, the temperature of the second reaction can be 70 °C, 75 °C, 80 °C, 85 °C or 90 °C, and the time can be 16 hours, 20 hours, 24 hours, 32 hours, 40 hours or 48 hours, or can be any value within the range of 16 hours to 48 hours.

[0096] The dispersion solvents used in the present invention can all be replaced with toluene, cyclohexane, dichloroethane, dimethyl sulfoxide, tetrahydrofuran, N,N-dimethylformamide, acetonitrile, isopropanol, ethanol, methanol or water.

[0097] The following will, in conjunction with examples, elaborate on some embodiments of the present invention. Without conflict, the features in the following examples and the examples can be combined with each other. The raw materials used in the present invention can be obtained through commercial purchase without special instructions.

[0098] The magnetite used in the following examples and comparative examples is a nanoscale magnetite powder synthesized by the solvothermal method.

[0099] Example 1

[0100] This example provides an HLB / WAX hybrid magnetic bead, and the preparation process is as follows:

[0101] 1. Take the nanoscale magnetite powder and ultrasonically disperse it in an 80% ethanol aqueous solution, then add tetraethyl orthosilicate (TEOS) which is 2 times the addition amount of magnetite. After stirring and reacting at room temperature for 5 h, continue to add 3-(trimethoxysilyl)propyl methacrylate (MEMO) which is 3 times the addition amount of magnetite. Raise the temperature to 80 °C and stir and react at this temperature for 8 h, then discharge the material. Wash the product with absolute ethanol to obtain a silica-modified magnetic core (Fe 3 O 4 @SiO 2)。

[0102] 2. HLB Magnetic Bead Synthesis

[0103] Take Fe 3 O 4 @SiO 2 After dispersing it in dichloroethane to make a 1.25% magnetic core suspension, add a monomer solution with vinylbenzyl chloride (VBC): divinylbenzene (DVB): glycidyl methacrylate (GMA) at a ratio of 2:1:1 to the magnetic core suspension, and the amount of initiator AIBN is 3.3% of the monomer addition amount. During stirring, add the magnetic core suspension to an aqueous PVA solution (concentration 2.25 wt%), discharge the product after reacting for 8 h, and obtain HLB magnetic beads after washing alternately with absolute ethanol and water.

[0104] 3. Disperse the HLB magnetic beads in an ethylenediamine / ethanol solution to make a 4.2% magnetic bead suspension, and the amount of ethylenediamine is 2 times the addition amount of HLB magnetic beads. Then raise the temperature to 80 °C and stir the magnetic bead suspension at this temperature for 24 h before discharging, and obtain HLB / WAX hybrid magnetic beads after washing the product with absolute ethanol.

[0105] Example 2

[0106] This example provides an HLB / WAX hybrid magnetic bead, and the preparation process is as follows:

[0107] 1. The same step as in Example 1.

[0108] 2. The same step as in Example 1.

[0109] 3. Disperse the HLB magnetic beads in an ethanolamine / ethanol solution to make a 4.2% magnetic bead suspension, and the amount of ethanolamine is 4 times the addition amount of HLB magnetic beads. Then raise the temperature to 80 °C and stir the magnetic bead suspension at this temperature for 24 h before discharging, and obtain HLB / WAX hybrid magnetic beads after washing the product with absolute ethanol.

[0110] Example 3

[0111] This example provides an HLB / WAX hybrid magnetic bead, and the preparation process is as follows:

[0112] 1. The same step as in Example 1.

[0113] 2. The same step as in Example 1.

[0114] 3. Disperse the HLB magnetic beads in an N-methylethanolamine / ethanol solution to make a 4.2% magnetic bead suspension, and the amount of N-methylethanolamine is 5 times the addition amount of HLB magnetic beads. Then raise the temperature to 80 °C and stir the magnetic bead suspension at this temperature for 24 h before discharging, and obtain HLB / WAX hybrid magnetic beads after washing the product with absolute ethanol.

[0115] Example 4

[0116] This example provides an HLB / WAX hybrid magnetic bead, and the preparation process is as follows:

[0117] 1. The same step as in Example 1.

[0118] 2. The same step as in Example 1.

[0119] 3. Different from this step in Example 1, the dosage of N-methylethanolamine is 0.5 times the addition amount of HLB magnetic beads. The remaining steps are the same as those in Example 1 and will not be elaborated here.

[0120] Example 5

[0121] This example provides an HLB / WAX hybrid magnetic bead, and the preparation process is as follows:

[0122] 1. The same step as in Example 1.

[0123] 2. The same step as in Example 1.

[0124] 3. Different from this step in Example 1, the dosage of N-methylethanolamine is 10 times the addition amount of HLB magnetic beads. The remaining steps are the same as those in Example 1 and will not be elaborated here.

[0125] Example 6

[0126] This example provides an HLB / WAX hybrid magnetic bead, and the preparation process is as follows:

[0127] 1. The same step as in Example 1.

[0128] 2. Take Fe 3 O 4 @SiO 2 After dispersing in dichloroethane to make a 1.25% magnetic core suspension, a monomer solution of styrene (ST):DVB:GMA = 2:1:1 is added to the magnetic core suspension, and the dosage of initiator AIBN is 3.3% of the monomer addition amount. During stirring, the magnetic core suspension is added to an aqueous PVA solution (concentration 2.25 wt%), and after reacting for 8 h, the product is discharged. After washing alternately with absolute ethanol and water, HLB magnetic beads are obtained.

[0129] 3. The same step as in Example 1.

[0130] Example 7

[0131] This example provides an HLB / WAX hybrid magnetic bead, and the preparation process is as follows:

[0132] 1. The same step as in Example 1.

[0133] 2. Take Fe 3 O 4 @SiO 2 After dispersing it in dichloroethane to make a 1.25% magnetic core suspension, a monomer solution with ST:DVB of 1:1 was added to the magnetic core suspension, and the amount of initiator AIBN was 3.3% of the monomer addition amount. During stirring, the magnetic core suspension was added to an aqueous PVA solution (concentration 2.25 wt%), and after reacting for 8 h, the product was discharged. After washing alternately with absolute ethanol and water, HLB magnetic beads were obtained.

[0134] 3. The same steps as in Example 1.

[0135] Comparative Example 1

[0136] This comparative example provides an HLB / WAX hybrid magnetic bead, and the preparation process is as follows:

[0137] 1. The same steps as in Example 1.

[0138] 2. The same steps as in Example 1.

[0139] 3. The HLB magnetic beads were dispersed in a triethylamine / ethanol solution to make a 4.2% magnetic bead suspension, and the amount of triethylamine was 4 times the addition amount of product B. Subsequently, the temperature was raised to 80 °C and the magnetic bead suspension was stirred at this temperature for 24 h and then discharged. After washing the product with absolute ethanol, HLB / WAX hybrid magnetic beads were obtained.

[0140] Test Example 1

[0141] The magnetic properties of the HLB / WAX hybrid magnetic beads in the examples and comparative examples were characterized, and a vibrating sample magnetometer was used to characterize the saturation magnetization intensity of the magnetic beads. The obtained saturation magnetization intensities are shown in Table 1 below.

[0142] Table 1

[0143]

[0144]

[0145] The hysteresis loops obtained in Examples 1-7 and Comparative Example 1 are as Figure 1 and Figure 2 shown. From Figure 1 and Figure 2 it can be seen that no obvious hysteresis phenomenon occurred during the test of the magnetic beads, indicating that the magnetic beads all have superparamagnetism.

[0146] Test Example 2

[0147] The HLB properties of the HLB magnetic beads obtained in step 2 of Example 1 were characterized using infrared spectroscopy. The obtained infrared spectrum is as Figure 3As shown. Among them, the -C-H stretching vibration peak is located at 2922.98 cm -1 ; the =C-H stretching vibration peak is located at 3010.45 cm -1 , combined with the benzene ring C=C stretching vibration peak located at 1603.96 cm -1 -1445.92 cm -1 , the presence of the lipophilic functional group benzene ring can be determined; the stretching vibration peak of C=O is located at 1723.63 cm -1 , and the stretching vibration peak of C-O is located at 1181.99 cm -1 . Both of these functional groups come from GMA, thus proving the presence of the polar component GMA in the structure of product B.

[0148] Test Example 3

[0149] Quantitative analysis was carried out on the density of amino functional groups carried by the HLB / WAX hybrid magnetic beads obtained in the examples and comparative examples. Quantitative analysis was carried out using acid-base titration. The specific process is as follows:

[0150] 1. Weigh 0.1 g of dry HLB / WAX hybrid magnetic beads, disperse them in 20 mL of 0.1 M hydrochloric acid and mix for 1 h. Then, separate the magnetic beads and the hydrochloric acid supernatant with a magnetic stand, and take 5 mL of the hydrochloric acid supernatant into a beaker, and titrate it with 0.1 M sodium hydroxide solution to determine the content of remaining hydrogen ions in the hydrochloric acid supernatant.

[0151] The amino density (unit: mmol / g) of the HLB / WAX hybrid magnetic beads can be obtained by calculation using Formula 1.

[0152]

[0153] Formula 1: Calculation formula for amino density

[0154] The data obtained are shown in Table 2 below.

[0155] Table 2

[0156]

[0157] As can be seen from Table 2, ethanolamine, as a primary amine substance, has a small steric hindrance effect and can efficiently enter the polymer shell of the magnetic beads to participate in the reaction. Therefore, the amine group density of the magnetic beads in Example 2 modified with ethanolamine is higher than that of Examples 3, 4, and 5 modified with the secondary amine substance N-methylethanolamine. Although in Example 5, the addition amount of N-methylethanolamine has been increased to 10 times, the excessive addition amount will instead reduce the reaction efficiency, resulting in an amine group modification amount less than that when 5 times of N-methylethanolamine is added in Example 4. The amine compounds used in Examples 1, 6, and 7 are all ethylenediamine, a binary primary amine substance. However, due to the different monomer components used in synthesizing the polymer shell, the amine group modification amounts are different. The amine group density of the magnetic beads in Example 1 is the highest among the three; in Example 2, when the monomer VBC is replaced by ST, the amine group density of the magnetic beads decreases; in Example 6, when the monomer GMA is removed, the monomer components for synthesizing the polymer shell are only DVB and ST, and at this time the amine group density of the magnetic beads is the lowest, indicating that both monomer VBC and GMA contribute to the amine group modification of ethylenediamine. Comparing Example 1 and Example 2, although both ethylenediamine and ethanolamine are primary amine substances, the modification ability of ethylenediamine is inferior to that of ethanolamine because, as a binary primary amine substance, ethylenediamine generates a larger steric hindrance effect than ethanolamine, resulting in ethylenediamine being unable to effectively enter the polymer shell, making the amine group density of the magnetic beads in Example 1 inferior to that in Example 2. In Comparative Example 1, triethylamine, as a tertiary amine substance, has a large steric hindrance and cannot undergo amine group modification with the monomer GMA, so its amine group modification amount cannot be measured.

[0158] Test Example 4

[0159] The specific surface areas of the HLB / WAX hybrid magnetic beads of the examples and comparative examples were measured using a specific surface area analyzer, and the obtained specific surface areas are shown in Table 3 below.

[0160] Table 3

[0161]

[0162] As can be seen from Table 3, there is no obvious difference in the specific surface areas of the products of Examples 2-5, 7 and Comparative Example 1 and the HLB magnetic beads, while the specific surface areas of Examples 1 and 6 have a relatively obvious increase compared to the HLB magnetic beads; the specific surface area of the product of Example 7 is inferior to that of the product of Example 6, and the specific surface area of the product of Example 1 is higher than that of the product of Example 6, further demonstrating that monomer VBC is more conducive to the amine group modification of ethylenediamine than monomer ST.

[0163] In specific applications, the HLB / WAX hybrid magnetic beads have a wide range of applications in multiple fields. The following are the main application fields of the HLB / WAX hybrid magnetic beads:

[0164] Physicochemical Safety Analysis of Food and Cosmetics: HLB / WAX mixed magnetic beads can be used for the analysis of chemical components in food and cosmetics to ensure their safety and compliance.

[0165] Clinical In Vitro Small Molecule Biomarker Research: In clinical research, such magnetic beads can be used to extract and analyze small molecule biomarkers in biological samples, which is very important for disease diagnosis and treatment monitoring.

[0166] Forensic Toxicology Detection: In the field of forensic medicine, HLB / WAX mixed magnetic beads can be used to detect toxins and other harmful substances in the body.

[0167] Environmental Detection: Such magnetic beads can also be used for the detection and analysis of pollutants in environmental samples to evaluate environmental quality and monitor pollution conditions.

[0168] Nucleic Acid Extraction: HLB / WAX mixed magnetic beads are also applied in the field of nucleic acid extraction, especially in the extraction of DNA and RNA.

[0169] Protein Purification and Immunochromatography: This is one of the applications with the highest demand for HLB / WAX mixed magnetic beads. They can be used for protein purification and immunochromatography, including chemiluminescent immunoassay technology (CLIA) with magnetic microparticles, microfluidic magnetic immunosensing analysis technology (MIA), fluorescence immunoassay technology (FIA), single molecule immunoassay technology, etc.

[0170] Finally, it should be noted that: The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: They can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A HLB / WAX hybrid magnetic bead, characterized in that: It includes a magnetic core, a silicon dioxide coating layer, a polymer coating layer and an amine-modified layer; The silicon dioxide coating layer includes a first coating layer and a second coating layer.

2. The HLB / WAX hybrid magnetic beads according to claim 1, characterized in that: The amino modified layer includes amino functional groups; Based on the mass of the HLB / WAX mixed magnetic beads, the density of the amine functional groups is 0.01 to 3 mmol / g.

3. The HLB / WAX hybrid magnetic beads according to claim 1, characterized in that: The polymer in the polymer coating layer contains active functional groups; Preferably, the active functional group includes at least one of an epoxy group, a carboxyl group, a carbonyl group and a halogenated hydrocarbon group.

4. The HLB / WAX hybrid magnetic beads according to claim 1, characterized in that: The material of the magnetic core is ferroferric oxide; Preferably, the particle size of the ferrosoferric oxide is 100-700 nm.

5. A method for preparing HLB / WAX hybrid magnetic beads according to any one of claims 1 to 4, characterized in that: The following steps are involved: A. In an ethanol aqueous solution of ferroferric oxide, a first silane coupling agent is added to form a first coating layer, and then a second silane coupling agent is added to form a second coating layer to obtain Fe3O4@SiO2; B. After the Fe3O4@SiO2 is made into a suspension, a monomer solution and an initiator are added to obtain a mixed solution; the mixed solution is added to a stabilizer solution for a first reaction to obtain HLB magnetic beads; C. Add the HLB magnetic beads into an amine compound solution to carry out a second reaction to obtain HLB / WAX mixed magnetic beads.

6. The preparation method according to claim 5, characterized in that: The structural formula of the amine compound in the amine compound solution is R1-NH-R2; Wherein, R1 is a substituted or unsubstituted hydrocarbon group, and R2 is a hydrogen atom, a substituted or unsubstituted hydrocarbon group.

7. The preparation method according to claim 6, characterized in that: The hydrocarbon group is a C1 to C10 hydrocarbon group; Preferably, the hydrocarbon group comprises an alkyl group, an alkenyl group or an alkynyl group; Preferably, the substituted hydrocarbon group includes at least one of a halogenated hydrocarbon group, a hydroxyl hydrocarbon group, a carboxylic acid hydrocarbon group, an ester hydrocarbon group, a keto hydrocarbon group, an ether hydrocarbon group, a nitrile hydrocarbon group, a sulfonic acid hydrocarbon group and a phosphoric acid hydrocarbon group.

8. The preparation method according to any one of claims 5 to 7, characterized in that: The first silane coupling agent includes at least one of acetyltrimethoxysilane, tetraethoxysilane, γ-aminopropyltriethoxysilane, 3-glycidyloxypropyltrimethoxysilane, and 3-aminopropyltriethoxysilane; Preferably, the mass of the first silane coupling agent is 0.5 to 2.5 times the mass of ferrosoferric oxide; Preferably, the temperature for forming the first coating layer is 20 to 40° C. and the time is 4 to 6 hours; Preferably, the second silane coupling agent includes at least one of 3-(trimethoxysilyl)propyl methacrylate, isobutyl (trimethoxy) silane, octyl trimethoxy silane, and octadecyl trimethoxy silane; Preferably, the mass of the second silane coupling agent is 1.5 to 4 times the mass of ferrosoferric oxide; Preferably, the temperature for forming the second coating layer is 70-90° C. and the time is 8-10 hours.

9. The preparation method according to any one of claims 5 to 7, characterized in that: In step B, the mass concentration of the suspension is 0.5-1.5%; Preferably, the amount of the initiator is 2-5% of the monomer mass; Preferably, the concentration of the stabilizer solution is 2-4%; Preferably, the stabilizer in the stabilizer solution includes at least one of sodium dodecyl sulfate, sodium dodecylbenzene sulfonate, gelatin, carboxymethyl cellulose, chitosan, polyvinyl alcohol, polyvinyl pyrrolidone, aluminum oxide, and magnesium hydroxide; Preferably, the first reaction time is 6 to 10 hours.

10. The preparation method according to any one of claims 5 to 7, characterized in that: In step C, the amount of the amine compound is 0.5 to 10 times the mass of the HLB magnetic beads; Preferably, in the reaction system for the second reaction, the concentration of HLB magnetic beads is 3 to 6 wt %; Preferably, the temperature of the second reaction is 70-90° C. and the time is 16-48 hours.

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