Hlb / wax hybrid magnetic beads and a method for preparing the same

Abstract

The application provides an HLB / WAX mixed type magnetic bead and a preparation method thereof, and particularly relates to the technical field of solid-phase extraction magnetic beads. The HLB / WAX mixed type magnetic bead comprises a magnetic core, a silica coating layer, a polymer coating layer and an amine group modification layer; the silica coating layer comprises a first coating layer and a second coating layer. The HLB / WAX mixed type magnetic bead significantly widens its potential application field by combining the two characteristics of HLB and WAX, not only enhances the magnetic response of the magnetic bead, makes it more efficient in the separation and extraction process, but also introduces additional functional groups through the amine group modification layer, improves the selective adsorption capacity of different polar and ionic compounds, so that the HLB / WAX mixed type magnetic bead has higher extraction efficiency and selectivity in the fields of drug monitoring, environmental analysis and biological sample processing, and provides a more efficient tool for the research and application in the related fields.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of solid phase extraction magnetic beads, in particular to an HLB / WAX mixed magnetic bead and a preparation method thereof. BACKGROUND

[0002] Liquid chromatography-tandem mass spectrometry (LC-MS / MS) is a powerful tool for analyzing small molecules, especially suitable for clinical monitoring of drugs and their metabolites. In the analysis of biological samples, sample processing 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 the sample through an extraction column filled with solid adsorbent, and the specific components in the sample are retained on the solid phase, and then using appropriate solvents to elute impurities first and then elute target substances, thereby achieving purification and concentration of target substances. However, the SPE procedure is complex and mostly manual operation, resulting in large deviations in parameters and affecting repeatability. In addition, the use of a large amount of elution solvent also makes the cost of SPE relatively high.

[0003] In order to overcome the limitations of traditional SPE, solid phase extraction magnetic beads (SPE magnetic beads) have been developed. SPE magnetic beads are composed of superparamagnetic nanomagnetic cores and functionalized polymer shells, and are specifically bound to target substances through binding sites on the polymer shell and are enriched in a magnetic field. After the magnetic field is removed, the extracted substances are released, achieving purification and concentration of target substances. The magnetic responsiveness of the magnetic beads has the potential for high automation, and when combined with LC-MS / MS, it can develop into a fully automated analyzer for pretreatment and detection, thereby improving the accuracy and efficiency of analysis.

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

[0005] Therefore, the present application is proposed. SUMMARY

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

[0007] The second purpose of the present application is to provide a preparation method of an HLB / WAX mixed magnetic bead.

[0008] To solve the above technical problems, the application adopts the following technical solutions:

[0009] The first aspect of the application provides an HLB / WAX mixed magnetic bead, comprising a magnetic core, a silica coating layer, a polymer coating layer and an amine group modification layer.

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

[0011] Further, the amine group modification layer comprises an amine group functional group.

[0012] The density of the amine group functional group is 0.01-3 mmol / g based on the mass of the HLB / WAX mixed magnetic bead.

[0013] Further, the polymer in the polymer coating layer contains an active functional group.

[0014] Preferably, the active functional group comprises at least one of an epoxy group, a carboxyl group, a carbonyl group and a halogenated hydrocarbon group.

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

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

[0017] The second aspect of the application provides a preparation method of the HLB / WAX mixed magnetic bead, comprising the following steps:

[0018] 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;

[0019] 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 to perform a first reaction, to obtain an HLB magnetic bead;

[0020] C. The HLB magnetic bead is added to an amine compound solution to perform a second reaction, to obtain an HLB / WAX mixed magnetic bead.

[0021] Further, the structure formula of the amine compound in the amine compound solution is R1-NH-R2.

[0022] Wherein, R1 is a substituted or unsubstituted hydrocarbon group, and 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 comprises an alkyl group, an alkenyl group or an alkinyl group.

[0025] Preferably, the substituted hydrocarbon group comprises at least one of halogenated hydrocarbon group, hydroxyl hydrocarbon group, carboxylic acid hydrocarbon group, ester hydrocarbon group, ketone hydrocarbon group, ether hydrocarbon group, nitrile hydrocarbon group, sulfonic acid hydrocarbon group and phosphoric acid hydrocarbon group.

[0026] Further, the first silane coupling agent comprises at least one of acetyl trimethylsilane, tetraethoxysilane, γ-aminopropyl triethoxysilane, 3-glycidyloxypropyl trimethoxysilane and 3-aminopropyl triethoxysilane.

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

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

[0029] Preferably, the second silane coupling agent comprises at least one of 3-(trimethoxysilyl) methyl methacrylate, isobutyl (trimethoxy) silane, octyl trimethoxysilane and octadecyl trimethoxysilane.

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

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

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

[0033] Preferably, the amount 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 comprises at least one of sodium dodecyl sulfate, sodium dodecyl benzene sulfonate, gelatin, carboxymethyl cellulose, chitosan, polyvinyl alcohol, polyvinyl pyrrolidone, aluminum oxide and magnesium hydroxide.

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

[0037] Further, in step C, the amount of the amine compound is 0.5-10 times the mass of the HLB magnetic bead.

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

[0039] Preferably, the second reaction has a temperature of 70-90 DEG C and a time of 16-48 hours.

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

[0041] The HLB / WAX hybrid magnetic beads provided by the present application significantly expand the potential application field by combining the HLB (hydrophilic-lipophilic balance) and weak anion exchange (WAX) two characteristics. The structure of the magnetic beads includes a magnetic core, a silica coating layer, a polymer coating layer and an amine group modification 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 process, but also introduces additional functional groups through the amine group modification layer, improving the selective adsorption capacity of different polar and ionic compounds.

[0042] The preparation method provided by the present application first enhances the stability and chemical resistance of the magnetic beads by preparing a silica coating layer (Fe3O4@SiO2) with a double-layer structure, and provides a good foundation for subsequent functionalization. Then, the HLB magnetic beads are obtained by coating a polymer on the surface of Fe3O4@SiO2, ensuring that the surface of the magnetic beads has good hydrophilicity and lipophilicity, and can effectively selectively adsorb different polar target substances. Finally, by carrying out a second reaction between the HLB magnetic beads and an amine compound solution, the weak anion exchange characteristic is introduced, so that the magnetic beads can electrostatically adsorb ionic compounds with opposite charges, thereby expanding its application potential in ionic compound extraction and separation. The preparation method precisely controls the reaction conditions and material composition to ensure that the specific surface area and magnetic responsiveness of the magnetic beads are in the best state, so that 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 more efficient tools for related research and application. BRIEF DESCRIPTION OF DRAWINGS

[0043] In order to more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the following will briefly introduce the drawings needed to be used in the specific embodiments or prior art description. Obviously, the drawings described below are some embodiments of the present application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor.

[0044] Figure 1 To obtain the hysteresis loop of test example 1;

[0045] Figure 2 To obtain the hysteresis loop of test example 1;

[0046] Figure 3The infrared spectrum of the HLB magnetic beads. DETAILED DESCRIPTION

[0047] In order to make the purposes, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below in combination with the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, rather than all the embodiments. The components of the embodiments of the present application described and shown herein can be arranged and designed in various different configurations.

[0048] A first aspect of the present application provides an HLB / WAX hybrid magnetic bead, comprising a magnetic core, a silica coating layer, a polymer coating layer, and an amine group modification layer.

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

[0050] The HLB / WAX hybrid magnetic bead provided by the present application significantly broadens its potential application fields by combining the properties 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 group modification 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 bead, making it more efficient in separation and extraction processes, but also introduces additional functional groups through the amine group modification layer, improving the selective adsorption capacity for different polar and ionic compounds.

[0051] Further, the amine group modification layer includes amine group functional groups.

[0052] The density of the amine group functional groups is 0.01-3 mmol / g, based on the mass of the HLB / WAX hybrid magnetic bead. Within this density range, the adsorption efficiency of the magnetic bead to target biomolecules is enhanced, while good dispersibility and resuspendability are ensured, avoiding aggregation phenomena, thereby exhibiting excellent performance in biomolecule extraction, purification, and analysis, improving experimental efficiency and the reliability of the results.

[0053] Typically but not limitedly, the density of the amine group functional groups may 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 any value within the range of 0.01 mmol / g to 3 mmol / g.

[0054] Further, the polymer in the polymer coating layer contains active functional groups.

[0055] Preferably, the active functional groups include at least one of an epoxy group, a carboxyl group, a carbonyl group and a halogenated hydrocarbon group, which can react with the amine compound to enable the magnetic beads to carry amine functional groups.

[0056] Further, the material of the magnetic core is ferroferric oxide.

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

[0058] The second aspect of the present application provides a preparation method of the HLB / WAX mixed magnetic beads, comprising the following steps:

[0059] A. In the ferroferric oxide ethanol aqueous solution, 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;

[0060] 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 to perform a first reaction, to obtain HLB magnetic beads;

[0061] C. The HLB magnetic beads are added to an amine compound solution to perform a second reaction, to obtain HLB / WAX mixed magnetic beads.

[0062] The preparation method provided by the present application first prepares a silica coating layer (Fe3O4@SiO2) with a double-layer structure, which enhances the stability and chemical resistance of the magnetic beads and provides a good foundation for subsequent functionalization. Then, the Fe3O4@SiO2 surface is coated with a polymer to obtain HLB magnetic beads, which ensures that the surface of the magnetic beads has good hydrophilicity and lipophilicity, and can effectively and selectively adsorb target substances of different polarity. Finally, by performing a second reaction of the HLB magnetic beads with an amine compound solution, weak anion exchange characteristics are introduced, so that the magnetic beads can electrostatically adsorb ionic compounds with opposite charges, thereby widening its application potential in ionic compound extraction and separation. The preparation method precisely controls the reaction conditions and material composition to ensure that the specific surface area and magnetic responsiveness of the magnetic beads are in the best state, so that the HLB / WAX mixed magnetic beads have higher extraction efficiency and selectivity in the fields of drug monitoring, environmental analysis and biological sample processing, and provide more efficient tools for related research and application.

[0063] Further, the amine compound in the amine compound solution has the structural formula of R1-NH-R2.

[0064] Wherein, R1 is a substituted or unsubstituted hydrocarbon group, and R2 is a hydrogen atom, a substituted or unsubstituted hydrocarbon group.

[0065] It should be noted that the amine compounds are primary amine compounds or secondary amine compounds.

[0066] In amine compounds, due to the difference in steric hindrance effect, their ability to participate in reactions also varies. Tertiary amine compounds are generally difficult to participate in the modification reaction of the polymer shell of magnetic beads due to the greater steric hindrance effect. In contrast, the steric hindrance effect of primary amine compounds is smaller, which enables them to more effectively penetrate and enter the polymer shell of magnetic beads, thereby participating 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 group functional group provides more active sites for the magnetic beads, enhancing their functionality and efficiency in various applications, especially compared to secondary amine compound-modified magnetic beads, which have a relatively low amine group density due to the greater steric hindrance effect, affecting their participation and effectiveness in reactions.

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

[0068] C1 to C10 hydrocarbon groups include a series of alkyl groups starting from methyl (-CH3) to decyl (-C 10 H 21 ) consisting of 1 to 10 carbon atoms. These hydrocarbon groups gradually increase in branched isomers as the number of carbon atoms increases, such as ethyl (-C2H5), propyl (-C3H7) and its isopropyl (-CH(CH3)2), butyl (-C4H9) and its isobutyl (-CH2CH(CH3)2), tert-butyl (-C(CH3)3), etc. As the carbon number increases, such as pentyl (-C5H 11 ), 13 ), heptyl (-C7H 15 ), octyl (-C8H 17 ), nonyl (-C9H 19 ), and decyl (-C 10 H 21 ), their structures become more complex, including more branched isomers, such as isoamyl (-CH2CH2CH(CH3)2), neopentyl (-CH(CH3)CH2CH3), tert-pentyl (-C(CH3)3), iso-hexyl (-CH2CH2CH2CH(CH3)2), tert-hexyl (-CH2CH(CH3)CH2CH2CH3), 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 hydroxyl hydrocarbon group, a carboxylic acid hydrocarbon group, an ester hydrocarbon group, a ketone hydrocarbon group, an ether hydrocarbon group, a nitrile hydrocarbon group, a sulfonic acid hydrocarbon group, and a phosphoric acid hydrocarbon group.

[0071] Further, the first silane coupling agent comprises at least one of acetyl trimethylsilane, tetraethoxysilane, γ-aminopropyl triethoxysilane, 3-glycidyloxypropyl trimethoxysilane, and 3-aminopropyl triethoxysilane.

[0072] The first coating layer formed by the first silane coupling agent is dense and can well coat the magnetic beads, reducing the direct contact of the magnetic core with the external environment, thereby avoiding oxidation or wear of the magnetic core under chemical reaction or physical action and ensuring the stability of the magnetic performance 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 ferroferric oxide.

[0074] Typically but not limitedly, the mass of the first silane coupling agent can be 0.5 times, 1 times, 1.5 times, 2 times the mass of the ferroferric 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℃, and the time is 4 to 6h.

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

[0077] Preferably, the second silane coupling agent comprises at least one of 3-(trimethoxysilyl) methyl propyl methacrylate, isobutyl (trimethoxy) silane, octyl trimethoxysilane, and octadecyl trimethoxysilane. The second coating layer formed by the second silane coupling agent mainly functions to perform surface modification, so that the magnetic beads can be better combined with the polymer.

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

[0079] Typically but not limitedly, 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 ferroferric 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℃, and the time is 8 to 10h.

[0081] Typically but not exclusively, 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 exclusively, 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 amount of the initiator is 2-5% of the mass of the monomer.

[0085] Typically but not exclusively, the amount 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 exclusively, 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 dodecyl benzene sulfonate, gelatin, carboxymethyl cellulose, chitosan, polyvinyl alcohol, polyvinyl pyrrolidone, aluminum oxide, magnesium hydroxide, which mainly functions to provide a stable reaction environment, when the magnetic core suspension containing the monomer is mixed in the stabilizer solution to form droplets, the stabilizer forms a physical barrier outside the droplets to prevent the adhesion or fusion between the droplets, thereby ensuring the sphericity and particle size of the magnetic beads, 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 for the first reaction is 6-10h.

[0089] Typically but not exclusively, the time for 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 amount of the amine compound is 0.5-10 times the mass of the HLB magnetic beads.

[0091] Typically but not exclusively, the amine compound can be used in an amount of 0.5 times, 1 times, 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 any value within the range of 0.5 to 10 times.

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

[0093] Typically but not exclusively, the concentration of the HLB magnetic beads can be 3 wt%, 4 wt%, 5 wt% or 6 wt%, or 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 exclusively, 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 h, 20 h, 24 h, 32 h, 40 h or 48 h, or any value within the range of 16 h to 48 h.

[0096] The dispersing solvent used in the present application can be replaced by toluene, cyclohexane, dichloroethane, dimethyl sulfoxide, tetrahydrofuran, N,N-dimethylformamide, acetonitrile, isopropanol, ethanol, methanol or water.

[0097] Some embodiments of the present application will be described in detail below with reference to examples. The examples and features in the examples below can be combined with each other without conflict, provided that the combinations do not conflict. The raw materials used in the present application can be purchased from the market, unless otherwise specified.

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

[0099] Example 1

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

[0101] 1. Nanoscale ferroferric oxide powder is ultrasonically dispersed in 80% aqueous ethanol solution, then tetraethoxysilane (TEOS) is added in an amount of 2 times the amount of ferroferric oxide, and stirred at room temperature for 5 h, then 3-(trimethoxysilyl) methyl propyl methacrylate (MEMO) is added in an amount of 3 times the amount of ferroferric oxide, the temperature is raised to 80°C and stirred at this temperature for 8 h, then the product is washed with anhydrous ethanol to obtain a silica-modified magnetic core (Fe3O4@SiO2).

[0102] 2. HLB magnetic beads synthesis

[0103] After the Fe3O4@SiO2dispersed in dichloroethane to make 1.25% of the magnetic core suspension, to the magnetic core suspension, add vinyl benzyl chloride (VBC): divinyl benzene (DVB): glycidyl methacrylate (GMA) monomer solution of 2:1:1, the amount of initiator AIBN is 3.3% of the monomer added. In the process of stirring the magnetic core suspension into the PVA aqueous solution (concentration of 2.25wt%), after 8h reaction, the product is obtained after washing with anhydrous ethanol and water alternately to obtain HLB magnetic beads.

[0104] 3. The HLB magnetic beads are dispersed in ethylenediamine / ethanol solution to make 4.2% of the magnetic bead suspension, and the amount of ethylenediamine is 2 times of the added amount of HLB magnetic beads. Then warm up to 80℃ and stir the magnetic bead suspension at this temperature for 24h after reaction, then discharge, and the product is washed with anhydrous ethanol to obtain HLB / WAX mixed magnetic beads.

[0105] Example 2

[0106] This example provides a kind of HLB / WAX mixed magnetic beads, and the preparation process is as follows:

[0107] 1. The same as the step of example 1.

[0108] 2. The same as the step of example 1.

[0109] 3. The HLB magnetic beads are dispersed in ethanolamine / ethanol solution to make 4.2% of the magnetic bead suspension, and the amount of ethanolamine is 4 times of the added amount of HLB magnetic beads. Then warm up to 80℃ and stir the magnetic bead suspension at this temperature for 24h after reaction, then discharge, and the product is washed with anhydrous ethanol to obtain HLB / WAX mixed magnetic beads.

[0110] Example 3

[0111] This example provides a kind of HLB / WAX mixed magnetic beads, and the preparation process is as follows:

[0112] 1. The same as the step of example 1.

[0113] 2. The same as the step of example 1.

[0114] 3. The HLB magnetic beads are dispersed in N-methyl ethanolamine / ethanol solution to make 4.2% of the magnetic bead suspension, and the amount of N-methyl ethanolamine is 5 times of the added amount of HLB magnetic beads. Then warm up to 80℃ and stir the magnetic bead suspension at this temperature for 24h after reaction, then discharge, and the product is washed with anhydrous ethanol to obtain HLB / WAX mixed magnetic beads.

[0115] Example 4

[0116] The embodiment provides an HLB / WAX mixed type magnetic bead, and a preparation process is as follows:

[0117] 1. The step is the same as that in the embodiment 1.

[0118] 2. The step is the same as that in the embodiment 1.

[0119] 3. Different from the step in the embodiment 1, the amount of N-methylethanolamine is 0.5 times the amount of HLB magnetic beads. The remaining steps are the same as those in the embodiment 1, and details are not described herein again.

[0120] Embodiment 5

[0121] The embodiment provides an HLB / WAX mixed type magnetic bead, and a preparation process is as follows:

[0122] 1. The step is the same as that in the embodiment 1.

[0123] 2. The step is the same as that in the embodiment 1.

[0124] 3. Different from the step in the embodiment 1, the amount of N-methylethanolamine is 10 times the amount of HLB magnetic beads. The remaining steps are the same as those in the embodiment 1, and details are not described herein again.

[0125] Embodiment 6

[0126] The embodiment provides an HLB / WAX mixed type magnetic bead, and a preparation process is as follows:

[0127] 1. The step is the same as that in the embodiment 1.

[0128] 2. After Fe3O4@SiO2 is dispersed in dichloroethane to prepare a 1.25% magnetic core suspension, a monomer solution with a ratio of styrene (ST) : DVB : GMA being 2:1:1 is added into the magnetic core suspension, and the amount of initiator AIBN is 3.3% of the amount of monomers. In the stirring process, the magnetic core suspension is added into a PVA aqueous solution (the concentration is 2.25 wt%), and after reaction for 8 hours, the HLB magnetic beads are obtained after being washed by anhydrous ethanol and water alternately.

[0129] 3. The step is the same as that in the embodiment 1.

[0130] Embodiment 7

[0131] The embodiment provides an HLB / WAX mixed type magnetic bead, and a preparation process is as follows:

[0132] 1. The step is the same as that in the embodiment 1.

[0133] 2. After preparing a 1.25% magnetic core suspension by dispersing Fe3O4@SiO2 in dichloroethane, a monomer solution of ST:DVB at a ratio of 1:1 was added to the magnetic core suspension, and the amount of initiator AIBN was 3.3% of the amount of monomer added. The magnetic core suspension was added to a PVA aqueous solution (2.25 wt%) during stirring, and the reaction was carried out for 8 h. After washing with anhydrous ethanol and water alternately, HLB magnetic beads were obtained.

[0134] 3. The same as the step in Example 1.

[0135] Comparative Example 1

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

[0137] 1. The same as the step in Example 1.

[0138] 2. The same as the step in Example 1.

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

[0140] Test Example 1

[0141] The magnetic properties of the HLB / WAX mixed magnetic beads of the examples and comparative examples were characterized. The saturation magnetization of the magnetic beads was characterized using a vibrating sample magnetometer, and the obtained saturation magnetization is shown in Table 1.

[0142] Table 1

[0143]

[0144]

[0145] The hysteresis loops of the magnetic beads obtained in Examples 1-7 and Comparative Example 1 are shown in Figure 1 and Figure 2 It can be seen from Figure 1 and Figure 2 that the magnetic beads did not show obvious hysteresis during the test, indicating that the magnetic beads all have superparamagnetic properties.

[0146] Test Example 2

[0147] The HLB properties of the HLB magnetic beads obtained in Example 1, Step 2, were characterized by infrared spectroscopy. The obtained infrared spectrum is shown in Figure 3 The -C-H stretching vibration peak is located at 2922.98 cm -1; = C-H stretching vibration peak at 3010.45 cm -1 , in combination with the benzene ring C=C stretching vibration peak at 1603.96 cm -1 -1445.92 cm -1 , the presence of the lipophilic functional group benzene ring can be determined; the C=0 stretching vibration peak is at 1723.63 cm -1 , and the C-O stretching vibration peak is at 1181.99 cm -1 , both of which functional groups are from GMA, thereby proving the existence of the polar component GMA in the structure of product B.

[0148] Test Example 3

[0149] The amine group density carried by the HLB / WAX mixed magnetic beads obtained from the examples and comparative examples was quantitatively analyzed. The quantitative analysis was performed using acid-base titration method, and the specific process was as follows:

[0150] 1. 0.1 g of dry HLB / WAX mixed magnetic beads was weighed, dispersed in 20 mL of 0.1 M hydrochloric acid and mixed for 1 h, then the magnetic beads and the hydrochloric acid supernatant were separated using a magnetic stand, and 5 mL of the hydrochloric acid supernatant was taken into a beaker. 0.1 M sodium hydroxide solution was used for titration to determine the content of residual hydrogen ions in the hydrochloric acid supernatant.

[0151] The amine group density of the HLB / WAX mixed magnetic beads (unit: mmol / g) can be calculated using Formula 1.

[0152]

[0153] Formula 1: Amine group density calculation formula

[0154] The obtained data is 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, so the amine group density of the magnetic beads of Example 2 modified by using ethanolamine is higher than that of Examples 3, 4 and 5 modified by using a secondary amine substance N-methylethanolamine. Although in Example 5, the addition amount of N-methylethanolamine has been increased to 10 times, too high addition amount will reduce the reaction efficiency, so that the amine group modification amount is less than that when the addition amount of N-methylethanolamine in Example 4 is 5 times. The amine compounds used in Examples 1, 6 and 7 are all ethylenediamine, a binary primary amine substance, but due to the difference in monomer components used for synthesizing the polymer shell, the amine group modification amounts are different. The amine group density of the magnetic beads of Example 1 is the highest among the three; in Example 2, when monomer VBC is replaced by ST, the amine group density of the magnetic beads decreases; in Example 6, when monomer GMA is removed, the monomer components used for synthesizing the polymer shell are only DVB and ST, at this time, the amine group density of the magnetic beads is the lowest, which indicates that monomers VBC and GMA are both helpful for amine group modification of ethylenediamine. Comparing Examples 1 and 2, although both ethylenediamine and ethanolamine are primary amine substances, the modification capacity of ethylenediamine is less than that of ethanolamine, because ethylenediamine, as a binary primary amine substance, has a larger steric hindrance effect than ethanolamine, which leads to that ethylenediamine cannot effectively enter the polymer shell, so that the amine group density of the magnetic beads of Example 1 is less than that of Example 2. In Comparative Example 1, triethylamine, as a tertiary amine substance, has a large steric hindrance and cannot be modified with monomer GMA, so the amine group modification amount cannot be measured.

[0158] Test Example 4

[0159] The specific surface area analyzer was used to measure the specific surface area of the HLB / WAX mixed type magnetic beads of the examples and comparative examples, and the specific surface area obtained is shown in Table 3.

[0160] Table 3

[0161]

[0162] As can be seen from Table 3, the specific surface area of the products of Examples 2-5, 7 and Comparative Example 1 has no obvious difference with that of the HLB magnetic beads, while the specific surface area of Examples 1 and 6 has been obviously improved compared with that of the HLB magnetic beads; the specific surface area of the product of Example 7 is less than that of the product of Example 6, while the specific surface area of the product of Example 1 is higher than that of the product of Example 6, which further demonstrates that monomer VBC is more helpful than monomer ST for amine group modification of ethylenediamine.

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

[0164] Food and cosmetic physicochemical safety analysis: HLB / WAX hybrid magnetic beads can be used for chemical component analysis in food and cosmetics to ensure their safety and compliance.

[0165] Clinical in vitro small molecule marker research: In clinical research, these magnetic beads can be used to extract and analyze small molecule markers 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 hybrid magnetic beads can be used to detect toxins and other harmful substances in the body.

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

[0168] Nucleic acid extraction: HLB / WAX hybrid magnetic beads also have applications in nucleic acid extraction, especially in the extraction of DNA and RNA.

[0169] Protein purification and immunochromatography: This is one of the highest demand applications of HLB / WAX hybrid magnetic beads, which can be used for protein purification and immunochromatography, including magnetic microparticle chemiluminescence immunoassay technology (CLIA), microfluidic magnetic sensitive immunoassay 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 application, and not to limit them; although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing embodiments, or make equivalent replacement to part 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 application.

Claims

1. An HLB / WAX hybrid magnetic bead, characterized in that, The magnetic core, a silica coating layer, a polymer coating layer, and an amine group modification layer are included. The silica coating layer includes a first coating layer and a second coating layer. The preparation method of the HLB / WAX mixed magnetic beads includes the following steps: 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, thereby obtaining 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 to perform a first reaction, thereby obtaining HLB magnetic beads; The monomer solution is formed by mixing vinyl benzyl chloride, divinyl benzene, and glycidyl methacrylate; C. The HLB magnetic beads are added to an amine compound solution to perform a second reaction, thereby obtaining HLB / WAX mixed magnetic beads; The amine compound is one of ethylenediamine, ethanolamine, and N-methyl ethanolamine; the amount of the amine compound is 0.5 to 10 times the mass of the HLB magnetic beads.

2. The HLB / WAX hybrid magnetic bead of claim 1, wherein, The density of the amine group functional group is 0.01 to 3 mmol / g based on the mass of the HLB / WAX mixed magnetic beads.

3. The HLB / WAX hybrid magnetic bead of claim 1, wherein, The material of the magnetic core is ferroferric oxide.

4. The HLB / WAX hybrid magnetic bead of claim 1, wherein, The particle size of the ferroferric oxide is 100 to 700 nm.

5. The HLB / WAX hybrid magnetic bead according to any one of claims 1 to 4, wherein, The first silane coupling agent includes at least one of acetyl trimethoxysilane, tetraethoxysilane, γ-aminopropyl triethoxysilane, 3-glycidyloxypropyl trimethoxysilane, and 3-aminopropyl triethoxysilane.

6. The HLB / WAX hybrid magnetic bead according to any one of claims 1 to 4, wherein, The mass of the first silane coupling agent is 0.5 to 2.5 times the mass of the ferroferric oxide.

7. The HLB / WAX hybrid magnetic bead according to any one of claims 1 to 4, wherein, The temperature for forming the first coating layer is 20 to 40℃, and the time is 4 to 6 h.

8. The HLB / WAX hybrid magnetic bead according to any one of claims 1 to 4, wherein, The second silane coupling agent includes at least one of 3-(trimethoxysilyl) methyl methacrylate, isobutyl (trimethoxy) silane, octyl trimethoxysilane, and octadecyl trimethoxysilane.

9. The HLB / WAX hybrid magnetic bead according to any one of claims 1 to 4, wherein, The mass of the second silane coupling agent is 1.5 to 4 times the mass of the ferroferric oxide.

10. The HLB / WAX hybrid magnetic bead according to any one of claims 1 to 4, wherein, The temperature for forming the second coating layer is 70 to 90℃, and the time is 8 to 10 h.

11. The HLB / WAX hybrid magnetic bead according to any one of claims 5-7, wherein, In step B, the mass concentration of the suspension is 0.5 to 1.5%.

12. The HLB / WAX hybrid magnetic bead according to any one of claims 1 to 4, wherein, The amount of the initiator is 2 to 5% of the mass of the monomer.

13. The HLB / WAX hybrid magnetic bead according to any one of claims 1 to 4, wherein, The concentration of the stabilizer solution is 2 to 4 wt%.

14. The HLB / WAX hybrid magnetic bead according to any one of claims 1 to 4, wherein, The stabilizer in the stabilizer solution includes at least one of sodium dodecyl sulfate, sodium dodecyl benzene sulfonate, gelatin, carboxymethyl cellulose, chitosan, polyvinyl alcohol, polyvinyl pyrrolidone, aluminum oxide, and magnesium hydroxide.

15. The HLB / WAX hybrid magnetic bead according to any one of claims 1 to 4, wherein, The time of the first reaction is 6 to 10 h.

16. The HLB / WAX hybrid magnetic bead according to any one of claims 1-4, wherein, In the reaction system for the second reaction, the concentration of the HLB magnetic beads is 3 to 6 wt%.

17. The HLB / WAX hybrid magnetic bead according to any one of claims 1-4, wherein, The temperature of the second reaction is 70 to 90℃, and the time is 16 to 48 h.

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