Amphiphilic C18 hybrid solid-phase extraction magnetic beads, their preparation methods and applications

Abstract

This invention relates to the field of solid-phase extraction materials technology, and discloses a surface-amphiphilic C18 hybrid solid-phase extraction magnetic bead, its preparation method, and its application. The method involves preparing modified solid-phase extraction magnetic beads through free radical polymerization of double bonds, lipophilic monomers, hydrophilic monomers, and C18 monomers modified on the surface of the magnetic beads. The double bonds on the magnetic bead surface participate in the reaction, allowing the polymer to effectively and tightly encapsulate the magnetic beads, preventing the loss and oxidation of magnetic materials. Simultaneously, this hybrid functional coating significantly improves the hydrophobicity of a single C18 bonded phase, markedly increasing the hydrophilic wetting properties of the magnetic bead surface. The coating thickness can be controlled by adjusting the ratio of double-bonded magnetic beads to the other monomers, thereby controlling the adsorption capacity, mass transfer efficiency, stability, and magnetic responsiveness within an optimal range. Furthermore, the specific surface properties can be controlled by adjusting the ratio of lipophilic monomers, hydrophilic monomers, and C18 monomers, thus facilitating the extraction of vitamins from water-soluble systems.

Description

Technical Field

[0001] This invention relates to the field of solid phase extraction materials technology, and in particular to a surface amphiphilic C18 hybrid solid phase extraction magnetic bead, its preparation method, and its application. Background Technology

[0002] Solid-phase extraction (SPE), a traditional extraction technique, boasts advantages such as high separation efficiency, high recovery rate, and stable operation, making it widely applicable. In the 1990s, Waters invented an amphiphilic mesoporous polymeric SPE material for extracting small organic molecules from biological fluids and bio-extraction solutions. Its key features include the combined effects of size exclusion, van der Waals forces, polar dipoles, and ion exchange to achieve excellent extraction and purification. However, it is limited by the shortcomings of traditional SPE columns, still unable to overcome the risks of column pressure instability due to differences in feed flow rates and column clogging. Therefore, SPE magnetic beads were developed. SPE magnetic beads not only offer higher adsorption recovery rates and are easily automated for high-throughput analysis, significantly increasing recovery rates, but also effectively overcome the risks of unstable recovery rates and clogging inherent in traditional SPE columns.

[0003] Vitamins, as essential micronutrients for maintaining life, are mainly divided into two categories: fat-soluble and water-soluble. Fat-soluble vitamins primarily include A, D, E, and K, while water-soluble vitamins include the B vitamins and vitamin C. Currently, technologies for detecting fat-soluble vitamins mainly focus on A, D2, D3, and E. However, while patent CN108195956A discloses a method for extracting vitamin E from food using a C18 column, its application is limited by the use of organic solvent systems. Since most biological fluids or metabolic solutions are water-soluble, this significantly restricts its applicability.

[0004] C18 solid-phase extraction material, as a general-purpose reversed-phase adsorbent, has many advantages. However, its strong hydrophobic surface makes it difficult to disperse effectively in aqueous solutions, thus hindering its efficient extraction of target analytes from complex water sample systems. Furthermore, C18 packing materials often use a silica matrix as a support. After surface acidification, a large amount of Si-OH groups can be obtained, which, when bonded to a C18 coupling agent, form C18 packing material. However, a significant amount of ungrafted Si-OH groups remain on the surface, readily reacting with alkaline substances and greatly reducing column efficiency and resolution. Summary of the Invention

[0005] To address the technical problems in existing technologies, such as the difficulty in extracting vitamins and applying C18 solid-phase extraction materials in water-soluble systems, and the ease with which the groups of C18 fillers react with alkaline substances, thus reducing column efficiency and separation, this invention provides a surface-amphiphilic C18 hybrid solid-phase extraction magnetic bead, its preparation method, and its application. This improves the application range and extraction recovery rate of C18 solid-phase extraction materials, effectively enhances the extraction rate of vitamins in water-soluble systems, allows for elution pH ranges from 0 to 14, and is also suitable for highly polar elution systems, showing broad application prospects.

[0006] The first aspect of this invention provides a method for preparing surface amphiphilic C18 hybrid solid-phase extraction magnetic beads, comprising: S1, the surface of the nano-magnetic core is modified with double bonds to obtain double-bonded magnetic beads; S2, a mixture containing the double-bonded magnetic beads obtained in step S1, lipophilic monomers, hydrophilic monomers, C18 monomers and initiators is subjected to a free radical polymerization reaction to obtain surface amphiphilic C18 mixed solid-phase extraction magnetic beads. The mass ratio of the double bond type magnetic beads, lipophilic monomer, hydrophilic monomer and C18 monomer is 1:(0.5-20):(0.5-20):(1-20).

[0007] Preferably, the lipophilic monomer includes at least one of styrene, substituted styrene, divinylbenzene, substituted divinylbenzene, and acrylate.

[0008] Preferably, the hydrophilic monomer includes at least one of hydroxyethyl methacrylate, ethylene glycol dimethacrylate, acrylamide, triacetyl oxalic acid, and N-vinylpyrrolidone.

[0009] Preferably, the C18 monomer includes at least one of octadecene, oleic acid, and oleylamine.

[0010] Preferably, the mass ratio of the double bond magnetic beads, lipophilic monomer, hydrophilic monomer and C18 monomer is 1:(1-10):(1-15):(1-10), and more preferably 1:(1-5):(2-10):(1-5).

[0011] The second aspect of the present invention provides an amphiphilic C18 polymer coating, which is obtained by free radical polymerization of lipophilic monomers, hydrophilic monomers and C18 monomers under an initiator, wherein the mass ratio of the lipophilic monomers, hydrophilic monomers and C18 monomers is (0.025-20):(0.025-20):1.

[0012] The third aspect of the present invention provides a surface amphiphilic C18 mixed solid-phase extraction magnetic bead, wherein the preparation method of the surface amphiphilic C18 mixed solid-phase extraction magnetic bead is selected from the preparation method of the surface amphiphilic C18 mixed solid-phase extraction magnetic bead provided by the present invention. Alternatively, the surface amphiphilic C18 hybrid solid-phase extraction magnetic beads include a core and a shell, the shell including a base layer and a functional layer; the base layer covers the surface of the core, the functional layer covers the surface of the base layer, and the functional layer includes the amphiphilic C18 polymer coating provided by the present invention.

[0013] The fourth aspect of the present invention provides the use of surface amphiphilic C18 mixed solid-phase extraction magnetic beads prepared by the method provided by the present invention and / or the surface amphiphilic C18 mixed solid-phase extraction magnetic beads provided by the present invention for preparing reagents for extracting target molecules from samples.

[0014] The fifth aspect of the present invention provides a method for extracting target molecules from a sample, comprising: contacting a sample with surface amphiphilic C18 mixed solid-phase extraction magnetic beads prepared using the method of the present invention and / or surface amphiphilic C18 mixed solid-phase extraction magnetic beads provided by the present invention to obtain solid-phase extraction magnetic beads adsorbing target molecules; magnetically separating the solid-phase extraction magnetic beads adsorbing target molecules; and eluting the target molecules.

[0015] In summary, this invention has the following beneficial technical effects: The method for preparing modified solid-phase extraction magnetic beads through free radical polymerization of double bonds, lipophilic monomers, hydrophilic monomers, and C18 monomers modified on the surface of magnetic beads allows the polymer to effectively and tightly encapsulate the magnetic beads, preventing the loss and oxidation of magnetic materials. Simultaneously, this hybrid functional coating significantly improves the hydrophobicity of a single C18 bonded phase, markedly increasing the hydrophilic wetting properties of the magnetic bead surface, effectively broadening the application range of single C18 solid-phase extractants, and providing more ideas for functional material design. Furthermore, the method allows for the reaction of double-bonded magnetic beads with other monomers (lipophilic monomers, hydrophilic monomers, and C18 monomers). The thickness of the coating can be controlled by adjusting the ratio of oleophilic monomers, hydrophilic monomers, and C18 monomers, thereby controlling the adsorption capacity, mass transfer efficiency, stability, and magnetic responsiveness to be within an optimal range. The specific surface properties can be controlled by adjusting the ratio of oleophilic monomers, hydrophilic monomers, and C18 monomers, thereby controlling the surface properties to facilitate the extraction of vitamins in water-soluble systems. Compared with C18 fillers, the method of this invention first effectively weakens the residual Si-OH (because this invention uses a polymerization method, utilizing Si-C=C), and at the same time changes the carrier to polymer-based spheres, utilizing the oleophilic and hydrophilic parts of the polymer monomers to effectively adjust the surface hydrophilic and hydrophobic properties, effectively adapting to the elution system. Basically, the elution pH range can be 0~14, and it can also be applied to elution systems with high polarity. Attached Figure Description

[0016] Figure 1 : Test diagram of the wetting properties of the surface of amphiphilic C18 hybrid solid phase extraction magnetic beads; Figure 2 Comparison of the extraction effects of solid-phase extraction magnetic beads on molecules of different polarities; Figure 3 Figure 1 shows the adsorption results of vitamin A by solid-phase extraction magnetic beads in the examples and comparative examples. Detailed Implementation

[0017] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention.

[0018] When numerical ranges are given in the specific embodiments, it should be understood that, unless otherwise stated in this invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the specific embodiments, this invention may be implemented using any prior art methods, apparatus, and materials similar to or equivalent to those described in the specific embodiments, based on the knowledge of those skilled in the art and the description of this invention.

[0019] The first aspect of this invention provides a method for preparing surface amphiphilic C18 hybrid solid-phase extraction magnetic beads, comprising: S1, the surface of the nano-magnetic core is modified with double bonds to obtain double-bonded magnetic beads; S2, a mixture containing the double-bonded magnetic beads obtained in step S1, lipophilic monomers, hydrophilic monomers, C18 monomers and initiators is subjected to a free radical polymerization reaction to obtain surface amphiphilic C18 mixed solid-phase extraction magnetic beads. The mass ratio of the double bond type magnetic beads, lipophilic monomer, hydrophilic monomer and C18 monomer is 1:(0.5-20):(0.5-20):(1-20).

[0020] This invention relates to a method for preparing modified solid-phase extraction magnetic beads through free radical polymerization of double bonds, lipophilic monomers, hydrophilic monomers, and C18 monomers modified on the surface of magnetic beads. The double bonds modified on the surface of the magnetic beads participate in the reaction, enabling the polymer to effectively and tightly encapsulate the magnetic materials on the surface, preventing the loss and oxidation of magnetic substances. At the same time, this hybrid functional coating greatly improves the hydrophobicity of the single C18 bonded phase and significantly increases the hydrophilic wetting properties of the magnetic bead surface. The thickness of the coating can be controlled by adjusting the ratio of double-bonded magnetic beads to the other monomers (lipophilic monomers, hydrophilic monomers, and C18 monomers), thereby controlling the adsorption capacity, mass transfer efficiency, stability, and magnetic responsiveness to be within an optimal range. The specific surface properties can be controlled by adjusting the ratio of lipophilic monomers, hydrophilic monomers, and C18 monomers, thereby controlling the surface properties to facilitate the extraction of vitamins from water-soluble systems.

[0021] By selecting the type of magnetic beads and monomers and using appropriate mass ratios, the surface amphiphilic C18 mixed solid-phase extraction magnetic beads prepared in this invention have good extraction effects on vitamins A, D and E with large differences in hydrophilicity; and have good recovery rates for samples with different vitamin concentrations.

[0022] In some implementations, step S1 includes: S11, nanomagnetic cores prepared by solvothermal method.

[0023] Superparamagnetic nanoparticles can be prepared using a solvothermal method, endowing solid-phase extraction magnetic beads with excellent magnetic response. The solvothermal method is a commonly used method in this field, and those skilled in the art can prepare the nanomagnetic core using conventional techniques.

[0024] In some implementations, step S1 includes: S12, the nano-magnetic core is modified with silicon oxide to obtain silicon-coated magnetic beads; S13, the silicon layer coated magnetic beads obtained in step S12 are subjected to surface functional group modification to obtain double bond type magnetic beads.

[0025] The above provides a method for preparing double-bonded magnetic beads. The double bond modification can be prepared by the well-known Stber method. For example, the surface of double-bonded magnetic beads obtained by hydrolysis of organosilicon esters and silane coupling agents has a large number of abundant hydroxyl groups and double bond groups, which are provided by silane hydrolysis and silane coupling agents, respectively.

[0026] In some embodiments, the lipophilic monomer includes at least one of styrene, substituted styrene, divinylbenzene, substituted divinylbenzene, and acrylate.

[0027] Preferably, the lipophilic monomer is divinylbenzene.

[0028] In some embodiments, the hydrophilic monomer includes at least one of hydroxyethyl methacrylate, ethylene glycol dimethacrylate, acrylamide, triacetyl oxalic acid, and N-vinylpyrrolidone.

[0029] Preferably, the hydrophilic monomer is N-vinylpyrrolidone.

[0030] In some embodiments, the C18 monomer includes at least one of octadecene, oleic acid, and oleylamine.

[0031] Preferably, the C18 monomer is octadecene.

[0032] In some embodiments, the initiator includes at least one of azobisisobutyronitrile, azobisisoheptanenitrile, and benzoyl peroxide.

[0033] Preferably, the initiator is azobisisobutyronitrile.

[0034] In some embodiments, the mass ratio of the double bond magnetic beads, lipophilic monomer, hydrophilic monomer, and C18 monomer is 1:(1-10):(1-15):(1-10).

[0035] Preferably, the mass ratio of the double bond magnetic beads, lipophilic monomer, hydrophilic monomer and C18 monomer is 1:(1-5):(2-10):(1-5).

[0036] In some embodiments, the initiator accounts for 0.1-5% of the mass fraction of the mixture.

[0037] Preferably, the initiator accounts for 0.1-2% of the mass fraction of the mixture.

[0038] In some embodiments, the reaction temperature of step S2 is 30-100°C.

[0039] Preferably, the reaction temperature in step S2 is 30-70°C.

[0040] In some embodiments, the reaction time of step S2 is 1-48 hours.

[0041] Preferably, the reaction time of step S2 is 2-24 hours.

[0042] In some embodiments, step S2 includes: cooling the reaction mixture to a lower temperature, washing it with an alcohol solvent, and obtaining surface amphiphilic C18 mixed solid-phase extraction magnetic beads.

[0043] In some embodiments, the nanomagnetic core is a magnetic iron oxide.

[0044] Preferably, the nanomagnetic core is at least one of Fe3O4, Fe2O3, and NiFe2O4.

[0045] In some embodiments, the particle size of the nanomagnetic core is 300-600 nm.

[0046] Preferably, the particle size of the nanomagnetic core is 350-550 nm.

[0047] More preferably, the particle size of the nanomagnetic core is 400-500 nm.

[0048] In some embodiments, step S12 includes: reacting the nanomagnetic core with silicone grease under the action of a weak alkali to obtain silicon-coated magnetic beads.

[0049] In some embodiments, step S13 includes: under the protection of a protective gas, the silicon-coated magnetic beads obtained in step S12 react with a silane coupling agent under the action of an alkaline substance to obtain double-bonded magnetic beads.

[0050] In some embodiments, the silicone grease includes at least one of methyl orthosilicate, ethyl orthosilicate, propyl orthosilicate, and butyl orthosilicate.

[0051] In some embodiments, the ratio of the nanomagnetic core to the silicone grease is 1 g:(0.1-10) ml.

[0052] Preferably, the ratio of the nanomagnetic core to the silicone grease is 1 g:(0.1-5) ml.

[0053] In some embodiments, the weak base comprises ammonia water with a mass concentration of 1-20%.

[0054] Preferably, the mass concentration of the ammonia solution is 1-10%.

[0055] In some embodiments, the reaction temperature of step S12 is 20-60°C.

[0056] Preferably, the reaction temperature in step S12 is 20-50°C.

[0057] In some embodiments, the reaction time of step S12 is 1-24 h.

[0058] Preferably, the reaction time of step S12 is 12-18 h.

[0059] In some embodiments, the silane coupling agent includes at least one of 3-(trimethoxysilyl)methacrylate, vinyltrimethoxysilane, and vinyltriethoxysilane.

[0060] In some embodiments, the ratio of silicon-coated magnetic beads to silane coupling agent obtained in step S12 is 1 g:(0.1-10) ml.

[0061] Preferably, the ratio of silicon-coated magnetic beads to silane coupling agent obtained in step S12 is 1 g:(0.1-5) ml; In some embodiments, the alkaline substance includes ammonia water with a mass concentration of 5-20%.

[0062] Preferably, the mass concentration of the ammonia solution is 5-10%.

[0063] In some embodiments, the volume ratio of the silane coupling agent to the alkaline substance is 1:(1-10).

[0064] Preferably, the volume ratio of the silane coupling agent to the alkaline substance is 1:(1-5).

[0065] In some embodiments, the protective gas includes an inert gas or nitrogen.

[0066] Preferably, the protective gas is nitrogen.

[0067] In some embodiments, the reaction temperature of step S13 is 50-100°C.

[0068] Preferably, the reaction temperature in step S13 is 70-90°C.

[0069] In some embodiments, the reaction time of step S12 is 1-48 h.

[0070] Preferably, the reaction time of step S12 is 1-20 h.

[0071] More preferably, the reaction time of step S12 is 4-16 h.

[0072] The second aspect of the present invention provides an amphiphilic C18 polymer coating, which is obtained by free radical polymerization of lipophilic monomers, hydrophilic monomers and C18 monomers under an initiator, wherein the mass ratio of the lipophilic monomers, hydrophilic monomers and C18 monomers is (0.025-20):(0.025-20):1.

[0073] In some embodiments, the lipophilic monomer includes at least one of styrene, substituted styrene, divinylbenzene, substituted divinylbenzene, and acrylate.

[0074] Preferably, the lipophilic monomer is divinylbenzene;

[0075] In some embodiments, the hydrophilic monomer includes at least one of hydroxyethyl methacrylate, ethylene glycol dimethacrylate, acrylamide, triacetyl oxalic acid, and N-vinylpyrrolidone.

[0076] Preferably, the hydrophilic monomer is N-vinylpyrrolidone.

[0077] In some embodiments, the C18 monomer includes at least one of octadecene, oleic acid, and oleylamine.

[0078] Preferably, the C18 monomer is octadecene.

[0079] In some embodiments, the initiator includes at least one of azobisisobutyronitrile, azobisisoheptanenitrile, and benzoyl peroxide.

[0080] Preferably, the initiator is azobisisobutyronitrile; In some embodiments, the mass ratio of the lipophilic monomer, the hydrophilic monomer, and the C18 monomer is (0.1-20):(0.1-20):1.

[0081] Preferably, the mass ratio of the lipophilic monomer, the hydrophilic monomer, and the C18 monomer is (0.2-5):(0.4-10):1.

[0082] The third aspect of the present invention provides a surface amphiphilic C18 mixed solid-phase extraction magnetic bead, wherein the preparation method of the surface amphiphilic C18 mixed solid-phase extraction magnetic bead is selected from the preparation method of the surface amphiphilic C18 mixed solid-phase extraction magnetic bead provided by the present invention. Alternatively, the surface amphiphilic C18 hybrid solid-phase extraction magnetic beads include a core and a shell, the shell including a base layer and a functional layer; the base layer covers the surface of the core, the functional layer covers the surface of the base layer, and the functional layer includes the amphiphilic C18 polymer coating provided by the present invention.

[0083] In some embodiments, the core is a magnetic iron oxide.

[0084] Preferably, the core is at least one of Fe3O4, -Fe2O3, and NiFe2O4.

[0085] In some implementations, the kernel has a particle size of 300-600 nm.

[0086] Preferably, the particle size of the core is 350-550 nm.

[0087] More preferably, the particle size of the core is 400-500 nm.

[0088] In some embodiments, the thickness of the substrate is 1-200 nm.

[0089] In some implementations, the base layer includes double bond modifications.

[0090] In some embodiments, the thickness of the functional layer is 10-100 nm.

[0091] The fourth aspect of the present invention provides the use of the surface amphiphilic C18 mixed solid-phase extraction magnetic beads prepared by the present invention and / or the surface amphiphilic C18 mixed solid-phase extraction magnetic beads provided by the present invention for preparing reagents for extracting target molecules from samples.

[0092] The fifth aspect of the present invention provides a method for extracting target molecules from a sample, comprising: contacting a sample with surface amphiphilic C18 mixed solid-phase extraction magnetic beads prepared in the present invention and / or surface amphiphilic C18 mixed solid-phase extraction magnetic beads provided in the present invention to obtain solid-phase extraction magnetic beads adsorbing target molecules; magnetically separating the solid-phase extraction magnetic beads adsorbing target molecules; and eluting the target molecules.

[0093] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available. Test methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or according to the conditions recommended by the respective manufacturers.

[0094] The following method was used to prepare surface amphiphilic C18 hybrid solid-phase extraction magnetic beads: 1. Preparation of nanomagnetic cores: 9.45 g FeCl36H2O and 6.52 g FeCl24H2O were dissolved in 400 mL ethylene glycol by stirring to form a homogeneous solution. Then, 16.65 g anhydrous sodium acetate, 2.33 g polyethylene glycol 4000, and 4.2 g sodium citrate were added and stirred until dissolved. The mixture was heated to 120℃ in an oil bath and maintained for 2 h. After cooling to 80℃, it was transferred to a hydrothermal reactor and placed in an oven at 200℃ for 10 h. After naturally cooling to room temperature, the supernatant was filtered off, and the mixture was washed three times each with anhydrous ethanol and deionized water, and finally dispersed in deionized water.

[0095] 2. Silicon coating on the surface of the nano-magnetic cores: 5 g of the nano-magnetic cores prepared in the previous step were weighed and uniformly dispersed in 250 mL of 80% (v / v) ethanol solution. Then, 3.75 mL of tetraethyl silicate was introduced into the solution, and the mixture was mechanically stirred at 25 °C for 30 min. Then, 3.75 mL of ammonia water was added, and the temperature was raised to 40 °C to continue the reaction for 15 h. After the product was washed with ethanol and water by magnetic adsorption, magnetic microparticles with a silicon coating were obtained.

[0096] 3. Surface double bond modification: 5 g of the silica-coated magnetic microparticles were weighed and uniformly dispersed in 250 mL of 80% (v / v) ethanol solution. Then, 5 mL of 3-(trimethoxysilyl)propyl methacrylate (KH570) was introduced into the solution. The mixture was mechanically stirred at 25 °C for 30 min, and then 5 mL of ammonia water was added. The temperature was raised to 50 °C and the reaction was continued for 15 h. After the product was washed with ethanol and water by magnetic adsorption, magnetic microspheres with double bonds on the surface were obtained.

[0097] 4. Amphiphilic polymer coating: 5 g of the modified double-bonded magnetic beads were uniformly dispersed in 250 mL of acetonitrile. N2 was bubbled into the solution for 30 min to remove dissolved oxygen. Then, under mechanical stirring, lipophilic monomers (divinylbenzene), hydrophilic monomers (N-vinylpyrrolidone), C18 monomers (octadecene), and initiator (0.205 g of azobisisobutyronitrile) were introduced. The temperature was raised to 70 °C and the reaction was carried out under Serving conditions for 20 h. After the reaction was completed, the magnetic separation was achieved by washing with acetonitrile and ethanol, respectively. The magnetic separation was then carried out by drying at 60 °C to obtain a dry powder, which is the surface amphiphilic C18 mixed solid-phase extraction magnetic bead.

[0098] In the amphiphilic polymer coating, the amounts of lipophilic monomers, hydrophilic monomers, and C18 monomers in each embodiment are shown in Table 1, and the amounts of lipophilic monomers, hydrophilic monomers, and C18 monomers in each comparative example are shown in Table 2.

[0099] Table 1 lipophilic monomer / g hydrophilic monomer / g C18 monomer / g Example 1 2.5 2.5 5 Example 2 100 100 100 Example 3 5 5 5 Example 4 50 75 50 Example 5 5 10 5 Example 6 25 50 25 Example 7 15 40 20 Table 2 lipophilic monomer / g hydrophilic monomer / g C18 monomer / g Comparative Example 1 0 0 20 Comparative Example 2 0 40 20 Comparative Example 3 15 0 20 Comparative Example 4 15 40 0 Comparative Example 5 2 2 120 Comparative Example 6 150 2 120 The wetting properties of surface amphiphilic C18 hybrid solid-phase extraction magnetic beads are characterized by the water contact angle, and the specific test method is as follows: A dispersion of surface-amphiphilic C18 mixed-type solid-phase extraction magnetic beads was uniformly coated onto a glass slide. After the microspheres were completely dry, their water contact angle was measured at room temperature using an XG-CAM contact angle meter. The droplet volume for each measurement was approximately 3 L, and five parallel tests were performed, with the average value taken. The shortest distance between measurement points on the same sample was 10 mm. The results are as follows: Figure 1 As shown.

[0100] In this study, a represents Comparative Example 1, b represents Comparative Example 2, c represents Comparative Example 3, and d represents Example 7. The static water contact angle indicates that the C18 monomer itself exhibits hydrophobic properties on the magnetic bead surface. The introduction of the hydrophilic monomer modifies the hydrophilicity of the magnetic bead surface, decreasing the static water contact angle from 135.0 in the Comparative Example to 90.0. The introduction of the lipophilic monomer increases the water contact angle on the magnetic bead surface to 145, while the surface contact angle of Example 7 is 115.0. This demonstrates the changing trend of the hydrophilic and lipophilic monomers on the wettability of the magnetic bead surface, and also illustrates the amphiphilic characteristics of the amphiphilic C18 mixed-type solid-phase extraction magnetic beads of Example 7.

[0101] The solid-phase extraction effects of vitamins A, D, and E are used to illustrate the application effect of surface amphiphilic C18 mixed solid-phase extraction magnetic beads.

[0102] Vitamin A, D, and E sample pretreatment: Weigh 100 mg of the surface-amphiphilic C18 mixed-type solid-phase extraction magnetic beads obtained in the above examples and comparative examples, and disperse them in 1000 L of methanol. Add 10 L of magnetic bead dispersion, 100 L of deionized water, 100 L of serum sample containing 64 ng / ml vitamin A, 64 ng / ml vitamin D, and 64 ng / ml vitamin E standards, 100 L of deionized water, and 100 L of methanol sequentially to a 96-well plate of an automated sample pretreatment system. The automated sample pretreatment system operates as follows: transfer the magnetic beads from the dispersion to deionized water and equilibrate by shaking for 10 s; then transfer the magnetic beads to the vitamin serum sample and mix by shaking for 15 s; next, transfer the vitamin-adsorbed magnetic beads to deionized water and wash by shaking for 10 s; then transfer the magnetic beads to methanol and elute by shaking for 10 s; finally, transfer the magnetic beads to the next well. The sample supernatant from the elution step is the vitamin A, D, and E sample extracted by magnetic beads. The sample can be filtered through a 0.22 μm filter membrane or directly loaded onto HPLC-MS.

[0103] Chromatographic conditions for the detection of vitamins A, D, and E in samples: Column: Waters ACQUITY UPLC BEH Shield RP18 (2.150 mm, 1.7 m).

[0104] Testing instrument: Shimadzu 8060LC-MS / MS.

[0105] Mobile phase: Mobile phase A: 0.1% formic acid aqueous solution; Mobile phase B: pure acetonitrile; Elution gradient: 0-2 min 40% B, 2-4 min 40-60% B, 4-5 min 60-100% B, 5-7 min 100% B, 7-7.5 min 100-40% B, 7.5-9 min 40% B; Column temperature: 40℃; Flow rate: 0.3 mL / min; Injection volume: 5 L.

[0106] Mass spectrometry conditions for vitamin A, D, and E sample detection: Ion source: Electrospray ionization (ESI) source, in positive ion mode, ion source temperature: 500℃; atomizing gas flow rate: 3.0 L / min; drying gas flow rate: 10.0 L / min; heating gas flow rate: 10.0 L / min; detection is performed using MRM multiple reaction monitoring mode.

[0107] The results of solid-phase extraction pretreatment tests for vitamins A, D, and E are shown in Table 3 and Figure 2 As shown, the mass spectrometry peak areas of substances extracted from vitamins A, D, and E samples by magnetic beads were compared. In mass spectrometry detection, the peak area value and concentration value are linearly related, and the amount of detected substances in the sample can be directly compared using the peak area value. Comparative Example 1 contains only the C18 component, which has a very strong adsorption for low-polarity vitamin E, but poor adsorption effect for relatively high-polarity vitamin A. Comparative Example 2, which added a hydrophilic monomer, and Comparative Example 3, which added a lipophilic monomer, showed increased adsorption amounts for vitamins A and E, respectively, and correspondingly decreased adsorption effects for molecules of opposite polarity. The surface amphiphilic C18 mixed solid-phase extraction magnetic beads of Example 7 have a synergistic effect of hydrophilicity, lipophilicity, and C18, and can adsorb vitamins A, D, and E simultaneously, indicating that the surface amphiphilic C18 mixed solid-phase extraction magnetic beads can adsorb molecules in a wider polar to non-polar range.

[0108] Table 3 sample Comparative Example 1 Comparative Example 2 Comparative Example 3 Example 7 Vitamin A 710 49884 3581 1591345 Vitamin D 1185542 584236 1246846 1178563 Vitamin E 1676546 1223564 1698349 1665985 To further illustrate the role of the hydrophilic monomer, lipophilic monomer, and C18 monomer described in this invention, and to highlight the application effect of the surface amphiphilic C18 mixed solid phase extraction magnetic beads, vitamin A was selected as the target molecule, and the extraction effect was comprehensively verified on the samples of Examples 1-7 and Comparative Examples 1-6 respectively.

[0109] The specific operation and detection methods are the same as above, the difference being that the samples used are serum samples containing different concentrations (1, 4, 16, 32, and 64 ng / ml) of vitamin A. The peak area results of the magnetic bead extraction of vitamin A samples from different examples and comparative examples are shown in Table 4. Figure 3 .

[0110] Table 4 Standard concentration ng / ml Example 1 Example 2 Example 3 Example 4 Example 5 Example 6 Example 7 Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 Comparative Example 5 Comparative Example 6 1 11330 14163 7939 21118 15959 21920 25514 10326 20699 9255 2383 9841 7957 4 37350 46498 25965 69208 53264 72709 84818 34067 68336 30125 8181 32297 27509 16 150132 186639 104112 273135 210482 287812 341638 137856 273809 118936 35867 128834 107219 32 314472 390605 220877 576214 443852 602082 708381 288348 572391 249682 68439 268367 225556 64 654762 844015 523726 1240137 1018311 1371164 1597573 657945 1239200 552983 123736 647362 485672 As shown in the figure, the recovery rates of the C18 solid-phase extraction magnetic beads prepared in this invention remained basically consistent for samples of different concentrations, indicating that the magnetic beads can adapt to the application requirements of samples with different concentrations. The extraction effects of vitamin A varied significantly among different examples and comparative examples, with Example 7 and examples with similar addition parameters showing the highest signal values ​​for vitamin A extraction. The ratio of C18 monomer to hydrophilic monomer plays a decisive role in the extraction effect of vitamin A; without the addition of C18 and / or hydrophilic monomer, the extraction effect was relatively poor.

[0111] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent modifications or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing surface amphiphilic C18 hybrid solid-phase extraction magnetic beads, characterized in that, Including the following steps: S1, the surface of the nano-magnetic core is modified with double bonds to obtain double-bonded magnetic beads; S2, a mixture containing double-bonded magnetic beads obtained in step S1, lipophilic monomers, hydrophilic monomers, and C18 monomers undergoes free radical polymerization under an initiator to obtain surface amphiphilic C18 mixed solid-phase extraction magnetic beads. The mass ratio of the double bond type magnetic beads, lipophilic monomer, hydrophilic monomer and C18 monomer is 1:(0.5-20):(0.5-20):(1-20).

2. The method according to claim 1, wherein, Step S1 includes: S11, preparation of nanomagnetic cores by solvothermal method; And / or, step S1 includes: S12, the nano-magnetic core is modified with silicon oxide to obtain silicon-coated magnetic beads; S13, the silicon layer coated magnetic beads obtained in step S12 are modified with surface functional groups to obtain double bond type magnetic beads; And / or, the lipophilic monomer includes at least one of styrene, substituted styrene, divinylbenzene, substituted divinylbenzene, and acrylate; And / or, the hydrophilic monomer includes at least one of hydroxyethyl methacrylate, ethylene glycol dimethacrylate, acrylamide, triacetyl oxalic acid, and N-vinylpyrrolidone; And / or, the C18 monomer includes at least one of octadecene, oleic acid, and oleylamine; And / or, the initiator includes at least one of azobisisobutyronitrile, azobisisoheptanenitrile, and benzoyl peroxide; And / or, the mass ratio of the double bond type magnetic beads, lipophilic monomers, hydrophilic monomers and C18 monomers is 1:(1-10):(1-15):(1-10); And / or, the initiator accounts for 0.1-5% of the mass fraction of the mixture; And / or, the reaction temperature of step S2 is 30-100℃; And / or, the reaction time of step S2 is 1-48 hours; And / or, step S2 includes: cooling the reaction mixture to a lower temperature, washing it with an alcohol solvent, and obtaining surface amphiphilic C18 mixed-type solid-phase extraction magnetic beads.

3. The method according to claim 1 or 2, wherein, The nano-magnetic core is a magnetic iron oxide; And / or, the particle size of the nanomagnetic core is 300-600 nm; And / or, step S12 includes: reacting the nano-magnetic core with organosilicon grease under the action of a weak alkali to obtain silicon-coated magnetic beads; And / or, step S13 includes: under the protection of a protective gas, the silicon-coated magnetic beads obtained in step S12 react with a silane coupling agent under the action of an alkaline substance to obtain double-bonded magnetic beads.

4. The method according to claim 3, wherein, The silicone grease includes at least one of methyl orthosilicate, ethyl orthosilicate, propyl orthosilicate, and butyl orthosilicate. And / or, the ratio of the nanomagnetic core to the silicone grease is 1g:(0.1-10)ml; And / or, the weak base includes ammonia solution with a mass concentration of 1-20%; And / or, the reaction temperature of step S12 is 20-60℃; And / or, the reaction time of step S12 is 1-24 hours; And / or, the silane coupling agent comprises at least one of 3-(trimethoxysilyl)methacrylate, vinyltrimethoxysilane, and vinyltriethoxysilane; And / or, the ratio of silicon-coated magnetic beads to silane coupling agent obtained in step S12 is 1g:(0.1-10)ml; And / or, the alkaline substance includes ammonia water, the mass concentration of which is 5-20%; And / or, the volume ratio of the silane coupling agent to the alkaline substance is 1:(1-10); And / or, the protective gas includes an inert gas or nitrogen; And / or, the reaction temperature of step S13 is 50-100℃; And / or, the reaction time of step S12 is 1-48h.

5. An amphiphilic C18 polymer coating, characterized in that, The amphiphilic C18 polymer coating is obtained by free radical polymerization of lipophilic monomers, hydrophilic monomers, and C18 monomers under an initiator, wherein the mass ratio of the lipophilic monomers, hydrophilic monomers, and C18 monomers is (0.025-20):(0.025-20):

1.

6. The amphiphilic C18 polymer coating according to claim 5, wherein, The lipophilic monomer includes at least one of styrene, substituted styrene, divinylbenzene, substituted divinylbenzene, and acrylate; And / or, the hydrophilic monomer includes at least one of hydroxyethyl methacrylate, ethylene glycol dimethacrylate, acrylamide, triacetyl oxalic acid, and N-vinylpyrrolidone; And / or, the C18 monomer includes at least one of octadecene, oleic acid, and oleylamine; And / or, the initiator includes at least one of azobisisobutyronitrile, azobisisoheptanenitrile, and benzoyl peroxide; And / or, the mass ratio of the lipophilic monomer, hydrophilic monomer and C18 monomer is (0.1-20):(0.1-20):

1.

7. A surface-amphiphilic C18 hybrid solid-phase extraction magnetic bead, characterized in that, The preparation method of the surface amphiphilic C18 hybrid solid phase extraction magnetic beads is selected from the method described in any one of claims 1-4; Alternatively, the surface amphiphilic C18 hybrid solid-phase extraction magnetic beads include a core and a shell, the shell including a base layer and a functional layer; the base layer covers the surface of the core, the functional layer covers the surface of the base layer, and the functional layer includes the amphiphilic C18 polymer coating as described in claim 5 or 6.

8. The surface amphiphilic C18 hybrid solid-phase extraction magnetic beads according to claim 7, wherein, The core is a magnetic iron oxide; And / or, the particle size of the core is 300-600 nm; And / or, the thickness of the base layer is 1-200 nm; And / or, the base layer includes double bond modifications; And / or, the thickness of the functional layer is 10-100 nm.

9. Use of the surface amphiphilic C18 hybrid solid-phase extraction magnetic beads as described in claim 7 or 8 for preparing a reagent for extracting target molecules from a sample.

10. A method for extracting target molecules from a sample, characterized in that, include: The surface amphiphilic C18 hybrid solid-phase extraction magnetic beads as described in claim 7 or 8 are contacted with the sample to obtain solid-phase extraction magnetic beads that adsorb the target molecules; the solid-phase extraction magnetic beads that adsorb the target molecules are magnetically separated; and the target molecules are eluted.

Images