Magnetic surface molecularly imprinted polymer and preparation method thereof

Abstract

The application belongs to the technical field of molecular imprinting polymers, and provides a magnetic surface molecular imprinting polymer, which comprises the following preparation raw materials: 5-hydroxytryptamine, a functional monomer, a porogen, a crosslinking agent, a four-iron oxide magnetic carrier coated with a silica layer and an initiator. The application also provides a preparation method of the magnetic surface molecular imprinting polymer. The magnetic surface molecular imprinting polymer prepared by the application has a high specific surface area, good biocompatibility and stability, and excellent magnetic sensitivity. The magnetic surface molecular imprinting polymer not only makes up for the disadvantages of traditional molecular imprinting polymers, such as difficult elution, template leakage and slow mass transfer rate, but also solves the problems of difficult separation and time consumption. The magnetic surface molecular imprinting polymer can be directly used as an adsorbent for solid-phase microextraction, can greatly increase the selectivity of adsorption, can improve the separation and enrichment efficiency, can simplify the experimental process, can reduce the experimental cost, and has easy-to-control preparation conditions and good stability. The magnetic surface molecular imprinting polymer can effectively and highly selectively separate and enrich trace 5-HT in a sample.

Description

Technical Field

[0001] This invention relates to the field of molecularly imprinted polymer technology, and in particular to a magnetic surface molecularly imprinted polymer and its preparation method. Background Technology

[0002] Serotonin (5-HT) is mainly found in the gastrointestinal tract, platelets, and central nervous system of mammals, and regulates their physiological functions as a neurotransmitter and hormone in most major organ systems, including the gastrointestinal tract and central nervous system. Many studies have shown that 5-HT can serve as a biomarker, playing an important role in various diseases such as autism spectrum disorder, attention deficit hyperactivity disorder, coronary microvascular dysfunction, atherosclerotic cardiovascular disease, chronic heart failure, and vascular complications of diabetes.

[0003] Current quantitative research on 5-HT primarily focuses on the central nervous system. However, studies have indicated that variations in trace 5-HT levels in peripheral body fluids also influence various mental illnesses, making quantitative research equally important. The concentration of 5-HT in peripheral body fluid samples is extremely low compared to cerebrospinal fluid samples, and complex matrix interference exists, preventing accurate quantification using conventional detection methods due to detection limits. Therefore, a rapid and efficient sample pretreatment method is needed to effectively separate and enrich trace 5-HT in peripheral body fluid samples, thereby improving the sensitivity and accuracy of analytical methods.

[0004] Molecular imprinting is a polymer preparation technique that exhibits high affinity and selectivity for specific compounds. Due to their advantages such as good specificity, simple preparation, and good reproducibility, molecularly imprinted polymers have wide applications in chiral separation, chromatographic separation, biosensors, catalyst screening, and solid-phase extraction. Magnetic surface molecularly imprinted polymers, due to their imprinting polymerization on magnetic carrier surfaces, possess the advantages of abundant imprinting sites and thin surface molecularly imprinted layers, overcoming the shortcomings of traditional molecularly imprinted polymers such as difficult elution, template leakage, and slow mass transfer rates. Furthermore, the external magnetic field can be directly controlled to rapidly separate and recover magnetic surface molecularly imprinted polymers from complex matrices without the need for additional centrifugation or filtration, simplifying the separation process. Therefore, selecting magnetic surface molecularly imprinted polymers as adsorbents for magnetic solid-phase microextraction to enrich trace amounts of 5-HT in peripheral body fluid samples from complex matrices shows promising application prospects. Summary of the Invention

[0005] The purpose of this invention is to provide a magnetic surface molecularly imprinted polymer and its preparation method to address the shortcomings of existing technologies.

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

[0007] This invention provides a magnetic surface molecularly imprinted polymer, comprising the following raw materials:

[0008] 5-hydroxytryptamine, functional monomers, porogens, crosslinking agents, ferric oxide magnetic carriers coated with silica layers, and initiators;

[0009] The molar ratio of 5-hydroxytryptamine, functional monomer, and crosslinking agent is 1:3.8-4.2:16-18;

[0010] The molar volume ratio of 5-hydroxytryptamine to the porogen is 1 mmol: 180-220 mL;

[0011] The mass ratio of the magnetite carrier coated with silicon dioxide layer to the initiator is 550-650:28-32;

[0012] The molar ratio of 5-hydroxytryptamine to the initiator is 1 mmol: 28–32 mg.

[0013] Preferably, the functional monomer comprises methacrylic acid and acrylamide, wherein the molar ratio of methacrylic acid and acrylamide is 1:0.9 to 1.1; the porogen is toluene, the crosslinking agent is ethylene glycol dimethacrylate, and the initiator is azobisisopropionitrile.

[0014] Preferably, the method for preparing the silicon dioxide-coated magnetite includes the following steps:

[0015] 1) FeCl3·6H2O, FeCl2·4H2O and water were mixed, and ammonia and oleic acid were added in sequence to react and then dried to obtain magnetic nanoparticles of iron oxide.

[0016] 2) Mix magnetite nanoparticles, styrene, 3-(trimethoxysilyl)propyl methacrylate and cyclohexane to obtain an oil phase; mix sodium dodecyl sulfonate, disodium hydrogen phosphate, sodium dihydrogen phosphate and water to obtain an aqueous phase; after ultrasonic mixing of the oil phase and the aqueous phase, add potassium persulfate to carry out a polymerization reaction to obtain magnetite magnetic nanospheres.

[0017] 3) After mixing the magnetic nanospheres of iron oxide and anhydrous ethanol, ammonia and tetraethyl silicate were added to react and obtain the magnetic carrier of iron oxide coated with a silicon dioxide layer.

[0018] Preferably, the volume fraction of ammonia in step 1) is 25-28%; the molar ratio of FeCl3·6H2O to FeCl2·4H2O is 1:1.5-4; the volume ratio of water, ammonia, and oleic acid is 180-220:18-22:0.6-1; and the molar volume ratio of FeCl3·6H2O to water is 0.004mol:180-220mL.

[0019] Preferably, in step 1), when ammonia is added for reaction, the reaction temperature is 45–55°C and the reaction time is 25–35 min; when oleic acid is added for reaction, the reaction time is 2–4 h; and the drying temperature is 60–80°C and the drying time is 8–12 h.

[0020] Preferably, in step 2), the mass-to-volume ratio of the magnetic nanoparticles of iron oxide, styrene, 3-(trimethoxysilyl)propyl methacrylate, and cyclohexane is 1–1.4 g : 3.5–4.5 mL : 1.5–2.5 mL : 1–2 mL; the mass-to-volume ratio of sodium dodecyl sulfonate, disodium hydrogen phosphate, sodium dihydrogen phosphate, and water is 0.4–0.5 g : 0.2–0.25 g : 0.1–0.15 g : 120–160 mL; and the mass ratio of the magnetic nanoparticles of iron oxide to sodium dodecyl sulfonate is 1–1.4 : 0.4–0.5.

[0021] Preferably, in step 2), the mass ratio of potassium persulfate to sodium dodecyl sulfonate is 0.05–0.07:0.4–0.5; the polymerization temperature is 65–75°C; the polymerization time is 10–14 h; and the polymerization is carried out in a nitrogen atmosphere.

[0022] Preferably, in step 3), the mass-to-volume ratio of the magnetic nanospheres of iron oxide, anhydrous ethanol, ammonia, and tetraethyl silicate is 580–620 mg: 80–120 mL: 13–17 mL: 2–4 mL; the reaction temperature is 20–30 °C, and the reaction time is 11–13 h.

[0023] The present invention also provides a method for preparing the aforementioned magnetic surface molecularly imprinted polymer, comprising the following steps:

[0024] (1) After prepolymerizing 5-hydroxytryptamine, functional monomers and porogens, a crosslinking agent, a magnetic carrier coated with silica layer and an initiator are added to carry out a polymerization reaction to obtain a magnetic surface molecular imprinted polymer layer with 5-hydroxytryptamine.

[0025] (2) The magnetic surface molecular imprinted polymer layer containing 5-hydroxytryptamine is eluted to obtain the magnetic surface molecular imprinted polymer.

[0026] Preferably, the temperature of the prepolymerization reaction in step (1) is 24-26°C and the time of the prepolymerization reaction is 0.5-1.5h; the temperature of the polymerization reaction is 60-80°C and the time of the polymerization reaction is 11-13h; the eluent used for elution in step (2) contains methanol and acetic acid, and the volume ratio of methanol to acetic acid is 3-5:1.

[0027] The beneficial effects of this invention include the following:

[0028] 1) The magnetic surface molecularly imprinted polymer prepared by this invention not only has a high specific surface area, good biocompatibility and stability, but also excellent magnetic sensitivity.

[0029] 2) The magnetic surface molecularly imprinted polymer of the present invention not only makes up for the shortcomings of traditional molecularly imprinted polymers, such as difficult elution, template leakage and slow mass transfer rate, but also solves the problems of difficult separation and time consumption.

[0030] 3) This invention directly uses magnetic surface molecularly imprinted polymers as adsorbents for solid-phase microextraction, which not only greatly increases the selectivity of adsorption and improves the separation and enrichment efficiency, but also simplifies the experimental process, reduces experimental costs, and makes the preparation conditions easy to control and has good stability.

[0031] 4) The magnetic surface molecularly imprinted polymer of the present invention can be reused and is particularly suitable for the separation and enrichment of small molecules in biological samples. It has broad application prospects in the research of solid phase microextraction pretreatment methods.

[0032] 5) The magnetic surface molecularly imprinted polymer of the present invention can effectively and selectively separate and enrich trace amounts of 5-HT in the sample. Attached Figure Description

[0033] Figure 1 This is a flowchart illustrating the preparation process of the magnetic surface molecularly imprinted polymer of the present invention.

[0034] Figure 2 The images shown are transmission electron microscope (TEM) images of the magnetic materials prepared in Example 1. In the images, A is a TEM image of magnetite magnetic nanoparticles, B is a TEM image of magnetite magnetic nanospheres, C is a TEM image of magnetite magnetic carriers coated with a silicon dioxide layer, and D is a TEM image of a polymer with magnetic surface molecular imprinting.

[0035] Figure 3 The images shown are scanning electron microscope (SEM) images of the magnetic materials prepared in Example 1. In the images, A is a scanning electron microscope image of magnetite magnetic nanoparticles, B is a scanning electron microscope image of magnetite magnetic nanospheres, C is a scanning electron microscope image of magnetite magnetic carrier coated with a silicon dioxide layer, and D is a scanning electron microscope image of a polymer with magnetic surface molecular imprinting.

[0036] Figure 4The Fourier transform infrared spectra of the magnetic materials prepared in Example 1 are shown below. In this spectrum, A is the Fourier transform infrared spectrum of the magnetite magnetic nanoparticles, B is the Fourier transform infrared spectrum of the magnetite magnetic nanospheres, C is the Fourier transform infrared spectrum of the magnetite magnetic carrier coated with a silicon dioxide layer, and D is the Fourier transform infrared spectrum of the polymer with magnetic surface molecular imprinting.

[0037] Figure 5 X-ray diffraction patterns of the magnetic materials prepared in Example 1 are shown below. In the figure, A is the X-ray diffraction pattern of magnetite magnetic nanoparticles, B is the X-ray diffraction pattern of magnetite magnetic nanospheres, C is the X-ray diffraction pattern of magnetite magnetic carrier coated with silica layer, and D is the X-ray diffraction pattern of magnetic surface molecularly imprinted polymer.

[0038] Figure 6 The following are the thermogravimetric curves of the magnetic materials prepared in Example 1: A is the thermogravimetric curve of the magnetite magnetic nanoparticles, B is the thermogravimetric curve of the magnetite magnetic nanospheres, C is the thermogravimetric curve of the magnetite magnetic carrier coated with a silicon dioxide layer, and D is the thermogravimetric curve of the magnetic surface molecularly imprinted polymer.

[0039] Figure 7 The magnetic hysteresis loop diagrams are for the magnetic materials prepared in Example 1, where A is the magnetic hysteresis loop diagram of magnetite magnetic nanoparticles, B is the magnetic hysteresis loop diagram of magnetite magnetic nanospheres, C is the magnetic hysteresis loop diagram of magnetite magnetic carriers coated with silicon dioxide layer, and D is the magnetic hysteresis loop diagram of magnetic surface molecularly imprinted polymers.

[0040] Figure 8 The adsorption capacity diagrams are for the imprinted polymers of Examples 1 and Comparative Examples 1-9 under different porogens.

[0041] Figure 9 The adsorption capacity diagrams are for the imprinted polymers of Example 1, Comparative Examples 1, and Comparative Examples 10-21 under different amounts of crosslinking agent.

[0042] Figure 10 The adsorption capacity diagrams are for the imprinted polymers of Example 1, Comparative Examples 1, and Comparative Examples 22-29 under different initiator dosages.

[0043] Figure 11 The static isothermal adsorption diagrams are for the magnetic surface molecularly imprinted polymer of Example 1 and the magnetic surface non-molecularly imprinted polymer of Comparative Example 1.

[0044] Figure 12 The kinetic adsorption diagrams are for the magnetic surface molecularly imprinted polymer of Example 1 and the magnetic surface non-molecularly imprinted polymer of Comparative Example 1.

[0045] Figure 13 The selective adsorption diagrams are for the magnetic surface molecularly imprinted polymer of Example 1 and the magnetic surface non-molecularly imprinted polymer of Comparative Example 1.

[0046] Figure 14 The diagram shows the reusability of the magnetic surface molecularly imprinted polymer of Example 1 and the magnetic surface non-molecularly imprinted polymer of Comparative Example 1.

[0047] Figure 15 The image shows the magnetic solid-phase microextraction effect of the magnetic surface molecularly imprinted polymer in alternative serum in Example 1. In the image, A is a 0.1 ng / mL alternative serum solution, B is the supernatant of the 0.1 ng / mL alternative serum solution after magnetic separation by adsorption of MMIPs, and C is the solution after elution of MMIPs by magnetic separation.

[0048] Figure 16 The image shows the magnetic solid-phase microextraction effect of the non-molecularly imprinted polymer on the magnetic surface in comparative example 1 in alternative serum. In the image, A is the alternative serum solution with a concentration of 0.1 ng / mL, B is the supernatant of the alternative serum solution with a concentration of 0.1 ng / mL after magnetic separation by adsorption of MNIPs, and C is the solution after elution of MNIPs by magnetic separation. Detailed Implementation

[0049] This invention provides a magnetic surface molecularly imprinted polymer, comprising the following raw materials:

[0050] 5-hydroxytryptamine, functional monomers, porogens, crosslinking agents, ferric oxide magnetic carriers coated with silica layers, and initiators;

[0051] The molar ratio of 5-hydroxytryptamine, functional monomer, and crosslinking agent is 1:3.8-4.2:16-18;

[0052] The molar volume ratio of 5-hydroxytryptamine to the porogen is 1 mmol: 180-220 mL;

[0053] The mass ratio of the magnetite carrier coated with silicon dioxide layer to the initiator is 550-650:28-32;

[0054] The molar ratio of 5-hydroxytryptamine to the initiator is 1 mmol: 28–32 mg.

[0055] In this invention, the molar ratio of 5-hydroxytryptamine, functional monomer and crosslinking agent is 1:3.8-4.2:16-18, preferably 1:3.9-4.1:16.5-17.5, and more preferably 1:4:17;

[0056] The molar volume ratio of 5-hydroxytryptamine to porogen is 1 mmol: 180-220 mL, preferably 1 mmol: 185-215 mL, more preferably 1 mmol: 190-210 mL, and even more preferably 1 mmol: 200 mL;

[0057] The mass ratio of the magnetite magnetic carrier coated with silicon dioxide layer to the initiator is 550-650:28-32, preferably 570-630:28.5-31.5, more preferably 590-610:29-31, and even more preferably 600:30;

[0058] The molar ratio of 5-hydroxytryptamine to the initiator is 1 mmol: 28-32 mg, preferably 1 mmol: 29-31 mg, and more preferably 1 mmol: 30 mg.

[0059] In this invention, the functional monomer preferably comprises methacrylic acid and acrylamide, and the molar ratio of methacrylic acid and acrylamide is preferably 1:0.9 to 1.1, more preferably 1:1; the porogen is preferably toluene, the crosslinking agent is preferably ethylene glycol dimethacrylate, and the initiator is preferably azobisisopropionitrile.

[0060] The preparation flow chart of the magnetic surface molecularly imprinted polymer of the present invention is shown below. Figure 1 As shown.

[0061] In this invention, the preparation method of the magnetite carrier coated with a silicon dioxide layer includes the following steps:

[0062] 1) FeCl3·6H2O, FeCl2·4H2O and water were mixed, and then ammonia and oleic acid were added sequentially to react and dry, resulting in magnetic nanoparticles of iron oxide (Fe3O4). Figure 1 (Fe3O4 in)

[0063] 2) Magnetic nanoparticles of iron oxide, styrene, 3-(trimethoxysilyl)propyl methacrylate, and cyclohexane were mixed to obtain an oil phase; sodium dodecyl sulfonate, disodium hydrogen phosphate, sodium dihydrogen phosphate, and water were mixed to obtain an aqueous phase; the oil and aqueous phases were ultrasonically mixed, and potassium persulfate was added to carry out a polymerization reaction to obtain magnetic nanospheres of iron oxide. Figure 1 Fe3O4@poly);

[0064] 3) After mixing magnetite nanospheres and anhydrous ethanol, ammonia and tetraethyl silicate are added to react and obtain a magnetite carrier coated with a silica layer. Figure 1 (Fe3O4@poly@SiO2).

[0065] In this invention, the preferred method for mixing FeCl3·6H2O, FeCl2·4H2O and water in step 1) is ultrasonic mixing; after ultrasonic mixing, nitrogen gas is preferably introduced into the mixed solution for deoxygenation.

[0066] In this invention, the volume fraction of ammonia in step 1) is preferably 25-28%, more preferably 26%; the molar ratio of FeCl3·6H2O and FeCl2·4H2O is preferably 1:1.5-4, more preferably 1:2-3.5, and even more preferably 1:3; the volume ratio of water, ammonia, and oleic acid is preferably 180-220:18-22:0.6-1, more preferably 190-210:19-21:0.7-0.9, and even more preferably 200:20:0.8; the molar volume ratio of FeCl3·6H2O and water is preferably 0.004mol:180-220mL, more preferably 0.004mol:190-210mL, and even more preferably 0.004mol:200mL.

[0067] In this invention, when adding ammonia in step 1), it is preferably carried out under stirring conditions, and the stirring speed is preferably 280-320 r / min, more preferably 290-310 r / min, and even more preferably 300 r / min.

[0068] In this invention, when ammonia is added in step 1) to carry out the reaction, the reaction temperature is preferably 45-55°C, more preferably 48-52°C, and even more preferably 50°C; the reaction time is preferably 25-35 min, more preferably 28-32 min, and even more preferably 30 min; when oleic acid is added to carry out the reaction, the reaction time is preferably 2-4 h, more preferably 2.5-3.5 h, and even more preferably 3 h.

[0069] In this invention, the drying method in step 1) is preferably vacuum drying, the vacuum drying temperature is preferably 60-80℃, more preferably 65-75℃, and even more preferably 60℃; the vacuum drying time is preferably 8-12h, more preferably 9-11h, and even more preferably 10h.

[0070] In this invention, before drying in step 1), it is preferable to rinse the magnetic particles; the rinsing is preferably rinsing with anhydrous ethanol and cyclohexane, and the number of times the anhydrous ethanol rinsing and cyclohexane rinsing are performed independently is preferably 2 to 4 times, and more preferably 3 times.

[0071] In this invention, the preferred mass-to-volume ratio of the magnetic nanoparticles of iron oxide, styrene, 3-(trimethoxysilyl)propyl methacrylate, and cyclohexane in step 2) is 1–1.4 g.

[0072] 3.5–4.5 mL: 1.5–2.5 mL: 1–2 mL, more preferably 1.1–1.3 g: 3.8–4.2 mL:

[0073] The preferred mass-to-volume ratio of sodium dodecyl sulfonate, disodium hydrogen phosphate, sodium dihydrogen phosphate, and water is 0.4–0.5 g: 0.2–0.25 g: 0.1–0.15 g: 120–160 mL, more preferably 0.42–0.48 g: 0.22–0.24 g: 0.12–0.14 g: 130–150 mL, and more preferably 0.46 g: 0.23 g: 0.13 g: 140 mL. The preferred mass-to-volume ratio of the magnetite magnetic nanoparticles and sodium dodecyl sulfonate is 1–1.4: 0.4–0.5, more preferably 1.1–1.3: 0.42–0.48, and more preferably 1.2: 0.44–0.46.

[0074] In this invention, the preferred order of mixing the magnetic nanoparticles of iron oxide, styrene, 3-(trimethoxysilyl)propyl methacrylate and cyclohexane in step 2) is to first mix the styrene and 3-(trimethoxysilyl)propyl methacrylate, and then add the magnetic nanoparticles of iron oxide and cyclohexane for ultrasonication.

[0075] In this invention, after mixing sodium dodecyl sulfonate, disodium hydrogen phosphate, sodium dihydrogen phosphate and water in step 2), it is preferable to adjust the pH value of the solution; when adjusting the pH value of the solution, the reagent used is preferably a phosphoric acid solution, and the volume fraction of the phosphoric acid solution is preferably 80-85%, more preferably 82-85%, and even more preferably 85%; it is preferable to adjust the pH value of the solution to 6-8, and more preferably 7.

[0076] In this invention, the mass ratio of potassium persulfate to sodium dodecyl sulfonate in step 2) is preferably 0.05-0.07:0.4-0.5, more preferably 0.06:0.45; the temperature of the polymerization reaction is preferably 65-75°C, more preferably 68-72°C, and more preferably 70°C; the time of the polymerization reaction is preferably 10-14 h, more preferably 11-13 h, and more preferably 12 h; the polymerization reaction is preferably carried out in a nitrogen atmosphere.

[0077] In this invention, after the polymerization reaction in step 2) is completed, the product is preferably washed and vacuum dried sequentially; the washing reagent is preferably deionized water, the number of washings is preferably 2 to 4 times, more preferably 3 times; the vacuum drying temperature is preferably 50 to 70°C, more preferably 55 to 65°C, more preferably 60°C, and the vacuum drying time is preferably 7 to 12 hours, more preferably 9 to 11 hours, more preferably 10 hours.

[0078] In this invention, the preferred mass-to-volume ratio of the magnetite nanospheres, anhydrous ethanol, ammonia, and tetraethyl silicate in step 3) is 580–620 mg: 80–120 mL: 13–17 mL: 2–4 mL, more preferably 590–610 mg: 90–110 mL: 14–16 mL: 2.5–3.5 mL, and even more preferably 600 mg: 100 mL: 15 mL: 3 mL; the preferred reaction temperature is 20–30 °C, more preferably 22–28 °C, and even more preferably 24–26 °C; the preferred reaction time is 11–13 h, more preferably 11.5–12.5 h, and even more preferably 12 h.

[0079] In this invention, when adding ammonia and tetraethyl silicate in step 3), it is preferably carried out under stirring conditions. The stirring speed is preferably 280-320 r / min, more preferably 290-310 r / min, and even more preferably 300 r / min. After the reaction is completed, the product is preferably washed and vacuum dried. The washing reagent is preferably deionized water, and the number of washings is preferably 4-6 times, more preferably 5 times. The vacuum drying temperature is preferably 60-80℃, more preferably 65-75℃, and even more preferably 70℃. The vacuum drying time is preferably 8-12 h, more preferably 9-11 h, and even more preferably 10 h.

[0080] The present invention also provides a method for preparing the aforementioned magnetic surface molecularly imprinted polymer, comprising the following steps:

[0081] (1) After prepolymerizing 5-hydroxytryptamine, functional monomers, and porogens, a crosslinking agent, a silica-coated magnetite carrier, and an initiator are added to carry out a polymerization reaction to obtain a magnetic surface molecularly imprinted polymer layer with 5-hydroxytryptamine. Figure 1 (Fe3O4@poly@SiO2@MIP);

[0082] (2) The magnetic surface molecular imprinted polymer layer containing 5-hydroxytryptamine was eluted to obtain the magnetic surface molecular imprinted polymer. Figure 1 MMIPs in (the context of the text).

[0083] In this invention, the preferred order of adding the crosslinking agent, the silicon dioxide-coated magnetite carrier, and the initiator in step (1) is to first add the crosslinking agent and the silicon dioxide-coated magnetite carrier for ultrasonication, and then add the initiator.

[0084] In this invention, the temperature of the prepolymerization reaction in step (1) is preferably 24-26°C, more preferably 25°C, and the time of the prepolymerization reaction is preferably 0.5-1.5h, more preferably 0.8-1.2h, and more preferably 1h; the temperature of the polymerization reaction is preferably 60-80°C, more preferably 65-75°C, and more preferably 70°C; the time of the polymerization reaction is preferably 11-13h, more preferably 11.5-12.5h, and more preferably 12h, and the polymerization reaction is preferably carried out in a nitrogen atmosphere; the eluent used for elution in step (2) is preferably composed of methanol and acetic acid, and the volume ratio of methanol to acetic acid is preferably 3-5:1, and more preferably 4:1.

[0085] In this invention, the polymerization reaction in step (1) is preferably carried out under stirring conditions, and the stirring speed is preferably 320-360 r / min, more preferably 330-350 r / min, and even more preferably 340 r / min.

[0086] In this invention, the elution in step (2) is preferably eluted until the eluent does not contain 5-hydroxytryptamine; after elution, the product is preferably washed and vacuum dried sequentially, the washing reagent is preferably methanol, the pH value of the product after washing is preferably 6-8, more preferably 7; the vacuum drying temperature is preferably 60-80℃, more preferably 65-75℃, more preferably 70℃; the vacuum drying time is preferably 8-12h, more preferably 9-11h, more preferably 10h.

[0087] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0088] Example 1

[0089] 0.004 mol FeCl3·6H2O, 0.008 mol FeCl2·4H2O, and 200 mL of deionized water were ultrasonically mixed for 15 min at a power of 80 W. Nitrogen gas was then introduced to remove oxygen. Then, 20 mL of 25% ammonia solution was added at 50 °C and a stirring speed of 300 r / min, and the mixture was reacted for 30 min. Afterward, 0.8 mL of oleic acid was added, and the reaction was continued for 3 h. After the reaction was complete, the resulting magnetic particles were washed four times with anhydrous ethanol and cyclohexane, and then vacuum-dried at -0.085 MPa and 70 °C for 10 h to obtain magnetite (Fe3O4) magnetic nanoparticles.

[0090] 4 mL of styrene and 2 mL of 3-(trimethoxysilyl)propyl methacrylate were mixed, and then 1.2 g of magnetite nanoparticles and 1.5 mL of cyclohexane were added and ultrasonically mixed for 15 min (ultrasonic power of 80 W) to obtain the oil phase. 0.45 g of sodium dodecyl sulfonate, 0.24 g of disodium hydrogen phosphate, 0.126 g of sodium dihydrogen phosphate and 150 mL of deionized water were mixed, and the pH of the solution was adjusted to 7 with 85% phosphoric acid solution to obtain the aqueous phase. The oil phase and aqueous phase were ultrasonically mixed for 15 min under 80 W power, and then poured into a three-necked flask at 70 °C under nitrogen atmosphere. 0.06 g of potassium persulfate was added and polymerized for 12 h. The obtained product was washed three times with deionized water and then vacuum dried at -0.085 MPa and 60 °C for 7 h to obtain magnetite magnetic nanospheres (Fe3O4@poly).

[0091] 600 mg of magnetite nanospheres were mixed with 100 mL of anhydrous ethanol. Then, 15 mL of 25% ammonia and 3 mL of tetraethyl silicate were added at 25 °C and a stirring speed of 300 r / min, and the mixture was reacted for 12 h. After the reaction, the product was washed five times with deionized water and then vacuum dried at -0.085 MPa and 70 °C for 10 h to obtain a magnetite carrier (Fe3O4@poly@SiO2) coated with a silica layer.

[0092] 0.5 mmol 5-hydroxytryptamine, 1 mmol methacrylic acid, 1 mmol acrylamide, and 100 mL toluene were prepolymerized at 25 °C for 1 h. Then, 8.75 mmol ethylene glycol dimethacrylate and 300 mg of silica-coated magnetite magnetic carrier were added and sonicated for 15 min (ultrasonic power 80 W). Then, 15 mg of azobisisopropionitrile was added, and polymerization was carried out at 70 °C, stirring speed 340 r / min, and nitrogen atmosphere for 12 h to obtain a magnetic surface molecularly imprinted polymer layer (Fe3O4@poly@SiO2@MIP) with 5-hydroxytryptamine. The magnetic surface molecularly imprinted polymer layer with 5-hydroxytryptamine was eluted with methanol and acetic acid in a volume ratio of 4:1 until the eluent was free of 5-hydroxytryptamine. The product was then washed with methanol until the pH reached 7 and vacuum dried at -0.085 MPa and 70 °C for 10 h to obtain magnetic surface molecularly imprinted polymers (MMIPs).

[0093] The transmission electron microscope images of Fe3O4, Fe3O4@poly, Fe3O4@poly@SiO2 and MMIPs prepared in this embodiment are shown below. Figure 2 As shown in A, B, C, and D, by Figure 2 It can be seen that the four magnetic materials Fe3O4, Fe3O4@poly, Fe3O4@poly@SiO2 and MMIPs are all spherical with gradually increasing size. MMIPs obtained after emulsion polymerization, silica encapsulation and imprinting polymerization have a core-shell structure. Figure 2 The D-scan showed that the thickness of the MIP was 10-20 nm, indicating that most of the selectively recognized sites were distributed on the surface of the magnetic carrier through the imprinting polymerization reaction. This accelerated the mass transfer rate during the binding and removal of the template molecule (5-hydroxytryptamine) with MMIPs, and largely solved the problems of template leakage and slow mass transfer rate in traditional bulk polymerization molecular imprinting polymers.

[0094] Scanning electron microscope images of Fe3O4, Fe3O4@poly, Fe3O4@poly@SiO2 and MMIPs prepared in this embodiment are shown below. Figure 3 As shown in A, B, C, and D, by Figure 3 As can be seen from D, MMIPs are regular spheres with a diameter of about 500 nm, with good dispersion, and their surface is relatively rough after imprinting modification.

[0095] The Fourier transform infrared spectra of Fe3O4, Fe3O4@poly, Fe3O4@poly@SiO2 and MMIPs prepared in this embodiment are shown below. Figure 4 As shown in A, B, C, and D, Figure 4 588cm in A -1The absorption peak at point B is attributed to the characteristic absorption peak of the Fe-O vibration in Fe3O4; the peak at 1360 cm⁻¹ in point B... -1 and 1560cm -1 The absorption peak at 950-1180 cm⁻¹ is attributed to the characteristic absorption peak of the benzene ring in styrene. -1 The broad absorption peak at [value missing] is attributed to the hydrolysis product of 3-(trimethoxysilyl) methacrylate, indicating that the polymer layer (poly) was successfully attached to the Fe3O4 surface; [value missing] at C [value missing] cm⁻¹ -1 The absorption peak at point D is attributed to the characteristic absorption peak of the stretching vibration of Si-O-Si in SiO2, indicating that Fe3O4@poly is coated with SiO2; the absorption peak at 1450 cm⁻¹ in D is also attributed to the characteristic absorption peak of the stretching vibration of Si-O-Si in SiO2, indicating that Fe3O4@poly is coated with SiO2; -1 The absorption peak at the point is attributed to the CN characteristic absorption peak of ethylene glycol dimethacrylate in the MMIPs imprinted layer, indicating that the imprinted polymer layer was successfully grafted onto the Fe3O4@poly@SiO2 surface to obtain MMIPs.

[0096] The X-ray diffraction patterns of Fe3O4, Fe3O4@poly, Fe3O4@poly@SiO2 and MMIPs prepared in this embodiment are shown below. Figure 5 As shown in A, B, C, and D, Figure 5 In sample A, the diffraction peaks of Fe3O4 at 30.0°, 35.5°, 43.2°, 53.8°, 57.2°, and 62.6° correspond to the 220, 311, 400, 422, 511, and 440 crystal planes, respectively. In samples B and C, the diffraction patterns of Fe3O4@poly and Fe3O4@poly@SiO2 show no significant changes. The diffraction peak intensities decrease slightly due to the encapsulation of the Fe3O4 layer by the polymer and SiO2 layers, indicating that the polymer and SiO2 layers are amorphous structures. In sample D, the diffraction pattern of MMIPs shows no significant difference compared to Fe3O4. The diffraction peak intensities decrease slightly due to the surface-grafted imprinted layer, indicating that the surface-imprinted polymerization reaction does not affect the crystal structure of Fe3O4.

[0097] The thermogravimetric curves of Fe3O4, Fe3O4@poly, Fe3O4@poly@SiO2 and MMIPs prepared in this embodiment are shown in the figures below. Figure 6 As shown in A, B, C, and D, by Figure 6 It can be seen that the weight loss rate of Fe3O4, Fe3O4@poly, Fe3O4@poly@SiO2 and MMIPs is less than 10% in the temperature range of 25 to 800℃, indicating that the MMIPs prepared by this invention have good thermal stability.

[0098] The hysteresis loop diagrams of Fe3O4, Fe3O4@poly, Fe3O4@poly@SiO2 and MMIPs prepared in this embodiment are shown below. Figure 7 As shown in A, B, C, and D, by Figure 7 It can be seen that within the magnetic field range of -25000 to 25000 G, the specific saturation magnetization values ​​of Fe3O4, Fe3O4@poly, Fe3O4@poly@SiO2, and MMIPs are 67.36, 46.32, 36.56, and 30.84 emu / g, respectively. With the introduction of the non-magnetic polymer shell, although the specific saturation magnetization gradually decreases to 30.84 emu / g, the imprinted polymer still maintains superparamagnetism. This indicates that the MMIPs prepared in this invention have good magnetic sensitivity characteristics and can be directly separated from the sample solution by controlling the external magnetic field, which greatly simplifies the sample processing procedure.

[0099] Example 2

[0100] 0.004 mol FeCl3·6H2O, 0.006 mol FeCl2·4H2O, and 180 mL of deionized water were ultrasonically mixed for 15 min at a power of 80 W. Nitrogen gas was then introduced to remove oxygen. Then, 18 mL of 26% ammonia solution was added at 45 °C and a stirring speed of 280 r / min, and the mixture was reacted for 25 min. Afterward, 0.6 mL of oleic acid was added, and the reaction was carried out for 2 h. After the reaction was completed, the resulting magnetic particles were washed three times with anhydrous ethanol and cyclohexane, and then vacuum-dried at -0.085 MPa and 60 °C for 12 h to obtain magnetite nanoparticles.

[0101] 3.5 mL of styrene and 1.5 mL of 3-(trimethoxysilyl)propyl methacrylate were mixed, and then 1 g of magnetite nanoparticles and 1 mL of cyclohexane were added and ultrasonically mixed for 15 min (ultrasonic power of 80 W) to obtain the oil phase. 0.4 g of sodium dodecyl sulfonate, 0.2 g of disodium hydrogen phosphate, 0.1 g of sodium dihydrogen phosphate and 120 mL of deionized water were mixed, and the pH of the solution was adjusted to 6 with 80% phosphoric acid solution to obtain the aqueous phase. The oil phase and aqueous phase were ultrasonically mixed for 15 min under 80 W power, and then poured into a three-necked flask at 65 °C under a nitrogen atmosphere. 0.05 g of potassium persulfate was added and polymerized for 10 h. The resulting product was washed twice with deionized water and then vacuum dried at -0.085 MPa and 50 °C for 12 h to obtain magnetite magnetic nanospheres.

[0102] 580 mg of magnetite nanospheres were mixed with 80 mL of anhydrous ethanol. Then, 13 mL of 28% ammonia and 2 mL of tetraethyl silicate were added at 20 °C and 280 r / min, and the mixture was reacted for 11 h. After the reaction, the product was washed four times with deionized water and then vacuum dried at -0.085 MPa and 60 °C for 8 h to obtain a magnetite carrier coated with a silica layer.

[0103] 0.5 mmol 5-hydroxytryptamine, 1 mmol methacrylic acid, 0.9 mmol acrylamide, and 90 mL toluene were prepolymerized at 24 °C for 0.5 h. Then, 8 mmol ethylene glycol dimethacrylate and 275 mg of silica-coated magnetite magnetic carrier were added and sonicated for 15 min (ultrasonic power 80 W). Then, 14 mg of azobisisopropionitrile was added, and polymerization was carried out at 60 °C, stirring speed 320 r / min, and nitrogen atmosphere for 11 h to obtain a magnetic surface molecularly imprinted polymer layer with 5-hydroxytryptamine. The magnetic surface molecularly imprinted polymer layer with 5-hydroxytryptamine was eluted with methanol and acetic acid in a volume ratio of 3:1 until the eluent was free of 5-hydroxytryptamine. The product was then washed with methanol until the pH reached 7, and then vacuum dried at -0.085 MPa and 60 °C for 8 h to obtain the magnetic surface molecularly imprinted polymer.

[0104] Example 3

[0105] 0.004 mol FeCl3·6H2O, 0.016 mol FeCl2·4H2O, and 220 mL of deionized water were ultrasonically mixed for 15 min at a power of 80 W. Nitrogen gas was then introduced to remove oxygen. 22 mL of 26% ammonia solution was added at 55 °C and a stirring speed of 320 r / min, and the mixture was reacted for 35 min. Then, 1 mL of oleic acid was added, and the reaction was carried out for 4 h. After the reaction, the magnetic particles were washed twice with anhydrous ethanol and cyclohexane, and then vacuum dried at -0.085 MPa and 80 °C for 8 h to obtain magnetite nanoparticles.

[0106] 4.5 mL of styrene and 2.5 mL of 3-(trimethoxysilyl)propyl methacrylate were mixed, and then 1.4 g of magnetite nanoparticles and 2 mL of cyclohexane were added and ultrasonically mixed for 15 min (ultrasonic power of 80 W) to obtain the oil phase. 0.5 g of sodium dodecyl sulfonate, 0.25 g of disodium hydrogen phosphate, 0.15 g of sodium dihydrogen phosphate and 160 mL of deionized water were mixed, and the pH of the solution was adjusted to 8 with 85% phosphoric acid solution to obtain the aqueous phase. The oil phase and aqueous phase were ultrasonically mixed for 15 min under 80 W power, and then poured into a three-necked flask at 75 °C under nitrogen atmosphere. 0.07 g of potassium persulfate was added and polymerized for 14 h. The obtained product was washed four times with deionized water and then vacuum dried at -0.085 MPa and 70 °C for 7 h to obtain magnetite magnetic nanospheres.

[0107] 620 mg of magnetite magnetic nanospheres were mixed with 120 mL of anhydrous ethanol. Then, under the conditions of 30 °C and 320 r / min, 17 mL of 28% ammonia water and 4 mL of tetraethyl silicate were added and the mixture was reacted for 13 h. After the reaction was completed, the product was washed 6 times with deionized water and then vacuum dried at -0.085 MPa and 75 °C for 12 h to obtain a magnetite magnetic carrier coated with a silica layer.

[0108] 1 mmol 5-hydroxytryptamine, 2 mmol methacrylic acid, 2.2 mmol acrylamide, and 220 mL toluene were prepolymerized at 26 °C for 1.5 h. Then, 18 mmol ethylene glycol dimethacrylate and 650 mg of silica-coated magnetite magnetic carrier were added and sonicated for 15 min (ultrasonic power 80 W). Then, 32 mg of azobisisopropionitrile was added, and polymerization was carried out at 80 °C, stirring speed 360 r / min, and nitrogen atmosphere for 13 h to obtain a magnetic surface molecularly imprinted polymer layer with 5-hydroxytryptamine. The magnetic surface molecularly imprinted polymer layer with 5-hydroxytryptamine was eluted with methanol and acetic acid in a volume ratio of 5:1 until the eluent was free of 5-hydroxytryptamine. The product was then washed with methanol until the pH reached 8, and then vacuum dried at -0.085 MPa and 80 °C for 12 h to obtain the magnetic surface molecularly imprinted polymer.

[0109] Example 4

[0110] 0.004 mol FeCl3·6H2O, 0.012 mol FeCl2·4H2O, and 210 mL of deionized water were ultrasonically mixed for 15 min at a power of 80 W. Nitrogen gas was then introduced to remove oxygen. Then, 20.5 mL of 25% ammonia solution was added at 48 °C and a stirring speed of 300 r / min, and the mixture was reacted for 28 min. Afterward, 0.7 mL of oleic acid was added, and the reaction was continued for 3.2 h. After the reaction was complete, the magnetic particles were washed three times with anhydrous ethanol and cyclohexane, and then vacuum dried at -0.085 MPa and 70 °C for 10 h to obtain magnetite nanoparticles.

[0111] 3.5 mL of styrene and 2.5 mL of 3-(trimethoxysilyl)propyl methacrylate were mixed, and then 1.25 g of magnetite nanoparticles and 1.4 mL of cyclohexane were added and ultrasonically mixed for 15 min (ultrasonic power of 80 W) to obtain the oil phase. 0.4 g of sodium dodecyl sulfonate, 0.22 g of disodium hydrogen phosphate, 0.13 g of sodium dihydrogen phosphate and 150 mL of deionized water were mixed, and the pH of the solution was adjusted to 7 with 82% phosphoric acid solution to obtain the aqueous phase. The oil phase and aqueous phase were ultrasonically mixed for 15 min under 80 W power, and then poured into a three-necked flask at 68 °C under a nitrogen atmosphere. 0.06 g of potassium persulfate was added, and polymerization was carried out for 12 h. The resulting product was washed three times with deionized water and then vacuum dried at -0.085 MPa and 65 °C for 9 h to obtain magnetite magnetic nanospheres.

[0112] 615 mg of magnetite magnetic nanospheres were mixed with 100 mL of anhydrous ethanol. Then, 15 mL of 25% ammonia water and 3 mL of tetraethyl silicate were added at 25 °C and a stirring speed of 300 r / min, and the mixture was reacted for 12 h. After the reaction was completed, the product was washed 5 times with deionized water and then vacuum dried at -0.085 MPa and 75 °C for 11 h to obtain a magnetite magnetic carrier coated with a silica layer.

[0113] 1 mmol of 5-hydroxytryptamine, 2 mmol of methacrylic acid, 2 mmol of acrylamide, and 210 mL of toluene were prepolymerized at 25 °C for 1.2 h. Then, 18 mmol of ethylene glycol dimethacrylate and 600 mg of silica-coated magnetite magnetic carrier were added and sonicated for 15 min (ultrasonic power of 80 W). Then, 30 mg of azobisisopropionitrile was added, and polymerization was carried out at 70 °C, stirring speed of 340 r / min, and nitrogen atmosphere for 12 h to obtain a magnetic surface molecularly imprinted polymer layer with 5-hydroxytryptamine. The magnetic surface molecularly imprinted polymer layer with 5-hydroxytryptamine was eluted with methanol and acetic acid in a volume ratio of 4:1 until the eluent was free of 5-hydroxytryptamine. The product was then washed with methanol until the pH reached 7, and then vacuum dried at -0.085 MPa and 75 °C for 9.5 h to obtain the magnetic surface molecularly imprinted polymer.

[0114] Comparative Example 1

[0115] Omit 5-hydroxytryptamine in Example 1, and keep other conditions the same as in Example 1 to obtain magnetic surface non-molecularly imprinted polymers (MNIPs).

[0116] Comparative Examples 2-5

[0117] In Example 1, toluene was replaced with equal amounts of acetonitrile, dimethyl sulfoxide, tetrahydrofuran, and deionized water, respectively, while other conditions remained the same as in Example 1, to obtain magnetic surface molecularly imprinted polymers (MMIPs).

[0118] Comparative Examples 6-9

[0119] By replacing toluene in Comparative Example 1 with equal amounts of acetonitrile, dimethyl sulfoxide, tetrahydrofuran, and deionized water, and keeping other conditions the same as in Comparative Example 1, magnetic surface non-molecularly imprinted polymers (MNIPs) were obtained.

[0120] Under different porogens, the adsorption capacities of the imprinted polymers in Example 1 and Comparative Examples 1-9 are as follows: Figure 8 As shown, by Figure 8 It can be seen that when toluene is used as the porogen, the synthesized MMIPs have the highest adsorption capacity.

[0121] Comparative Examples 10-15

[0122] The amount of ethylene glycol dimethacrylate used in Example 1 was modified to 5 mmol, 6.25 mmol, 7.5 mmol, 10 mmol, 11.25 mmol and 12.25 mmol respectively, while other conditions were the same as in Example 1, to obtain magnetic surface molecularly imprinted polymers (MMIPs).

[0123] Comparative Examples 16–21

[0124] The amount of ethylene glycol dimethacrylate in Comparative Example 1 was modified to 5 mmol, 6.25 mmol, 7.5 mmol, 10 mmol, 11.25 mmol and 12.25 mmol respectively, while other conditions were the same as in Comparative Example 1, to obtain magnetic surface non-molecularly imprinted polymers (MNIPs).

[0125] The adsorption capacities of the imprinted polymers in Example 1, Comparative Examples 1, and Comparative Examples 10-21 were as follows, under different amounts of crosslinking agent: Figure 9 As shown, by Figure 9 It can be seen that when the molar ratio of 5-hydroxytryptamine, functional monomer and crosslinking agent is 1:4:17.5, the synthesized MMIPs have the highest adsorption capacity. If the amount of crosslinking agent is too large, a large number of adsorption sites will be "buried", thus leading to a decrease in adsorption capacity.

[0126] Comparative Examples 22-25

[0127] The amount of azobisisopropionitrile in Example 1 was modified to 5 mg, 10 mg, 20 mg and 25 mg respectively, while other conditions were the same as in Example 1, to obtain magnetic surface molecularly imprinted polymers (MMIPs).

[0128] Comparative Examples 26–29

[0129] The amount of azobisisopropionitrile in Comparative Example 1 was modified to 5 mg, 10 mg, 20 mg and 25 mg respectively, while other conditions were the same as in Comparative Example 1, to obtain magnetic surface non-molecularly imprinted polymers (MNIPs).

[0130] The adsorption capacities of the imprinted polymers in Example 1, Comparative Examples 1, and Comparative Examples 22-29 were as follows, under different amounts of initiator: Figure 10 As shown, by Figure 10 It is known that when the molar mass ratio of 5-hydroxytryptamine to initiator is 1 mmol: 30 mg, the prepared MMIPs have the highest adsorption capacity.

[0131] Under conditions of 30℃ and a flow rate of 100 r / min, 5 mg of MMIPs prepared in Example 1 and MNIPs prepared in Comparative Example 1 were added to 1 mL of acetonitrile solution in 10 groups (the concentrations of 5-HT in the 10 groups of acetonitrile solutions were 1 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, 70 μg / mL, and 80 μg / mL, respectively). After adsorption for 12 h, magnetic separation was performed, and the concentration of remaining 5-HT in the supernatant was determined by liquid chromatography-mass spectrometry. The adsorption capacity corresponding to different concentrations was calculated. The static isothermal adsorption behavior of MMIPs and MNIPs was studied using Langmuir (1-1) and Freundlich (1-2) isothermal adsorption models. The static isothermal adsorption curves of MMIPs and MNIPs are shown below. Figure 11 As shown, by Figure 11 It can be seen that the adsorption capacity of MMIPs for 5-HT is much greater than that of MNIPs. When the initial concentration of 5-HT is greater than 60 μg / mL, the adsorption capacity of MMIPs tends to be a constant of 8.4108 μg / mg. However, for MNIPs, when the initial concentration of 5-HT is greater than 30 μg / mL, the increase in adsorption capacity is not significant. This phenomenon indicates that, compared with MNIPs, there are a large number of structure-selective imprinted cavities on the surface of MMIPs.

[0132] Langmuir (1-1): Freundlich(1-2): log Q e =log C e / n+logK F Where Qe (μg / mg) is the adsorption capacity of MMIPs or MNIPs at adsorption equilibrium; Ce (μg / mL) is the solution concentration of MMIPs or MNIPs at adsorption equilibrium; Qm (μg / mg) is the maximum adsorption capacity of MMIPs or MNIPs; K L K is the Langmuir adsorption coefficient. F and n are Freundlich adsorption coefficients, and the fitting parameters for Langmuir and Freundlich are shown in Table 1.

[0133] Table 1 Fitting parameters for Langmuir and Freundlich

[0134]

[0135] Table 1 shows that the R-values ​​fitted by Langmuir and Freundlich are: 2The value of 0.9932 indicates that the adsorption of 5-HT by MMIPs is a monolayer adsorption process.

[0136] Five mg of MMIPs prepared in Example 1 and MNIPs prepared in Comparative Example 1 were added to 1 mL of acetonitrile solution in 10 groups (the concentration of 5-HT in the acetonitrile solution was 60 μg / mL). The kinetic adsorption model of MMIPs and MNIPs was studied by measuring the concentration of 5-HT in the supernatant during the adsorption time from 0 to 700 min. The kinetic adsorption diagrams of MMIPs and MNIPs are shown in the figure. Figure 12 As shown, by Figure 12 It can be seen that the adsorption capacity of both MMIPs and MNIPs increases relatively slowly after 180 min of adsorption, and the adsorption process basically reaches equilibrium at around 300 min. Furthermore, the adsorption capacity of MMIPs is consistently higher than that of MNIPs, with an imprinting efficiency of 2.55. Therefore, it can be concluded that the MMIPs of this invention, due to their recognition sites being located on the carrier surface, can reach adsorption equilibrium in a short time, and under the same conditions, their adsorption capacity is significantly higher than that of MNIPs.

[0137] The dynamics of MMIPs and MNIPs were further studied using quasi-first-order dynamic equations (1-3) and quasi-second-order dynamic equations (1-4), and the results are shown in Table 2.

[0138] The pseudo-first-order dynamic equation (1-3): ln(Q) e -Q t )=ln Q e -k1 t, quasi-second-order dynamic equation (1-4): Among them, Q e (μg / mg) represents the saturated adsorption capacity of MMIPs or MNIPs when adsorption equilibrium is reached; Q t (μg / mg) represents the adsorption capacity of MMIPs or MNIPs at different times; k1 (1 / min) represents the first-order reaction rate constant; and k2 (mg / (μg·min)) represents the second-order reaction rate constant.

[0139] Table 2. Kinetic results of MMIPs and MNIPs

[0140]

[0141] As shown in Table 2, the adsorption process of 5-HT by MMIPs is more in line with the pseudo-second-order kinetic model and is a rate-limited process.

[0142] The specificity and selectivity of MMIPs were evaluated by investigating their adsorption capacity for dopamine (DA), adrenaline (E), norepinephrine (NE), 5-hydroxytryptophan (5-HTP), and 5-hydroxyindoleacetic acid (5-HIAA), which have structures similar to 5-hydroxytryptamine (5-HT). A mixed solution with a concentration of 50 μg / mL for each substance was prepared. Then, 5 mg of MMIPs from Example 1 and MNIPs from Comparative Example 1 were added to 1 mL of the mixed solution, and adsorption was performed isothermally for 12 h. Magnetic separation was then performed, and the concentration of each substance in the supernatant was determined by high-performance liquid chromatography (HPLC). The adsorption capacity of MMIPs for each substance was calculated, and their selective adsorption performance for 5-HT was investigated. The results are as follows: Figure 13 As shown: MMIPs and MNIPs exhibit different adsorption capacities for dopamine, adrenaline, noradrenaline, 5-hydroxytryptophan, 5-hydroxyindoleacetic acid, and 5-hydroxytryptamine, and their imprinting efficiencies also differ. The highest adsorption capacity was observed for 5-hydroxytryptamine (QMMIPs: 5.290±0.143 μg / mg, QMNIPs: 1.603±0.054 μg / mg); followed by 5-hydroxyindoleacetic acid (QMMIPs: 2.883±0.089 μg / mg; QMNIPs: ...).

[0143] The highest concentrations of 5-hydroxytryptamine (5-HT) were 1.328±0.053 μg / mg, while the lowest concentrations of dopamine (QMMIPs: 1.620±0.083 μg / mg; QMNIPs: 1.43±0.069 μg / mg). This indicates that the specific recognition vacancies generated on the surface of the MMIPs of the present invention are perfectly matched to the shape, size, and spatial arrangement of 5-HT. DA, E, NE, 5-HTP, and 5-HIAA are not complementary to the recognition sites. Therefore, the MMIPs of the present invention have good specific adsorption performance for 5-hydroxytryptamine.

[0144] 5 mg of MMIPs from Example 1 and MNIPs from Comparative Example 1 were added to 1 mL of acetonitrile solution (the concentration of 5-hydroxytryptamine in the acetonitrile solution was 60 μg / mL). The MMIPs were then isothermally adsorbed for 6 h in a shaker at 30 °C and 100 r / min. Magnetic separation was then performed, and the concentration of 5-hydroxytryptamine in the supernatant was measured. The separated MMIPs were then washed with an eluent (methanol to acetic acid volume ratio of 4:1) to remove the adsorbed 5-hydroxytryptamine molecules until no 5-hydroxytryptamine was detected in the eluent. The MMIPs were then washed with methanol until neutral and vacuum-dried at -0.085 MPa and 65 °C for 10 h to obtain MMIPs free of 5-hydroxytryptamine molecules, completing one adsorption-elution experiment. The 5-hydroxytryptamine-free MMIPs were then subjected to another adsorption-elution experiment, with the same procedure as before. This adsorption-elution cycle was repeated 8 times, and the adsorption capacity of each cycle was calculated. The results are shown below. Figure 14 As shown, by Figure 14 It can be seen that MMIPs can be effectively recycled and reused. After 8 cycles, its saturated adsorption capacity loss is about 9.41%, but the adsorption capacity can still be guaranteed to be above 90%. This shows that MMIPs that selectively recognize 5-hydroxytryptamine have excellent reusability and high reuse rate.

[0145] 0.5 g bovine serum albumin was sonicated with 10 mL of phosphate buffer solution (a mixed solution purchased from Aladdin) at 80 W for 15 min to obtain blank serum. 5-HT standard powder was prepared into a standard stock solution with a concentration of 1 mg / mL using methanol solution. The standard stock solution was diluted with acetonitrile to obtain a working solution with a concentration of 8 ng / mL. 250 μL of the working solution was mixed with 5 mL of blank serum to obtain a substitute serum solution with a concentration of 0.4 ng / mL. 3 mL of the substitute serum solution was mixed with 9 mL of acetonitrile, and protein precipitation was performed by vortexing for 3 min, followed by centrifugation at 13000 r / min for 15 min to obtain a supernatant (adsorption solution) with a concentration of 0.1 ng / mL. 20 mg of MMIPs from Example 1 and 20 mg of MNIPs from Comparative Example 1 were mixed with 10 mL of adsorption solution and isothermally adsorbed for 5 h in a shaker at 28 °C and 100 r / min. Magnetic separation was then performed, and the residual concentration of 5-HT in the supernatant was determined. The 5-HT-adsorbed MMIPs and MNIPs were dispersed in 0.5 mL of a mixed solution of methanol and acetic acid (formic acid to acetic acid volume ratio 4:1), and then eluted by ultrasonication for 30 min at 80 W. Magnetic separation was then performed, and the eluent was collected. The 5-HT content in the eluent was determined by ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS / MS). The magnetic solid-phase microextraction (MS / MS) results of the MMIPs from Example 1 and the MNIPs from Comparative Example 1 in alternative serum are as follows: Figure 15 and Figure 16 As shown, by Figure 15 Figure A (adsorption solution: 0.1 ng / mL spiked blank serum after treatment) shows that the chromatographic peak area of ​​5-HT is very small, and the signal-to-noise ratio (S / N = 2.75) is very low. Therefore, it is difficult to reach the lower limit of LC-MS / MS quantification. Using MMIPs as solid-phase extraction adsorbents to enrich and determine the content of 5-HT from the trace blank serum system, no obvious chromatographic peak was detected in the supernatant B after solid-phase extraction, while the eluent C showed... Figure 16 Compared to MNIPs in C, MMIPs exhibit a distinct 5-HT chromatographic peak. This is because MMIPs specifically adsorb 5-HT, while MNIPs lack specific selectivity. The results indicate that MMIPs possess high affinity, high selectivity, and excellent adsorption capacity for 5-HT, making them suitable for the selective identification and enrichment of trace amounts of 5-HT.

[0146] The magnetic surface molecularly imprinted polymer prepared by this invention has a high specific surface area, good biocompatibility and stability, and excellent magnetic sensitivity. It not only overcomes the shortcomings of traditional molecularly imprinted polymers, such as difficult elution, template leakage, and slow mass transfer rate, but also solves the problems of difficult separation and time consumption. When used directly as an adsorbent for solid-phase microextraction, it can not only greatly increase the selectivity of adsorption and improve the separation and enrichment efficiency, but also simplify the experimental procedure and reduce the experimental cost. Moreover, the preparation conditions are easy to control and the stability is good. It can be reused and is particularly suitable for the separation and enrichment of small molecules in biological samples. It has broad application prospects in the research of solid-phase microextraction pretreatment methods. It can achieve effective and highly selective separation and enrichment of trace amounts of 5-HT in samples.

[0147] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A magnetic surface molecularly imprinted polymer, characterized in that, The preparation raw materials comprise: 5-hydroxytryptamine, functional monomer, porogen, crosslinking agent, four-iron oxide magnetic carrier coated with a silica layer and initiator; The molar ratio of the 5-hydroxytryptamine, functional monomer and crosslinking agent is 1:3.8-4.2:16-18; The molar volume ratio of the 5-hydroxytryptamine and porogen is 1 mmol:180-220 mL; The mass ratio of the four-iron oxide magnetic carrier coated with a silica layer and initiator is 550-650:28-32; The molar mass ratio of the 5-hydroxytryptamine and initiator is 1 mmol:28-32 mg; The functional monomer comprises methacrylic acid and acrylamide; and the porogen is toluene. The preparation method of the four-iron oxide magnetic carrier coated with a silica layer comprises the following steps: 1) mixing FeCl3·6H2O, FeCl2·4H2O and water, then sequentially adding ammonia and oleic acid to react and dry, to obtain four-iron oxide magnetic nanoparticles; 2) mixing the four-iron oxide magnetic nanoparticles, styrene, 3-(trimethoxysilyl) propyl methacrylate and cyclohexane to obtain an oil phase; mixing sodium dodecyl sulfonate, disodium hydrogen phosphate, sodium dihydrogen phosphate and water to obtain an aqueous phase; ultrasonic mixing the oil phase and aqueous phase, then adding potassium persulfate to carry out polymerization reaction, to obtain four-iron oxide magnetic nanospheres; 3) mixing the four-iron oxide magnetic nanospheres and anhydrous ethanol, then adding ammonia and tetraethyl silicate to react, to obtain the four-iron oxide magnetic carrier coated with a silica layer.

2. The magnetic surface molecularly imprinted polymer according to claim 1, wherein, The molar ratio of the methacrylic acid and acrylamide is 1:0.9-1.1; the crosslinking agent is ethylene glycol dimethacrylate, and the initiator is azobis isopropyl cyanide.

3. The magnetic surface molecularly imprinted polymer according to claim 2, wherein, In step 1), the volume fraction of the ammonia is 25-28%; the molar ratio of the FeCl3·6H2O and FeCl2·4H2O is 1:1.5-4; the volume ratio of the water, ammonia and oleic acid is 180-220:18-22:0.6-1; and the molar volume ratio of the FeCl3·6H2O and water is 0.004 mol:180-220 mL.

4. The magnetic surface-molecularly imprinted polymer according to claim 3, wherein Step 1) when the ammonia is added to react, the reaction temperature is 45-55℃, and the reaction time is 25-35 min; when the oleic acid is added to react, the reaction time is 2-4 h; the drying temperature is 60-80℃, and the drying time is 8-12 h. In step 2), the mass-volume ratio of the four-iron oxide magnetic nanoparticles, styrene, 3-(trimethoxysilyl) propyl methacrylate and cyclohexane is 1-1.4 g:3.5-4.5 mL:1.5-2.5 mL:1-2 mL; the mass-volume ratio of the sodium dodecyl sulfonate, disodium hydrogen phosphate, sodium dihydrogen phosphate and water is 0.4-0.5 g:0.2-0.25 g:0.1-0.15 g:120-160 mL; and the mass ratio of the four-iron oxide magnetic nanoparticles and sodium dodecyl sulfonate is 1-1.4:0.4-0.

5.

5. The magnetic surface-molecularly imprinted polymer according to claim 3 or 4, wherein ​ 6. The magnetic surface-molecularly imprinted polymer according to claim 5, wherein, Step 2) the mass ratio of the potassium persulfate to the sodium dodecyl sulfate is 0.05-0.07:0.4-0.5; the temperature of the polymerization reaction is 65-75℃, the time of the polymerization reaction is 10-14h, and the polymerization reaction is carried out in a nitrogen atmosphere.

7. The magnetic surface-molecularly imprinted polymer according to claim 6, wherein Step 3) the mass-volume ratio of the four-iron oxide magnetic nanospheres, the anhydrous ethanol, the ammonia water and the tetraethyl silicate is 580-620mg:80-120mL:13-17mL:2-4mL; the temperature of the reaction is 20-30℃, and the time of the reaction is 11-13h.

8. The method for preparing the magnetic surface molecularly imprinted polymer according to any one of claims 1-7, characterized in that, Comprising the following steps: (1) after pre-polymerization of 5-hydroxytryptamine, a functional monomer and a pore-forming agent, adding a crosslinking agent, a four-iron oxide magnetic carrier coated with a silica layer and an initiator for polymerization to obtain a magnetic surface molecularly imprinted polymer layer with 5-hydroxytryptamine; (2) eluting the magnetic surface molecularly imprinted polymer layer with 5-hydroxytryptamine to obtain a magnetic surface molecularly imprinted polymer.

9. The production method according to claim 8, characterized by, Step (1) the temperature of the pre-polymerization is 24-26℃, and the time of the pre-polymerization is 0.5-1.5h; the temperature of the polymerization is 60-80℃, and the time of the polymerization is 11-13h; step (2) the eluent used for elution comprises methanol and acetic acid, and the volume ratio of the methanol to the acetic acid is 3-5:1.

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