CN116253833A - Preparation method of dual-template MMPs for specifically adsorbing HC and P - Google Patents

Preparation method of dual-template MMPs for specifically adsorbing HC and P Download PDF

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CN116253833A
CN116253833A CN202310349763.6A CN202310349763A CN116253833A CN 116253833 A CN116253833 A CN 116253833A CN 202310349763 A CN202310349763 A CN 202310349763A CN 116253833 A CN116253833 A CN 116253833A
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template
mmips
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陈贵堂
肖伯贤
周思璇
王海翔
綦国红
杨志萍
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China Pharmaceutical University
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Abstract

The invention discloses a preparation method of double-template MMIPs for specifically adsorbing hydrocortisone HC and prednisolone P, which belongs to the technical field of polymer adsorption materials and comprises the following steps: preparation of Fe 3 O 4 A nanoparticle; preparation of Fe 3 O 4 @SiO 2 Composite particles; preparation of Fe 3 O 4 @SiO 2 -c=c; preparing double-template MMPs: mixing each template molecule (HC and P) with a functional monomer and a pore-forming agent respectively, dissolving, and imprinting at room temperature to obtain a first mixed solution; weighing Fe 3 O 4 @SiO 2 C=c, adding a pore-forming agent, uniformly dispersing, mixing with the first mixed solution, and sequentially adding a crosslinking agent and an initiator to perform polymerization reaction; and then collecting the product, drying and Soxhlet extracting to obtain the polymer with the specific cavity. The dual-template MMIPs prepared by the application can specifically adsorb, enrich and detect trace HC and P remained in food, and have the reusability of continuous use after elution.

Description

Preparation method of dual-template MMPs for specifically adsorbing HC and P
Technical Field
The invention belongs to the technical field of polymer adsorption materials, and particularly relates to a preparation method of a double-template Magnetic Molecularly Imprinted Polymer (MMIPs) for specifically adsorbing Hydrocortisone (HC) and prednisolone (P).
Background
Hydrocortisone (HC), also known as cortisol, is one of the adrenoglucocorticoid drugs and has mainly anti-inflammatory, immunosuppressive, antitoxic and antishock effects, but excessive or prolonged intake of hydrocortisone can cause serious side effects including obesity, blood sugar, blood pressure and elevated intraocular pressure, muscle paralysis, gastrointestinal ulcers and even bleeding perforations. Since hydrocortisone can be used as a veterinary drug, its residues in agricultural and sideline products are potentially dangerous to humans. The European Union, the United states, etc. prescribes a limiting standard of up to 10 mug/kg for residual hydrocortisone in milk. Prednisolone (P) also belongs to adrenoglucocorticoid medicines, is mainly used for resisting inflammation and allergy, and is used as veterinary medicine for treating animal inflammation. Similar to hydrocortisone, excessive or prolonged intake of prednisolone can also cause side effects such as abnormal bone metabolism, hypoadrenocortical function, muscle weakness, elevated blood glucose, blood lipid, sodium blood, etc. Therefore, prednisolone is also a veterinary drug residue subject of great concern in agricultural and sideline products, and the highest residue limit in domestic milk standards is 6 mug/kg.
Currently, the common methods for detecting the two substances include fluorescence photometry, gas chromatography, high performance liquid chromatography, gas chromatography tandem mass spectrometry, high performance liquid chromatography tandem mass spectrometry and the like, and the methods are complex in operation or require complex pretreatment steps of the to-be-detected substances, so that the detection speed is slow. The molecular imprinting technology is a novel pretreatment method, and utilizes Molecular Imprinting Polymers (MIPs) to adsorb substances to be detected for enrichment, and the principle is that functional monomers, cross-linking agents and template molecules are polymerized to obtain a polymer with a three-dimensional network structure, and then the template molecules are eluted, so that binding sites matched with the size and the shape of the template molecules can be left on the polymer, and the binding sites can adsorb target substances again to achieve the purpose of specific adsorption. The Magnetic Molecularly Imprinted Polymer (MMIPs) combined with the magnetic separation technology adds Fe on the basis of MIPs 3 O 4 The nano particles are used as magnetic cores, so that the whole polymer can be separated from the to-be-detected product through the action of an external magnetic field, the convenience of a molecular imprinting technology is further enhanced, and the energy conservation and the environmental protection are realized.
Therefore, the specificity sensitive detection of hydrocortisone and prednisolone in agricultural and sideline products is realized, and the method has quite important practical significance and application prospect for rapid detection of pesticide and veterinary drug residues in foods.
Disclosure of Invention
The invention aims to provide a preparation method of a dual-template magnetic molecularly imprinted polymer for specifically adsorbing Hydrocortisone (HC) and prednisolone (P), and the preparation method successfully synthesizes a three-dimensional structure with a specific cavity for a target compound, so that the hydrocortisone and the prednisolone have larger adsorption capacity and better specific adsorption capacity.
The invention aims at realizing the following technical scheme:
a preparation method of dual-template MMPs for specifically adsorbing HC and P comprises the following steps:
s1, preparing Fe with carboxyl on surface by improved hydrothermal method 3 O 4 A nanoparticle;
s2, preparing Fe 3 O 4 @SiO 2 Composite particles;
s3, preparing Fe 3 O 4 @SiO 2 -C＝C；
S4, preparing a dual-template magnetic molecularly imprinted polymer: mixing each template molecule with a functional monomer and a pore-forming agent respectively, dissolving, and imprinting at room temperature to obtain a first mixed solution; weighing Fe obtained in the step S3 3 O 4 @SiO 2 -c=c, adding a porogenic agent, uniformly dispersing the magnetic material by ultrasound, and mixing with the first mixed solution to obtain a second mixed solution; then adding a cross-linking agent and an initiator into the second mixed solution in sequence to carry out polymerization reaction; heating after deoxidizing the nitrogen; after the reaction, the product is collected by a magnetic field, dried and Soxhlet extracted to obtain a polymer with a specific cavity, wherein the template molecules are hydrocortisone HC and prednisolone P.
Further, the step S1 specifically includes: adding ferric chloride hexahydrate, trisodium citrate dihydrate and ethylene glycol into a beaker, magnetically stirring at room temperature until solid particles are dissolved, then adding anhydrous sodium acetate, and stirring until the solution is uniform; pouring the solution into a reactor, and reacting for 10 hours at 200 ℃; after the reaction, magnetic particles are separated by applying a magnetic field, repeatedly washed by deionized water and ethanol, and dried to obtain Fe with carboxyl on the surface 3 O 4 A nanoparticle;
the step S2 specifically includes: taking Fe synthesized in the step S1 3 O 4 Dispersing the nano particles in hydrochloric acid solution, carrying out ultrasonic treatment, collecting magnetic materials by using a magnet, and washing to be neutral; dispersing the washed particles in a mixture of ethanol and deionized waterAdding tetraethyl orthosilicate and 28wt% ammonia water solution into the mixed solution respectively, and stirring at normal temperature for reaction; separating, washing and drying after the reaction to obtain Fe 3 O 4 @SiO 2 Composite particles;
the step S3 specifically includes: taking Fe synthesized in the step S2 3 O 4 @SiO 2 Dispersing composite particles in methanol water solution added with silane coupling agent, stirring, separating, washing and drying to obtain Fe 3 O 4 @SiO 2 -C＝C。
Further, the silane coupling agent is gamma-methacryloxypropyl trimethoxysilane.
Further, the functional monomer is any one of acrylamide, methacrylic acid, 4-vinyl pyridine and itaconic acid.
Further, the pore-forming agent is any one of acetonitrile, ethanol, chloroform and tetrahydrofuran. The selection of the pore-forming agent not only affects the pre-polymerization of the functional monomer and the template molecule, but also has great influence on the selection of the eluting solvent, the preparation of the adsorption solution and the chromatographic conditions.
Further, the time for imprinting at room temperature is 3-24 h.
Further, the cross-linking agent is any one of ethylene glycol dimethacrylate, tetraethyl orthosilicate and divinylbenzene; the initiator is 2, 2-azo diisobutyronitrile.
In step S4, the addition ratio of the template molecule, the functional monomer and the crosslinking agent is 1:1-10:5-50.
Further, the temperature for polymerization reaction is 55-75 ℃; the reaction time is 3-24 h.
Further, the polymerization reaction is carried out at a temperature of 60 ℃; the polymerization reaction was carried out for 6 hours.
1. The preparation method of the dual-template MMIPs for specifically adsorbing hydrocortisone HC and prednisolone P comprises the steps of firstly pre-arranging enough functional monomers which can be anchored with template molecules to the greatest extent in space with the template molecules to form a pre-polymerized compound, mixing the pre-polymerized compound with a certain proportion of cross-linking agent and initiator, introducing inert gas, deoxidizing and sealing, and performing free radical polymerization by utilizing thermal initiation to obtain high-molecular polymer solid with larger volume, and obtaining polymer particle products with proper particle diameters after cleaning, drying, crushing, grinding and sieving; finally, removing the template molecules from the polymer structure through solvent extraction, thereby obtaining the highly cross-linked molecularly imprinted polymer with high specificity and adsorption capacity for hydrocortisone HC and prednisolone P. According to the invention, the three-dimensional structure with specific cavities for hydrocortisone HC and prednisolone P is successfully synthesized through the synthetic polymer, so that the hydrocortisone and the prednisolone have larger adsorption capacity and better specific adsorption capacity.
2. The magnetic molecularly imprinted polymer prepared by the invention can not only specifically adsorb, enrich and detect residual trace hydrocortisone and prednisolone in food, but also has stable and specific adsorption capacity on target compounds through a specific cavity with a fixed shape, so that the magnetic molecularly imprinted polymer can keep higher adsorption capacity even in multiple elution-adsorption operations, has reusability for continuous use after elution, is simple and convenient in separation means from a detected product, and has sufficient application potential in the field of food impurity detection.
Drawings
FIG. 1 is a graph of experimental criteria for linear relationship of Hydrocortisone (HC) and prednisolone (P) according to the present invention; wherein a is a hydrocortisone standard curve; b is a prednisolone standard curve;
FIG. 2 is a single-factor adsorption capacity histogram for the polymerization experimental feedstock screen of example 4;
FIG. 3 is a molar ratio one-factor adsorption capacity histogram of the polymerization experiment in example 4;
FIG. 4 is a graph of the time stamped single factor results of example 4;
FIG. 5 is a graph of the polymerization temperature one-factor results in example 4;
FIG. 6 is a graph of the polymerization time single-factor results in example 4;
FIG. 7 is a graph of the static adsorption test of the magnetic molecularly imprinted polymer in example 4;
FIG. 8 is a graph of the static adsorption fit of the magnetic molecularly imprinted polymer of example 4; wherein graph A, B is a Langmuir fit for HC and P, respectively, and graph C, D is a Freundlich fit for HC and P, respectively;
FIG. 9 is a graph of dynamic adsorption test of magnetic molecularly imprinted polymer in example 4;
FIG. 10 is a graph of the dynamic adsorption kinetics fit of the magnetic molecularly imprinted polymer of example 4; wherein graph A, B is a first order kinetic fit to HC and P, respectively, and graph C, D is a second order kinetic fit to HC and P, respectively;
FIG. 11 is a repetitive adsorption cycle bar chart in example 4;
FIG. 12 is a graph of the adsorption capacity of the selectivity experiment in example 4.
Detailed Description
For better understanding of the content of the patent of the present invention, the following detailed description of the embodiments of the present invention is provided for the purpose of providing detailed embodiments and specific operation procedures on the premise of the technical solution of the present invention, but the content of the present invention is not limited to the following examples.
The reagents, materials and instrument sources involved in the examples of the present invention are shown below:
reagent and material (check): acrylamide (AM, 99%), methacrylic acid (MAA, 98%), 4-vinylpyridine (4-VP, 96%), ethylene glycol dimethacrylate (EGDMA, 99%), divinylbenzene (DVB, 80%), 2-azobisisobutyronitrile (AIBN, 99%), hydrocortisone (HC, 98%), ursodeoxycholic acid (UA, 99%), γ -methacryloxypropyl trimethoxysilane (silane coupling agent, KH 570), prednisolone (Pre, 98%), itaconic acid (IA, 99%), acetonitrile (MeCN), trichloromethane, tetrahydrofuran (THF), tetraethyl orthosilicate (TEOS), hydrochloric acid, ethylene glycol and anhydrous sodium acetate, all available from national pharmaceutical chemicals limited; absolute ethanol and methanol were purchased from stannous biochemical engineering limited; glacial acetic acid, aqueous ammonia, and dihydrate, trisodium citrate are all purchased from south Beijing chemical agents, inc.; chromatographic acetonitrile was purchased from Shanghai Sixinbiotech Co. The above reagents were all analytically pure unless otherwise specified.
Self-matching reagent:
(1) Hydrocortisone standard solution: 5mg of hydrocortisone was fixed in a 10mL volumetric flask with acetonitrile to prepare a standard solution with a concentration of 500ug/mL, which was diluted to the desired concentration for subsequent experiments.
(2) Prednisolone standard solution: 5mg of prednisolone is fixed in a 10mL volumetric flask with acetonitrile to prepare a standard solution with the concentration of 500ug/mL, and the standard solution is diluted to the required concentration by a subsequent experiment.
The main instrument is as follows: fourier transform infrared spectrometer (FT-IR) model tensor 27 (Bruker, germany), scanning Electron Microscope (SEM), regulatory 8100 (japan); transmission electron microscopy, TGA4000, perkin elmer, usa; vibrating sample magnetometer VSM-7404 (lasesshore, U.S.A.); high Performance Liquid Chromatography (HPLC) lc-20at,Shimadzu Japan using an inorganic phosphorus C18 column (4.6I.DX250 mm,5mm, shimadzuGL, shanghai).
Example 1: preparation of Magnetic Molecularly Imprinted Polymers (MMIPs)
The specific preparation process of the dual-template Magnetic Molecularly Imprinted Polymer (MMIPs) for specifically adsorbing hydrocortisone and prednisolone is as follows:
1. improved hydrothermal method for preparing Fe with carboxyl on surface 3 O 4 A nanoparticle;
4.5g of ferric chloride hexahydrate, 1.245g of trisodium citrate dihydrate and 150ml of ethylene glycol were added to the beaker, magnetically stirred at room temperature until the solid particles dissolved, and 7.2g of anhydrous sodium acetate was slowly added to the mixed solution, magnetically stirred at 30 ℃ for 30 minutes. The solution was poured into a reactor and reacted at 200℃for 10 hours. After the reaction, the magnetic particles were separated by applying a magnetic field and repeatedly washed with deionized water and ethanol until the solution was clear. Drying the cleaned magnetic particles in a vacuum drying oven at 55deg.C for 6 hr to obtain Fe with carboxyl on surface 3 O 4 And (3) nanoparticles.
2. Preparation of Fe 3 O 4 @SiO 2 Composite particles;
0.75g of synthesized Fe is weighed 3 O 4 Nanoparticles were dispersed in 150ml of 0.1% (v/v) hydrochloric acid solution, sonicated for 10 minutes, the magnetic material was collected with a magnet and washed with deionized water until it was neutral. The washed particles were dispersed in 120ml of ethanol and 30ml of deionized water, 3.75ml of tetraethyl orthosilicate and 7.5ml of 28wt% aqueous ammonia solution were added to the solutions, respectively, and mechanically stirred at 30℃for 12 hours. After the reaction, the magnetic material was collected by applying a magnetic field and washed with deionized water and ethanol until the solution was clear. Drying the cleaned particles in a vacuum drying oven at 55deg.C for 12 hr to obtain Fe 3 O 4 @SiO 2 And (3) composite particles.
3. Preparation of Fe 3 O 4 @SiO 2 -C＝C；
0.5g of synthesized Fe is weighed 3 O 4 @SiO 2 This was dispersed in 100ml of 80% (v/v) aqueous methanol to which 2ml of gamma-methacryloxypropyl trimethoxysilane was added, and magnetically stirred at 70℃for 24 hours under nitrogen protection. After the reaction, the magnetic substance was separated by a magnetic field, and the product was washed with methanol. The cleaned material was dried in a vacuum oven at 55℃for 3h.
4. Preparation of Dual-template magnetic molecularly imprinted Polymer
Mixing 0.2mmol of hydrocortisone and prednisolone with 2mmol of acrylamide and 20ml of acetonitrile respectively, performing ultrasonic operation for 30min to dissolve the solid, and imprinting for a certain time at room temperature. Weigh 0.2g Fe 3 O 4 @SiO 2 -c=c, adding 20ml acetonitrile, sonicating for 30min, allowing the magnetic material to disperse homogeneously, and mixing with the solution with template molecules added as described before. To the mixed solution, 20mmol of ethylene glycol dimethacrylate and 50mg of N, N-azobisisobutyronitrile were sequentially added. After deoxygenation with nitrogen for 15min, magnetic stirring was performed at 60℃for 12h. After the reaction, the product was collected by magnetic field and dried in a vacuum oven at 55 ℃ for 6h. Soxhlet extraction of the dried product in 150ml of methanol/acetic acid=9/1 (v/v) solution at 80℃for 24 hours and more, until detection by UV spectrophotometry at 245nm and 240nmHC or P could not be detected to obtain a polymer with specific cavities.
Example 2: preparation of magnetic non-molecularly imprinted polymers (MNIPS)
The preparation method of this example was the same as that of example 1, except that the template molecule was not added.
Example 3: test of Linear relation
The hydrocortisone and prednisolone standard solutions are diluted to be 1, 2, 5, 10, 20, 50, 100, 125 and 250 mug/mL in a gradient manner, three parallel concentrations are set in each group, a high performance liquid chromatography ultraviolet detector is used for injecting samples with the concentrations for analysis, and a standard curve is drawn by taking the standard concentration as an abscissa (X, mug/mL) and the peak area of a chromatogram as an ordinate (Y). The chromatographic column is as follows: inertSustatin C18 column (4.6 i.D. times.250 mm,5 mm), mobile phase deionized water and acetonitrile, solvent ratio deionized water: acetonitrile=45:55 (V/V), flow rate was set at 1ml/min, column temperature 35 ℃, sample volume 10 μl. The ultraviolet detection wavelength was 254nm. The standard graph is shown in fig. 1.
As shown in FIG. 1, the results in FIG. A show that the concentration of HC and the peak area in the range of 1-500. Mu.g/mL have a good linear relationship, Y=24668X-71350 (R= 0.9996), the detection limit is 2.9ng/mL, the quantitative limit is 9.1ng/mL, and the effective measurement of HC under physiological conditions can be performed.
The results in panel B show that the concentration of P in the range of 1-500 μg/mL has a good linear relationship with peak area, y= 24192X-77584 (r=0.9997), the detection limit is 1.3ng/mL, the quantification limit is 4.7ng/mL, and effective measurement of P under physiological conditions can be performed.
Example 4: performance measurement
(one) static adsorption test
1. Single factor screening of functional monomers, cross-linking agents and porogens selected for the preparation of MIPs and NIPs
The experimental group of 11 different raw material combinations was named MIPs1 to MIPs11, and the following MIPs and the respective NIPs were prepared according to the preparation steps of the molecularly imprinted polymer, and according to the raw material combinations of the functional monomers, the crosslinking agent, and the porogen in table 1. 10mg of MIPs and NIPs were weighed out respectively, 2ml of hydrocortisone standard solution having a concentration of 50. Mu.g/ml was added thereto, and the mixture was stirred and shaken at 25℃for 3 hours. After the adsorption, the mixture was passed through a 0.22 μm organic phase microporous membrane and analyzed by high performance liquid chromatography.
The adsorption capacity (mg/g) of the polymer at which adsorption equilibrium was reached was noted Qe; the initial concentration (. Mu.g/ml) of the hydrocortisone standard solution was designated C 0 The concentration of hydrocortisone in the solution after reaching the adsorption equilibrium (μg/ml) was noted as Ce, the volume of the added standard solution (ml) was noted as V, and the mass of the added imprinted polymer (g) was noted as m, and the equilibrium adsorption capacity Qe was determined as follows:
Figure BDA0004161068620000071
the ratio of adsorption capacity of MMPs and MNIPS to template molecules is distinguished by the imprinting factors, and the larger the imprinting factors are, the more the adsorption capacity of MMPs is higher than that of MNIPS, and the more the adsorption capacity of MMPs to template molecules is specific.
Figure BDA0004161068620000072
Wherein QMIPs and QMINIPs (mg/g) represent adsorption capacities of MMIPs and MNIPs to respective template molecules.
The single factor combinations and Qe data used are shown in table 1 and the data histogram is shown in fig. 2.
Table 1: single factor table for screening polymerization experimental raw materials
Figure BDA0004161068620000073
Figure BDA0004161068620000081
As can be seen from Table 1 and FIG. 2, MIP 3, MIP 4, MIP 7 and MIP 9 have higher Qe values of 7.98mg/g, 8.065mg/g, 9.136mg/g, 8.126mg/g and 8.978mg/g, respectively, and can realize high adsorption capacity for HC.
Whereas MIP1, MIP 4, MIP5 and MIP 8 possess excellent IF values of 2.025, 1.813, 2.61 and 8.04, respectively, which can satisfy specific selectivity to HC. In order to obtain a polymer with high adsorption capacity and specific recognition capacity at the same time, the adsorption capacity and the IF value need to be comprehensively considered, and finally MIPs4 are selected, namely AM is taken as a functional monomer, EGDMA is taken as a cross-linking agent, and MeCN is taken as a pore-forming agent to be taken as an optimal raw material combination.
2. Determining the addition proportion of the combination of the screened functional monomer, the cross-linking agent and the template molecule
The preparation method comprises the steps of setting 8 groups of experimental groups with different molar ratios to be MIP 1-8, preparing corresponding MIPs, NIPs and Qe according to the raw material molar ratios in the table 2 by using the optimal raw material combination obtained in the static adsorption experiment 1, namely the combination of AM as a functional monomer, EGDMA as a crosslinking agent and MeCN as a pore-forming agent according to the preparation steps of the molecularly imprinted polymer. The data histogram is shown in fig. 3.
TABLE 2 Single factor table for mole ratio screening of polymerization experiments
Figure BDA0004161068620000082
From the analysis of Table 2 and FIG. 3, it can be concluded that too large a proportion of template molecules or too small a proportion of cross-linking agent can affect the production of MIPs, such as MIP 2, MIP 4, MIP5, and MIP 7, while too small a proportion of functional monomer to cross-linking agent can affect the cross-linking of NIPs, such as MIP5 and MIP 7.MIP Qe is 7.521-8.813mg/g, NIP Qe is 5.104-6.645mg/g, and MIP 6 is found to reach IF value of 1.727, and the molecular engram polymer is prepared according to the raw material molar ratio.
3. Study of the time of the MIPs bulk Pre-polymerization blotting (also called Pre-polymerization)
5 sets of blotting times (3 h,6h,12h,18h,24 h) were set, each set of experiments was run in triplicate, and the optimal blotting time was screened using Qe for HC as an indicator. The experimental results are shown in table 3 and fig. 4.
TABLE 3 time stamp single factor table
Figure BDA0004161068620000091
The pre-polymerization time affects the formation of template molecule-functional monomer complexes. The template molecule and the functional monomer are paired by intermolecular force, so that the template molecule and the functional monomer need to be sufficiently reacted by incubation for a proper time.
From the experimental results, qe shows a trend of rising and then falling along with the increase of the imprinting time, and the maximum adsorption capacity is 8.42mg/g when the imprinting time reaches 12h, so that the pre-polymerization time of template molecules and functional monomers is selected before the synthesis of the polymer is 12h.
4. Investigation of reaction temperature of bulk polymerization of MIPs
Because the structure of the reactant can be damaged due to the excessively high reaction temperature, the final product is hard, the reaction temperature is too low, the reaction is slow, the polymerization reaction is incomplete, and the adsorption effect is affected, 5 groups of reaction temperatures (55 ℃,60 ℃,65 ℃,70 ℃,75 ℃) are set, and each group of experiments are performed in parallel for three times, so that the optimum reaction temperature is screened by taking Qe of HC as an index. The experimental results are shown in table 4 and fig. 5.
TABLE 4 polymerization temperature one-factor Table
Figure BDA0004161068620000092
Figure BDA0004161068620000101
The reaction temperature affects how fast the reaction occurs and the nature of the reactants. It is therefore necessary to conduct experiments with the reaction temperature as a single variable. As can be seen from Table 4 and FIG. 5, the adsorption amount Qe of the polymer increases gradually as the reaction temperature increases, and then decreases gradually, and the maximum adsorption capacity is 8.453mg/g at 60 ℃. After exceeding 60 ℃, the Qe value is continuously reduced with the increase of the temperature, probably due to the fact that the temperature is too high, the generated product is hardened, and the rigidity of the formed crosslinked polymer is too high, so that the adsorption effect is reduced. According to the results, 60℃was finally selected as the optimal reaction temperature for polymer synthesis.
5. Study of reaction time of bulk polymerization of MIPs and NIPs
Since the ideal specific adsorption effect cannot be achieved completely due to the short reaction time, the excessive reaction time may cause the massive reactants to be too hard for subsequent treatment, and therefore, 5 groups of reaction time (3 h,6h,12h,18h and 24 h) are set, and the appropriate reaction time is screened by taking Qe of HC as an index, and experimental results are shown in Table 5 and FIG. 6.
TABLE 5 polymerization time one-factor Table
Figure BDA0004161068620000102
The experimental results are shown in table 5 and fig. 6. It can be seen that when the reaction time is 6 hours, qe reaches a maximum of 8.704mg/g, and the continuous increase of the reaction time Qe does not only cause a significant increase, but also gradually decreases with the increase of the polymerization reaction time, and the adsorption specificity of the polymer synthesized for 6 hours reaches a maximum, IF is 1.973, so that 6 hours is selected as the optimal reaction time for synthesizing the polymer.
6. Testing the maximum adsorption Capacity of double template MMPs and template-free MNIPS Polymer molecules on Hydrocortisone (HC) and prednisolone (P)
10mg of MMIPs and MNIPs are respectively weighed accurately, 2ml of mixed standard solution (1-250 mug/ml, obtained by re-diluting standard solution of HC and P with acetonitrile water solution and mixing the two solutions with the same concentration) with different concentrations is added, and stirring and shaking are carried out for 3 hours at 25 ℃. After the adsorption is finished, the mixed solution is passed through a 0.22 mu m organic phase microporous filter membrane and subjected to high performance liquid chromatography analysis to determine the residual hydrocortisone and prednisolone concentrations in the mixed solution. The experimental results are shown in table 6 and fig. 7.
Table 6 adsorption capacity meter for static adsorption experiment
Figure BDA0004161068620000111
As shown in FIG. 7, the dual-template magnetic molecularly imprinted polymer has good adsorption capacity on hydrocortisone and prednisolone and has a concentration of 125 mug ml -1 Before, as the initial concentration increases, the adsorption capacity tends to increase more, and the adsorption capacity increases more slowly after 125. Mu.g ml-1. At 125 mug ml-1, MMIPs have an adsorption capacity of 16.708mg/g for HC, an adsorption efficiency of 78.6%, an adsorption capacity of 16.708mg/g for Pre, an adsorption efficiency of 83.23%, and IF of 1.918 and 2.229 respectively. The graph shows that the thermodynamic curve growth rate of MNIPS is slower than that of MMIPs, and the difference between the adsorption capacity of MNIPS and the adsorption capacity of MMIPs gradually increases along with the increase of the initial concentration, so that the MMIPs have better specific adsorption capacity to target molecules, and the MMIPs form specific cavities of template molecules HC and P, so that the template molecules have higher adsorption capacity.
7. Static adsorption experimental data analysis
The binding pattern and adsorption process of the polymer were described using Langmuir and Freundlich fitting models for analysis:
Figure BDA0004161068620000112
freundlich fitting model: logQ e ＝(1/n)logC e +logK F
Wherein Qmax (mg/g) is the theoretical maximum adsorption capacity, K, of the polymer L Is the langmuir adsorption coefficient, KF is the Freundlich adsorption coefficient, and n is the Freundlich adsorption constant. The various coefficients in the two fitting curves of MMPs and MNIPs are shown in the following table, and the two fitting curves of MMPs and MNIPs on HC and P adsorption are shown in FIG. 8.
TABLE 7 Langmuir and Freundlich fitting Curve correlation coefficients of Hydrocortisone (HC) and prednisolone (P)
Figure BDA0004161068620000121
Table 7 and FIG. 8 show that the R of HC MMPs and MNIPs is known from the curve trend 2 The values are 0.9812 and 0.9454 respectively, the fitting degree of the dual-template magnetic molecularly imprinted polymer to the fitting curve of HC is the same as that of the fitting curve of L and F, and R of L in the fitting curve of prednisolone 2 The values were greater than F, so the fitted curve was more consistent with the L model, indicating that the adsorption of the polymer to hydrocortisone included both monolayer adsorption present on the surface and adsorption by hydrogen bonding, whereas prednisolone was predominantly monolayer adsorption, with recognition sites present on the molecular surface. Q of MMIPs according to the data of Table 7 max 、K L 、K F The n is higher than MNIPS, which proves that MMPs have better adsorption effect and better selective adsorption capability on template molecules hydrocortisone and prednisolone.
(II) dynamic adsorption test
1. 10mg of MMPs and MNIPs were weighed accurately, 2ml of mixed standard solution of hydrocortisone and prednisolone (each concentration 50. Mu.g/ml before mixing) was added, and the mixture was adsorbed at 25℃for different times (1-240 min). The mixed solution was then passed through a 0.22 μm organic phase microporous filter and then subjected to high performance liquid chromatography to determine the concentrations of residual HC and P in the liquid. The dynamic adsorption curves of MMPs and MNIPs to HC and P are shown in FIG. 9, and the experimental results are shown in Table 8.
Table 8 adsorption capacity table for dynamic adsorption experiments
Figure BDA0004161068620000122
/>
Figure BDA0004161068620000131
As can be seen from FIG. 9, the adsorption capacities of MMIPs of HC and P are rapidly increased within 5min, then the acceleration is slowed down within 5-30min, and the adsorption capacities are slowly increased within 30-90min until the adsorption equilibrium is reached within 90min, and the adsorption equilibrium time of MNIPs is 120min. Meanwhile, the adsorption capacities of MMIPs of HC and P are 8.412mg/g and 7.898mg/g respectively, and MNIPS adsorption capacities of HC and P are 3.778mg/g and 3.812mg/g respectively, which indicate that the polymer is successfully synthesized to have a three-dimensional structure with a specific cavity for a target compound, so that the polymer has larger adsorption capacities for HC and P, and better adsorption capacity for HC and P.
2. And fitting data of a dynamic adsorption experiment by using a primary dynamic model and a secondary dynamic model, so as to know the adsorption mechanism of the polymer and the change of the adsorption rate of the polymer.
First-order dynamics model: ln (Q) e -Q t )＝lnQ max1 -k 1 t
Second order kinetic model:
Figure BDA0004161068620000132
wherein Q is max1 And Q max2 (mg/g) theoretical maximum adsorption capacity of polymer in primary and secondary kinetic models, Q t (mg/g) is the adsorption capacity, k, of the polymer at time t 1 And k 2 (min -1 ) Is the first and second adsorption rate constants, and t (min) is the adsorption time. The various coefficients in the two kinetic models of MMIPs and MNIPs are shown in table 9, and the corresponding kinetic fit graphs are shown in fig. 10.
TABLE 9 first and second order kinetic fit curve correlation coefficients for Hydrocortisone (HC) and prednisolone (P)
Figure BDA0004161068620000133
Figure BDA0004161068620000141
Table 9 and FIG. 10 show that R of the fitted curve is analyzed 2 The values are known, HC andthe second-order kinetic curves of MMIPs and MNIPs of P have the best fitting performance compared with the first-order kinetic curves, which shows that the surface of the polymer has saturated adsorption sites, and adsorption reaches a certain degree, so that adsorption equilibrium is achieved. Q of MMPs in the secondary kinetic profile of P max2 、k 2 All are larger than MNIPs, which indicates that MMPs have more excellent selectivity on P and have larger adsorption effect.
(III) repeatability test
10mg MMPs and MNIPS are respectively placed into different centrifuge tubes, 2ml of hydrocortisone and prednisolone standard solution diluted to 50 mug/ml are added into the centrifuge tubes, the mixture is adsorbed for 2 hours in a constant temperature shaking table at 25 ℃, the supernatant is separated by using an external magnetic field after the completion, and the supernatant is filtered through a 0.22 mu m organic microporous filter membrane for testing the residual hydrocortisone and prednisolone content in the solution by HPLC. The magnetic molecularly imprinted polymer obtained by the applied magnetic field was collected, mixed with 2ml of a methanol/acetic acid (9:1, v/v) mixture, separated by the applied magnetic field after 5min, and dried. The above procedure was repeated six times in total.
The adsorption capacity Qe measured for each of the six repeated adsorption-elution-re-adsorption cycles is shown in the table, wherein Qe 0 Represents the unrepeated adsorption capacity, qe 1 The adsorption capacity after one repeated adsorption cycle is shown, and the results of the repeatability test are shown in fig. 11 and table 10:
table 10 adsorption capacity table for repeatability experiments
Figure BDA0004161068620000142
After MMPs and MNIPs of HC and P are respectively subjected to adsorption-elution-re-adsorption circulation operation, the adsorption capacity of the four polymers gradually decreases, and the adsorption capacity Qe of MMPs of HC and P is respectively reduced by 4.95% and 4.30% from the first repetition to the sixth repetition; MNIPs for HC and P decreased by 12.13% and 6.076% respectively from the first to the sixth repetition of adsorption capacity Qe.
From the results of MMIPs and MNIPs, it can be seen that the MMIPs recycling rate remains high after six times of repeated use, which indicates that MMIPs have higher stability and reusability than MNIPs, because MMIPs have specific cavities of a fixed shape, and can have stable and specific adsorption capacity for target compounds, so that the MMIPs can maintain high adsorption capacity even in multiple elution-adsorption operations. MNIPS has slightly lower repeatability than MMPs due to non-template blotting, and Qe 0 And are lower than MMPs, and the adsorption performance of the dual-mode MMPs is considered to be more excellent.
(IV) Selectivity test
A plurality of structural analogues similar to Hydrocortisone (HC) are selected to prepare a mixed solution for adsorption test, and the selected structural analogues comprise ursodeoxycholic acid (Canoic), ethinyl Estradiol (EE), deoxynivalenol (DON) and prednisolone (Prednis). 5 samples were mixed in a beaker to prepare mixed solutions each having a concentration of 50. Mu.g/ml, 10mg MMPs and MNIPs were accurately weighed, 2ml of the above mixed solution was added thereto, and the mixture was adsorbed at 25℃for 3 hours. The supernatant was separated by an external magnetic field, passed through a 0.22 μm organic phase microporous membrane, and then used for HPLC to detect the concentration of the above structural analog. The adsorption capacities of MMIPs and MNIPs for each compound were determined, and then the blotting factor IF was calculated to compare the adsorption capacities of the two.
The results of the selectivity experiments are shown in figure 12.
FIG. 12 shows that MMPs and MNIPs have the highest adsorption capacity for template molecules HC and P among the five compounds, and the imprinting factor IF reaches 2.11 and 2.307, respectively. Canoic, EE and DON have similar backbone and functional group structures as HC and P, but polymers have a lower recognition capacity for them. The polymer has no high adsorption capacity to other compounds, can selectively adsorb target molecules HC and P in the mixture, is a material with high selectivity and high specificity, and is suitable for accurately adsorbing HC and P in a complex matrix.
Example 5: application of Magnetic Molecularly Imprinted Polymers (MMIPs) in milk and milk powder
The raw material obtaining sources are as follows: the milk is Deya brand skimmed milk and is purchased in a Jiang Ningou Su fruit supermarket in Nanjing; the milk powder is a formula milk powder for children in the golden collar crown, and is purchased in the Jiang Ningou Su fruit supermarket in Nanjing.
The preparation method of MMPs in this example was the same as in example 1.
Dispersing 1g milk powder in a mixed solution of 45ml acetonitrile and 5ml deionized water, ultrasonically extracting for 10min, oscillating for 10min, centrifuging at a speed of not less than 5000r/min for 15min, and collecting supernatant. The collected supernatant was refrigerated in a refrigerator at 4-8 ℃ and left for a while, filtered again to collect the supernatant. Four standard solutions of HC and P mix with standard levels of 10, 20, 50 and 100ng/ml, respectively, were added to the blank matrix of the milk powder solution described above. To 20ml of the sample solution, 10mg of dual-template MMPs were added, and the pellet was collected by external magnetic field at 25℃for 40min of adsorption with shaking. The eluate was collected with an external magnetic field, eluted with 1ml of methanol/acetic acid (9:1, v/v) solution for 5 minutes, dried with nitrogen, and then redissolved in 1ml of acetonitrile. After passing through a 0.22 μm organic phase microporous filter membrane, loading and analyzing by HPLC, each standard adding level is measured in parallel for 6 times, and the average recovery, recovery rate and precision are obtained.
2g of milk was dispersed in 5ml of acetonitrile, vortexed and mixed for 5min, centrifuged at 10000r/min for 10min, and the supernatant was collected. The collected supernatant was refrigerated in a refrigerator at 4-8 ℃ and left for a while, filtered again to collect the supernatant. Four standard solutions of HC and P mix with standard levels of 10, 20, 50 and 100ng/ml, respectively, were added to the blank matrix of the milk solution. To 50ml of the sample solution, 10mg of dual-template MMPs were added, and the pellet was collected by external magnetic field at 25℃for 40min of adsorption with shaking. The eluate was collected with an external magnetic field, eluted with 1ml of methanol/acetic acid (9:1, v/v) solution for 5 minutes, dried with nitrogen, and then redissolved in 1ml of acetonitrile. After passing through a 0.22 μm organic phase microporous filter membrane, loading and analyzing by HPLC, each standard adding level is measured in parallel for 6 times, and the average recovery, recovery rate and precision are obtained.
The results of the experiment are shown in Table 11.
TABLE 11 sample recovery and precision test results
Figure BDA0004161068620000161
By comparing the results of the application of the two types of compounds in the samples, the recovery rate of the two sample treatment methods is more than 87%, the precision is within 4%, and the treatment method is proved to be feasible and can be directly applied to the enrichment, purification and detection of HC and P in the milk products.
The above description is only a preferred embodiment of the present invention and the above embodiments are not intended to limit the scope of the present invention, but the present invention is not limited to the above embodiments, and all equivalent modifications, equivalent substitutions and improvements made by those skilled in the art based on the present disclosure should be included in the scope of the present invention.

Claims (10)

1. The preparation method of the dual-template MMPs for specifically adsorbing HC and P is characterized by comprising the following steps of:
s1, preparing Fe with carboxyl on surface by improved hydrothermal method 3 O 4 A nanoparticle;
s2, preparing Fe 3 O 4 @SiO 2 Composite particles;
s3, preparing Fe 3 O 4 @SiO 2 -C＝C；
S4, preparing a dual-template magnetic molecularly imprinted polymer: mixing each template molecule with a functional monomer and a pore-forming agent respectively, dissolving, and imprinting at room temperature to obtain a first mixed solution; weighing Fe obtained in the step S3 3 O 4 @SiO 2 -c=c, adding a porogenic agent, uniformly dispersing the magnetic material by ultrasound, and mixing with the first mixed solution to obtain a second mixed solution; then adding a cross-linking agent and an initiator into the second mixed solution in sequence to carry out polymerization reaction; heating after deoxidizing the nitrogen; after the reaction, the product is collected by a magnetic field, dried and Soxhlet extracted to obtain a polymer with a specific cavity, wherein the templateThe molecules are hydrocortisone HC and prednisolone P.
2. The method for preparing dual-template MMIPs for specific adsorption of HC and P according to claim 1, wherein step S1 specifically comprises: adding ferric chloride hexahydrate, trisodium citrate dihydrate and ethylene glycol into a beaker, magnetically stirring at room temperature until solid particles are dissolved, then adding anhydrous sodium acetate, and stirring until the solution is uniform; pouring the solution into a reactor, and reacting for 10 hours at 200 ℃; after the reaction, magnetic particles are separated by applying a magnetic field, repeatedly washed by deionized water and ethanol, and dried to obtain Fe with carboxyl on the surface 3 O 4 A nanoparticle;
the step S2 specifically includes: taking Fe synthesized in the step S1 3 O 4 Dispersing the nano particles in hydrochloric acid solution, carrying out ultrasonic treatment, collecting magnetic materials by using a magnet, and washing to be neutral; dispersing the washed particles in a mixed solution of ethanol and deionized water, respectively adding tetraethyl orthosilicate and 28wt% ammonia water solution into the solution, and stirring at normal temperature for reaction; separating, washing and drying after the reaction to obtain Fe 3 O 4 @SiO 2 Composite particles;
the step S3 specifically includes: taking Fe synthesized in the step S2 3 O 4 @SiO 2 Dispersing composite particles in methanol water solution added with silane coupling agent, stirring, separating, washing and drying to obtain Fe 3 O 4 @SiO 2 -C＝C。
3. The method for preparing dual-template MMIPs for specific adsorption of HC and P according to claim 2, wherein the silane coupling agent is γ -methacryloxypropyl trimethoxysilane.
4. The method for preparing dual-template MMIPs for specific adsorption of HC and P according to claim 1, wherein the functional monomer is any one of acrylamide, methacrylic acid, 4-vinylpyridine, itaconic acid.
5. The method for preparing dual-template MMIPs for specific adsorption of HC and P according to claim 1, wherein the porogen is any one of acetonitrile, ethanol, chloroform, tetrahydrofuran.
6. The method for preparing dual-template MMIPs for specific adsorption of HC and P according to claim 1, wherein the time of blotting at room temperature is 3-24 h.
7. The method for preparing dual-template MMIPs for specific adsorption of HC and P according to claim 1, wherein the cross-linking agent is any one of ethylene glycol dimethacrylate, tetraethyl orthosilicate, divinylbenzene; the initiator is 2, 2-azo diisobutyronitrile.
8. The method for preparing dual-template MMPs for specifically adsorbing HC and P according to claim 1, wherein in the step S4, the addition ratio of the template molecule, the functional monomer and the crosslinking agent is 1:1-10:5-50.
9. The method for preparing dual-template MMIPs for specific adsorption of HC and P according to claim 1, characterized in that the polymerization reaction is carried out at a temperature of 55-75 ℃; the reaction time is 3-24 h.
10. The method for preparing dual-template MMIPs for specific adsorption of HC and P according to claim 9, characterized in that the polymerization reaction is carried out at a temperature of 60 ℃; the polymerization reaction was carried out for 6 hours.
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