CN111019070A

CN111019070A - Preparation method of zearalenone magnetic molecularly imprinted polymer

Info

Publication number: CN111019070A
Application number: CN201911325619.9A
Authority: CN
Inventors: 陈贵堂; 付含; 徐武
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2019-12-20
Filing date: 2019-12-20
Publication date: 2020-04-17
Anticipated expiration: 2039-12-20
Also published as: CN111019070B

Abstract

The invention discloses a preparation method of a zearalenone magnetic molecularly imprinted polymer, which comprises the following steps: (1) hydrothermal method for preparing Fe₃O₄A nanoparticle; (2) preparation of Fe₃O₄@SiO₂Microspheres; (3) fe₃O₄@SiO₂Vinylation of the microspheres; (4) fe₃O₄@SiO₂Preparation of-C ═ C molecularly imprinted polymers. The magnetic surface molecularly imprinted material prepared by the invention has good selectivity, strong specificity and strong magnetism.

Description

Preparation method of zearalenone magnetic molecularly imprinted polymer

Technical Field

The invention relates to a preparation method of a molecularly imprinted polymer, in particular to a preparation method of a zearalenone magnetic molecularly imprinted polymer.

Background

Zearalenone (Zearalenone, ZEA) is a mycotoxin produced by fusarium and gibberella. ZEA is found in many cereal crops worldwide, such as corn, barley, oats, wheat, rice and sorghum. Many reports indicate that zearalenone causes excessive estrogen in humans and animals, and that their adverse effects on animal and human reproduction are not negligible. To assess the risk of ZEA, sensitive detection of ZEA in cereals is required.

Disclosure of Invention

The purpose of the invention is as follows: the invention aims to provide a preparation method of a zearalenone magnetic molecularly imprinted polymer.

The technical scheme is as follows: the invention provides a preparation method of a zearalenone magnetic molecularly imprinted polymer, which comprises the following steps:

(1) hydrothermal method for preparing Fe₃O₄A nanoparticle;

(2) preparation of Fe₃O₄@SiO₂Microspheres;

(3)Fe₃O₄@SiO₂vinylation of the microspheres;

(4)Fe₃O₄@SiO₂preparation of-C ═ C molecularly imprinted polymers.

Further, the preparation method of the step (1) is as follows: FeCl is added₃·6H₂Dissolving O and trisodium citrate in ethylene glycol, adding sodium acetate into the solution system, stirring until the solution is uniform, heating for reaction, cooling to room temperature after the reaction is finished, washing, and drying to obtain Fe₃O₄And (3) nanoparticles.

Further, the preparation method of the step (2) is as follows: taking Fe₃O₄The method comprises the following steps of putting nano particles into a hydrochloric acid solution, carrying out ultrasonic treatment, then uniformly dispersing the nano particles into a mixed solution of ethanol and deionized water, adding concentrated ammonia water and tetraethyl orthosilicate, stirring the mixture at normal temperature to react, separating, washing and drying the mixture after the reaction is finished to obtain Fe₃O₄@SiO₂And (3) microspheres.

Further, the preparation method of the step (3) is as follows: mixing Fe₃O₄@SiO₂Dispersing the microspheres in 3- (methacryloyloxy) propyl trimethoxy silane (MPS) -containing methanol acetate solution, stirring, separating, washing, and drying to obtain vinyl Fe₃O₄@SiO₂-C ═ C microspheres.

Further, the preparation method of the step (4) is as follows: dissolving virtual template molecules warfarin and APTES into methanol, standing at room temperature to obtain a first mixed solution, and reacting vinylated Fe₃O₄@SiO₂Ultrasonically dispersing the-C microspheres into a mixed solution of methanol and distilled water, sequentially adding TEOS and concentrated ammonia water, stirring, adding the first mixed solution, stirring at room temperature, separating by using a magnet, washing, performing Soxhlet extraction after the reaction is finished, and eluting template molecules.

Has the advantages that: the invention prepares the magnetic zearalenone molecularly imprinted polymer by a hydrothermal method and a surface imprinting method, inspects the adsorption performance and the selectivity of the polymer to ZEA, and performs specific adsorption on the ZEA in an actual sample to obtain a specific adsorption material with high recovery rate and high precision. The magnetic surface molecular imprinting material prepared by the invention has good selectivity, strong specificity and strong magnetism, and avoids complex centrifugation and filtration operations. Ferroferric oxide prepared by a hydrothermal method is used as a molecular imprinting material after being modified, and the molecular imprinting polymer prepared by the surface imprinting method has strong specificity recognition capability on zearalenone. The preparation cost of the polymer is greatly reduced by using the zearalenone structural analogue warfarin as a template molecule.

Drawings

FIG. 1 is an infrared spectrum of various products;

FIG. 2 shows XRD spectra of different products;

FIG. 3 is a transmission electron microscope image of various products;

FIG. 4 is a graph of the static adsorption profile of a polymer;

FIG. 5 is a graph of a Scatchard fit of MMIPs;

FIG. 6 is a MNIPs Scatchard fit graph;

FIG. 7 is a graph of polymer dynamic adsorption;

FIG. 8 is a graph of a first order fit of dynamics;

FIG. 9 is a graph of a kinetic secondary fit;

FIG. 10 is a graph showing the results of the recycling experiment.

Detailed Description

Example 1

The preparation method of the zearalenone magnetic Molecularly Imprinted Polymer (MIP) comprises the following steps:

(1) hydrothermal method for preparing Fe₃O₄Nanoparticles

FeCl₃·6H₂O (1.38g) + trisodium citrate (0.5g) was dissolved in 40mL of ethylene glycol. To the above solution system was added 1.2g of sodium acetate, and the solution was stirred until homogeneous (about 0.5 h). The mixture is transferred into a 50mL hydrothermal reaction kettle to react for 10h at 200 ℃. And cooling to room temperature. Washed repeatedly three times with ethanol and water (vacuum dried at 55 ℃ C.).

(2)Fe₃O₄@SiO₂Preparation of microspheres

Accurately weighing 0.2g of magnetic Fe₃O₄Placing the nano particles in 100mL of 0.1mol/L hydrochloric acid solution, carrying out ultrasonic treatment for 10min, then uniformly dispersing the nano particles in 80mL of ethanol and 20mL of deionized water, adding 2mL of 28% concentrated ammonia water and 1mL of tetraethyl orthosilicate, stirring at 200rpm and normal temperature, reacting for 6h, adding a magnetic field for separation after the reaction is finished, repeatedly washing the nano particles with the deionized water and the methanol, and placing the nano particles in a vacuum drying oven for drying at 55 ℃.

(3)Fe₃O₄@SiO₂Preparation of-C ═ C microspheres

200mg of prepared Fe₃O₄@SiO₂The microspheres were dispersed in 100mL of 10% acetic acid methanol solution containing 0.6mL3- (methacryloyloxy) propyltrimethoxysilane (MPS), and subjected to magnetic separation by mechanical stirring at 50 ℃ for 24h vinylation, washed three times with deionized water methanol, and vacuum dried at 55 ℃.

(4)Fe₃O₄@SiO₂Preparation of-C ═ C Molecularly Imprinted Polymers (MIP) and non-molecularly imprinted polymers (NIP)

1mmol of a virtual moldDissolving warfarin and 2mL of APTES in 10mL of methanol, standing at room temperature for 3h, and accurately weighing 200mg of Fe₃O₄Ultrasonically dispersing the nano material into a mixed solution containing 30mL of methanol and 5mL of distilled water, sequentially adding 2mL of TEOS and 1mL of concentrated ammonia water, mechanically stirring for 5min, adding the solution, mixing, mechanically stirring for 1h at room temperature, separating by using a magnet after the reaction is finished, washing by using deionized water and methanol for a plurality of times, and performing Soxhlet extraction by using methanol/acetic acid (9: 1) for 24 hours to elute template molecules.

Example 2: preparation of non-molecularly imprinted polymers (NIP)

The preparation method of this example was the same as that of example 1 except that no template molecule was added.

The products of the 2 examples above were used for the following structural validation, performance or use determinations.

Example 3: structural identification

(1) Infrared spectroscopy

Fourier transform infrared spectroscopy (FT-IR) was used to examine the preparation of magnetic nanoparticles. FIG. 1 is an infrared spectrum, Fe₃O₄1612 and 1400cm in the curve^-1The peak at (A) is the stretching vibration peak of the citric acid carboxyl group, and 578 and 454cm^-1The peak at (A) is a typical stretching vibration peak of Fe-0, and it can be confirmed that carboxyl is in Fe₃O₄And (4) attaching the surface. At curve Fe₃O₄@SiO₂In 1085cm^-1The peak at (A) is a Si-O-Si stretching vibration peak. At curve Fe₃O₄-C ═ C at 1695cm^-1The absorption peak at (b) is a C ═ C bond absorption oscillation peak of MPS, which also confirmed that the carbon-carbon double bond is Fe₃O₄The surfaces of the nanoparticles are connected. In the curve MIPs, the characteristic absorption peak of N-H of APTES is 1548cm^-1. The tensile vibration peak of the C-H bond was 2927cm^-1Shows that APTES generates copolymerization reaction and the MIPs layer is successfully wrapped in Fe₃O₄And (4) the surface of the nanosphere.

(2) X-ray diffraction pattern

FIG. 2 shows Fe₃O₄(a scheme), Fe₃O₄@SiO₂(b scheme), Fe₃O₄-C ═ C (panel C), MNIPs (panel d), XRD spectrum of samples of MMIPs (panel e). In the range of 20-80 DEG, Fe₃O₄The five characteristic diffraction peaks of (a) appear in the five sample spectra at 30.10 °, 35.48 °, 43.38 °, 57.35 ° and 62.72 °, respectively. The positions of these diffraction peaks and Fe₃O₄The diffraction peak positions of PDF (card number: 65-3107) were consistent and no other impurity crystal peaks appeared, indicating that the crystal structure was not changed during the synthesis of the polymeric layer. The appearance of characteristic peaks in the XRD pattern indicates that the synthesized polymeric material is highly crystalline. With Fe₃O₄(a diagram) comparison of Fe₃O₄@SiO₂(b scheme), Fe₃O₄C (panel C), MNIPs (panel d) and MMIPs (panel e) have slightly increased peak widths and slightly decreased intensities due to the encapsulation of the silicon layer and the molecularly imprinted layer.

(3) Transmission Electron Microscope (TEM)

From FIG. 3(a. Fe. C₃O₄b.Fe₃O₄@SiO₂c.Fe₃O₄@Si0₂Mmips e.mmips), Fe synthesized by hydrothermal method₃O₄The nanoparticles have a particle size of about 300nm and are relatively uniform in shape and particle size. After silanization, the particle size changed significantly to about 350nm, with a wrapped silane layer of about 25 nm. Mixing Fe₃O₄@SiO₂After the microspheres are subjected to surface vinylation and double bond grafting, the particle size is not obviously changed. The particle size of MMIPs and MNIPs is about 450nm, and the thickness of the molecular imprinting layer is about 50nm, which indicates that the surface imprinting reaction is successful. In the figure, a is Fe synthesized by a hydrothermal method₃O₄In the drawing, b is Fe₃O₄Fe coated with silicon dioxide₃O₄@SiO₂FIG. c Fe₃O₄@SiO₂Surface vinylated Fe₃O₄@SiO₂-C ═ C diagram, and d and e respectively polymerise MNIPs and MMIPs prepared.

Example 4: determination of Properties or of uses

(1) Static adsorption

The concentration of the adsorption solution affects the adsorption amount of the polymer, and too high or too low concentration is not favorable for adsorption. In order to study the relationship of the adsorption amount with the change of the concentration of the adsorption solution, an adsorption isotherm is drawn. Eight groups of MMIPs and MNIPs obtained in the previous examples are accurately weighed according to the amount of 20mg of each part, and are respectively placed in 10mL centrifuge tubes, 2mL corn samples with different concentrations are respectively added with zearalenone solutions (1mg/L, 2mg/L, 5mg/L, 10mg/L, 20mg/L, 30mg/L, 40mg/L, 50mg/L, 60mg/L, 70mg/L, 80mg/L and 90mg/L), and the mixture is placed in an air shaking bed and shaken at room temperature for 90 min. Then centrifugating at 5000r/min for 15min, collecting supernatant, filtering with 0.22 μm filter membrane, and measuring with high performance liquid chromatograph. And calculating the adsorption capacity of the polymer according to a formula.

And (4) conclusion: as can be seen from FIG. 4, as the concentration increases, the adsorption amounts of MMIPs and MNIPs also increase when the concentration reaches 80mg/L, the equilibrium adsorption amount is basically 13.904mg/g, but the increase of MNIPs is lower than that of MMIPs, and the adsorption amount at the same concentration is much smaller than that of MMIPs. This indicates that the template molecule leaves imprinted pores in the imprinted polymer during imprinting, making its affinity for the template molecule much higher than that of the non-imprinted polymer.

Scatchard model

Qe(mg·g^-1) Represents the amount of zearalenone adsorbed by the polymer at equilibrium. ρ e (mg. L)^-1) Represents the residual zearalenone concentration in the solution at equilibrium; qmax (mg. g)^-1) Represents the theoretical maximum adsorption capacity of the polymer, and Kd represents the adsorption coefficient.

The number of binding sites of the material to the target was assessed by the Scatchard model. The Scatchard fit curve for MMIPs shown in figure 5 has two linear portions, indicating the presence of two types of binding sites on the surface of the MMIPs. The linear regression equations of the sections of FIG. 5 are Q, respectively_e/ρe＝-4.07523Qe+10.42501(R²0.991) and Q_e/ρe＝-0.12159Qe+1.86929(R²0.959). Kd and Q calculated from the formula_maxThe values are 0.245 mg.L on the left^-1And 2.55mg g^-18.22 mg. L on the right side^-1And 15.37mg g^-1. From the Kd values, it can be seen that MMIPs have non-specific and specific recognition sites for selective adsorption of zearalenone. In contrast, fig. 6 shows that the Scatchard fit curves for MNIPs show only one straight line. The corresponding linear regression equation is Q_e-0.01706Qe +0.14035(R2 0.993), with a high Kd value (58.6mg · L)^-1)，Q_maxThe value is low (8.22 mg. g)^-1). From the Kd values, it can be seen that MMIPs have two low affinity and high affinity binding sites that selectively adsorb zearalenone, whereas MNIPs have only one binding site with no affinity. These results demonstrate that the prepared MMIPs have selective binding ability with high adsorption capacity to zearalenone.

(2) Dynamic adsorption

The experimental method comprises the following steps: in order to examine the adsorption rate of the polymer prepared by the experiment on the target zearalenone added in an actual sample corn sample, 7 groups of 20mg MMIPs and MNIPs are accurately weighed respectively, placed in a 10mL centrifuge tube, 2mL of 50mg/L zearalenone solution is added respectively, the mixture is fixed on an air shaking table, the mixture is shaken at room temperature for different times (5min, 10min, 20min, 30min, 60min, 90min, 120min and 180min) and is respectively centrifuged for 15min at 5000r/min by a centrifuge, the supernatant is taken to pass through a 0.22 mu m filter membrane, the concentration of the zearalenone in the supernatant is measured on a high performance liquid chromatograph, and the adsorption capacity of the polymer is calculated according to a formula.

As can be seen from the kinetic adsorption curve in FIG. 7, the adsorption amount of MMIPs rapidly increased before 20 minutes and then decreased. The adsorption equilibrium was reached at 30 minutes, the adsorption quantity was 8.71mg/g and the saturation adsorption quantity was 8.81 mg/min. The adsorption behavior of MNIPs is essentially the same as that of MMIP, but the adsorption capacity is significantly lower. At 30 minutes, the adsorption capacity of MMIPs was 2.3 times that of MNIPs. This is due to the presence of specific recognition sites on the MMIPs, which allows for easier entry and exit of zearalenone.

first-order model In (Qe-Qt) ═ InQ_1c-k₁t

second-order model

Wherein Q_e(mg·g^-1) Is the adsorption capacity of the polymer in an equilibrium state; q_cTheoretical adsorption capacity; q_tThe adsorption amount at time t. k is a radical of₁And k₂First and second order adsorption coefficients, respectively.

TABLE 1

Fig. 8 is a first order kinetic fit curve and fig. 9 is a second order kinetic fit curve, and the fit results are shown in table 1. It can clearly be seen that the quasi-second order equations have better linearity and higher precision than the first order equations. Further, as shown in Table 1, Q_eAnd Q_2cRatio Q_eAnd Q_1cMore recently. These results indicate that the adsorption mechanism of MMIPs conforms to the quasi-second order model. It can be concluded that chemical interactions between molecules are the main cause of influencing the adsorption rate.

(3) Repeated utilization experiment

In order to evaluate the repeated utilization rate of the magnetic molecularly imprinted polymer, 20mg of the imprinted polymer is weighed and placed in 2mL of zearalenone standard solution with the concentration of 50 mug/mL, stirring and oscillating are carried out for 90min at room temperature, a magnet is additionally arranged for separation, Soxhlet extraction is carried out for 24h by using methanol and acetic acid (9: 1/v: v), so that adsorbed zearalenone is desorbed, the eluted material is dried in vacuum at 55 ℃ to constant weight, the same adsorption and desorption process is repeated for 8 times, and the change of the adsorption effect of the imprinted polymer is observed, which is shown in figure 10.

After the polymer material is used for a plurality of times, the adsorption quantity is slightly reduced, but the adsorption quantity can still be maintained to be about 8 mg/g. This indicates that the recognition sites of MMIPs can maintain stable and reusable properties after multiple regenerations. The zearalenone is eluted from the polymer by washing with a mixed solution of methanol and acetic acid. Therefore, the synthesized molecularly imprinted polymer has strong adsorption capacity and high stability, can realize the recycling of materials by a simple method, and greatly reduces the process of sample pretreatment compared with the traditional materials.

Claims

1. A preparation method of a zearalenone magnetic molecularly imprinted polymer is characterized by comprising the following steps: the method comprises the following steps:

(1) hydrothermal method for preparing Fe₃O₄A nanoparticle;

(2) preparation of Fe₃O₄@SiO₂Microspheres;

(3)Fe₃O₄@SiO₂vinylation of the microspheres;

(4)Fe₃O₄@SiO₂preparation of-C ═ C molecularly imprinted polymers.

2. The method for preparing a zearalenone magnetic molecularly imprinted polymer according to claim 1, characterized in that: the preparation method of the step (1) comprises the following steps: FeCl is added₃·6H₂Dissolving O and trisodium citrate in ethylene glycol, adding sodium acetate into the solution system, stirring until the solution is uniform, heating for reaction, cooling to room temperature after the reaction is finished, washing, and drying to obtain Fe₃O₄And (3) nanoparticles.

3. The method for preparing a zearalenone magnetic molecularly imprinted polymer according to claim 1, characterized in that: the preparation method of the step (2) is as follows: taking Fe₃O₄The method comprises the following steps of putting nano particles into a hydrochloric acid solution, carrying out ultrasonic treatment, then uniformly dispersing the nano particles into a mixed solution of ethanol and deionized water, adding concentrated ammonia water and tetraethyl orthosilicate, stirring the mixture at normal temperature to react, separating, washing and drying the mixture after the reaction is finished to obtain Fe₃O₄@SiO₂And (3) microspheres.

4. The method for preparing a zearalenone magnetic molecularly imprinted polymer according to claim 1, characterized in that: the preparation method of the step (3) is as follows: mixing Fe₃O₄@SiO₂Dispersing microspheres in 3- (methacryloyloxy) propyl trimethoxy silane (MPS) -containing methanol acetate solution, stirring, separating, washing, and dryingDrying to obtain vinylated Fe₃O₄@SiO₂-C ═ C nanospheres.

5. The method for preparing a zearalenone magnetic molecularly imprinted polymer according to claim 4, characterized in that: the preparation method of the step (4) is as follows: dissolving virtual template molecules warfarin and APTES into methanol, standing at room temperature to obtain a first mixed solution, and reacting vinylated Fe₃O₄@SiO₂Ultrasonically dispersing the-C microspheres into a mixed solution of methanol and distilled water, sequentially adding TEOS and concentrated ammonia water, stirring, adding the first mixed solution, stirring at room temperature, separating by using a magnet, washing, performing Soxhlet extraction after the reaction is finished, and eluting template molecules.