阅读说明：本技术 一种限进介质-分子印迹聚合物及其制备方法与应用 (Limited medium-molecularly imprinted polymer and preparation method and application thereof ) 是由 左海根 于 2021-08-11 设计创作，主要内容包括：本发明涉及生化材料技术领域,尤其涉及一种限进介质-分子印迹聚合物及其制备方法与应用。本发明先将TiO-(2)纳米线的表面苄基化,再将表面苄基化的TiO-(2)纳米线合成RAFT链转移试剂,最后再利用RAFT链转移试剂限进介质-分子印迹聚合物。该制备方法得到的限进介质-分子印迹聚合物分子量分布较窄、结构均匀的聚合物,具有较好的的吸附性能,应用于奶粉中雌二醇测试时,该材料能有效的排除奶粉中大分子杂质的干扰,而对雌二醇具有特异性吸附,可应用于检测奶粉中雌二醇残留量。(The invention relates to the technical field of biochemical materials, in particular to a limited entry medium-molecularly imprinted polymer and a preparation method and application thereof. Firstly, TiO is mixed 2 Surface benzylation of nanowires followed by surface benzylation of TiO 2 Synthesizing the RAFT chain transfer reagent by the nano wires, and finally limiting the RAFT chain transfer reagent into the medium-molecularly imprinted polymer. The limited entry medium-molecularly imprinted polymer obtained by the preparation method has narrow molecular weight distribution and uniform structure, has good adsorption performance, can effectively eliminate the interference of macromolecular impurities in milk powder when being applied to estradiol test in milk powder, has specific adsorption on estradiol, and can be applied to the detection of the residual quantity of estradiol in milk powder.)

1. A preparation method of a limited entry medium-molecularly imprinted polymer is characterized by comprising the following preparation steps:

(1)TiO₂surface benzylation of nanowires

Adding TiO into the mixture₂Dispersing the nano-wires in toluene, stirring, adding 4- (chloromethyl) phenyltrichlorosilane after stirring is finished, continuously stirring, adding triethylamine after stirring is finished, stirring and reacting under the protection of nitrogen, and performing suction filtration after the reaction is finished to obtain the TiO with the benzylated surface₂A nanowire;

(2)TiO₂nanowire synthesis RAFT chain transfer reagent

Adding phenyl magnesium bromide into tetrahydrofuran, stirring uniformly, heating, and adding carbon disulfide for reaction after heating is finished; after the reaction is finished, adding surface benzylated TiO₂Stirring the nanowires for reaction, washing and drying a product after the reaction to obtain the RAFT chain transfer reagent;

(3) preparation of limited entry media-molecularly imprinted polymer

Dissolving estradiol and methacrylic acid in acetonitrile/toluene, standing, adding trihydroxymethyl acrylate, azodiisobutyronitrile, glycidyl methacrylate and the RAFT chain transfer reagent prepared in the step (2) after standing is finished, reacting under the protection of nitrogen, performing suction filtration after the reaction is finished, washing and drying to obtain a polymerization product, performing Soxhlet extraction on the polymerization product, washing and drying, and grinding to obtain a molecularly imprinted polymer; and adding perchloric acid/water solution into the molecularly imprinted polymer, stirring, and washing and drying after stirring to obtain the limited entry medium-molecularly imprinted polymer prepared based on the RAFT method.

2. The method for preparing the limited entry media-molecularly imprinted polymer according to claim 1, wherein the TiO in the step (1)₂Nanowire, toluene, 4- (chloromethyl) phenyltrichlorosilane, triethylamineThe weight-to-volume ratio of (1 g) to (15-20 ml) to (0.8-1.2 ml) to (0.4-0.6 ml).

3. The method for preparing the limited entry media-molecularly imprinted polymer according to claim 1 or 2, wherein the step (1) of TiO is carried out₂The time for dispersing the nanowires in toluene and stirring is 25-35 min; stirring for 28-32 min after adding 4- (chloromethyl) phenyl trichlorosilane; and after triethylamine is added, stirring and reacting for 18-30 h under the protection of nitrogen.

4. The method for preparing limited entry media-molecularly imprinted polymer according to claim 1, wherein in the step (2), tetrahydrofuran, phenylmagnesium bromide, carbon disulfide and surface-benzylated TiO are used₂The volume-to-weight ratio of the nanowires is 6-10 (ml): 2-4 (ml): 4-6 (ml):1 (g).

5. The preparation method of the limited entry media-molecularly imprinted polymer according to claim 1 or 4, wherein the heating temperature in the step (2) is 45-55 ℃, the reaction time after the carbon disulfide is added is 0.5-1.5 h, and the TiO with the benzylated surface is added₂The stirring reaction time after the nanowire is 5-7 h.

6. The method for preparing a limited entry media-molecularly imprinted polymer according to claim 5, wherein the washing in the step (2) is sequentially washed with tetrahydrofuran for 2-3 times and then with ethanol for 5-7 times; the drying temperature is 60-80 ℃.

7. The preparation method of the limited entry media-molecularly imprinted polymer according to claim 1, wherein the volume weight ratio of the estradiol, the methacrylic acid, the acetonitrile/toluene, the trimethylol acrylate, the azodiisobutyronitrile, the glycidyl methacrylate and the RAFT chain transfer reagent in the step (3) is 0.25-0.3 (g), 0.4-0.6 (g), 140-160 (ml), 6-7.5 (g), 0.1-0.2 (g), 0.1-0.3 (ml): 1.0 to 2.0 (g).

8. The preparation method of the limited entry media-molecularly imprinted polymer according to claim 1, wherein the standing temperature in the step (3) is 3-5 ℃ and the standing time is 30-42 h; the reaction temperature is 65-75 ℃ under the protection of nitrogen, and the reaction time is 12-36 h; the Soxhlet extraction time is 36-60 h.

9. The limited entry medium-molecularly imprinted polymer prepared by the preparation method of any one of claims 1 to 8.

10. Use of the limiting mediator-molecularly imprinted polymer of claim 9 for the detection of residual estradiol levels in milk powder.

Technical Field

The invention relates to the technical field of biochemical materials, in particular to a limited entry medium-molecularly imprinted polymer and a preparation method and application thereof.

Background

The Molecular Imprinting technology is a process for preparing an imprinted Polymer (MIP) having specific recognition for a certain target molecule by studying the weak interaction between supramolecules in combination with Polymer chemistry. The molecular engram polymer is also called artificial receptor, and has the greatest advantage of high selectivity and adsorptivity for target molecules. Compared with the traditional biological macromolecule receptor, the artificial receptor has better stability to the change of temperature and pressure, and can also be used in the environments of acid, alkali, metal ions, organic solvents and the like. Meanwhile, the molecularly imprinted polymer also has the advantages of low preparation cost, long service life and the like. The molecular imprinting technology has wide application range, can identify biological macromolecules such as amino acid, protein, nucleotide 5 and the like, and can also identify chemical micromolecules in food, medicines and environmental samples. Has good application prospect in the fields of chromatographic separation, purification, chemical sensors, catalysis, drug delivery, biological antibodies, receptor systems and the like.

The limited material is a mixed mode extracting agent with proper pore size, can adsorb target and exclusion macromolecules at the same time, and avoids the denaturation and adsorption of the macromolecules on the outer surface of the material due to the hydrophilic modification of the outer surface. The inner surface is still hydrophobic or ion-exchange, thus ensuring that the target can be extracted in the presence of macromolecules.

The limited entry medium-molecularly imprinted material combines the advantages of the limited entry medium material and the molecularly imprinted material, has the functions of size exclusion of biomacromolecules and specificity recognition of target micromolecules, is concerned by people as a novel material, and is generally prepared by a free radical polymerization method in the traditional preparation method. But the polymer chain length, molecular weight and molecular structure of the polymerization product in the traditional free radical polymerization reaction are difficult to control, so that the appearance of the functional polymer layer is not uniform.

Therefore, the technical problem to be solved by those skilled in the art is how to provide a limited entry medium-molecularly imprinted polymer with narrow molecular weight distribution and uniform structure, and further improve the adsorption performance of the limited entry medium-molecularly imprinted material.

Disclosure of Invention

The invention aims to provide a limited entry medium-molecularly imprinted polymer with narrow molecular weight distribution and uniform structure, so as to improve the adsorption performance of the limited entry medium-molecularly imprinted material.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a preparation method of a limited entry medium-molecularly imprinted polymer, which comprises the following preparation steps:

(1)TiO₂surface benzylation of nanowires

Adding TiO into the mixture₂Dispersing the nano-wires in toluene, stirring, adding 4- (chloromethyl) phenyltrichlorosilane after stirring is finished, continuously stirring, adding triethylamine after stirring is finished, stirring and reacting under the protection of nitrogen, and performing suction filtration after the reaction is finished to obtain the TiO with the benzylated surface₂A nanowire;

(2)TiO₂nanowire synthesis RAFT chain transfer reagent

Adding phenyl magnesium bromide into tetrahydrofuran, stirring uniformly, heating, and adding carbon disulfide for reaction after heating is finished; after the reaction is finished, adding surface benzylated TiO₂Stirring the nanowires for reaction, washing and drying a product after the reaction to obtain the RAFT chain transfer reagent;

(3) preparation of limited entry media-molecularly imprinted polymer

Dissolving estradiol and methacrylic acid in acetonitrile/toluene, standing, adding trihydroxymethyl acrylate, azodiisobutyronitrile, glycidyl methacrylate and the RAFT chain transfer reagent prepared in the step (2) after standing is finished, reacting under the protection of nitrogen, performing suction filtration after the reaction is finished, washing and drying to obtain a polymerization product, performing Soxhlet extraction on the polymerization product, washing and drying, and grinding to obtain a molecularly imprinted polymer; and adding perchloric acid/water solution into the molecularly imprinted polymer, sealing and stirring, and after stirring is finished, washing and drying to obtain the limited access medium-molecularly imprinted polymer prepared based on the RAFT method.

Preferably, TiO in step (1)₂The weight-volume ratio of the nanowire, the toluene, the 4- (chloromethyl) phenyltrichlorosilane and the triethylamine is 1(g) to 15-20 (ml) to 0.8-1.2 (ml) to 0.4-0.6 (ml).

Preferably, step (1) TiO₂The time for dispersing the nanowires in toluene and stirring is 25-35 min; stirring for 28-32 min after adding 4- (chloromethyl) phenyl trichlorosilane; and after triethylamine is added, stirring and reacting for 18-30 h under the protection of nitrogen.

Preferably, in step (2), tetrahydrofuran, phenylmagnesium bromide, carbon disulfide, and surface-benzylated TiO₂The volume-to-weight ratio of the nanowires is 6-10 (ml): 2-4 (ml): 4-6 (ml):1 (g).

Preferably, the heating temperature in the step (2) is 45-55 ℃, the reaction time after the carbon disulfide is added is 0.5-1.5 h, and the surface benzylated TiO is added₂The stirring reaction time after the nanowire is 5-7 h.

Preferably, the washing in the step (2) is sequentially washed for 2-3 times by using tetrahydrofuran and then washed for 5-7 times by using ethanol; the drying temperature is 60-80 ℃.

Preferably, in the step (3), the volume weight ratio of the estradiol, the methacrylic acid, the acetonitrile/toluene, the trihydroxymethyl acrylate, the azobisisobutyronitrile, the glycidyl methacrylate and the RAFT chain transfer reagent is 0.25-0.3 (g): 0.4-0.6 (g): 140-160 (ml): 6-7.5 (g): 0.1-0.2 (g): 0.1-0.3 (ml): 1.0 to 2.0 (g).

Preferably, the standing temperature in the step (3) is 3-5 ℃, and the time is 30-42 h; the reaction temperature is 65-75 ℃ under the protection of nitrogen, and the reaction time is 12-36 h; the Soxhlet extraction time is 36-60 h.

Preferably, the volume ratio of acetonitrile to toluene in the acetonitrile/toluene solution in step (3) is 3: 1.

Preferably, the first washing in step (3) is: sequentially washing the mixture with methanol/acetic acid, water and methanol for 2-4 times respectively, and drying the washed mixture at the temperature of 65-75 ℃; wherein the volume ratio of methanol to acetic acid in the methanol/acetic acid solution is 9: 1.

Preferably, after the Soxhlet extraction in the step (3) is finished, washing the product to be neutral by using deionized water, and then washing the product for 2-4 times by using methanol; and then drying at 65-75 ℃.

Preferably, the weight-to-volume ratio of the molecularly imprinted polymer and the perchloric acid/aqueous solution in step (3) is 5(g): 90-110 (ml), stirring for 12-36 h, then washing with water, methanol and n-hexane for 2-3 times respectively in sequence, and drying at 65-75 ℃.

The invention also provides a limited entry medium-molecularly imprinted polymer prepared by the preparation method.

The invention also provides application of the limited entry medium-molecularly imprinted polymer in detection of the residual quantity of estradiol in milk powder.

According to the technical scheme, compared with the prior art, the invention has the following beneficial effects:

the limited entry medium-molecularly imprinted polymer obtained by the preparation method has narrow molecular weight distribution and uniform structure, the main aperture of the material is about 40nm, and the material has good adsorption performance, when the material is applied to estradiol test in milk powder, the material can effectively eliminate the interference of macromolecular impurities in the milk powder, has specific adsorption on estradiol, and can be applied to the detection of the residual quantity of estradiol in the milk powder.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.

FIG. 1 is a schematic diagram of the preparation of RAFT chain transfer reagent of example 2 of the present invention;

FIG. 2 is a schematic diagram of the synthesis process of the limited entry media-molecularly imprinted polymer of example 2 of the present invention;

FIG. 3 is an infrared spectrum of a limited entry medium-molecularly imprinted polymer prepared in example 2 of the present invention;

FIG. 4 is a diagram of the pore size distribution of the limited entry media-molecularly imprinted polymer prepared in example 2 of the present invention;

FIG. 5 is a selected ion flow graph of a sample made in example 2 of the present invention.

Detailed Description

The invention provides a preparation method of a limited entry medium-molecularly imprinted polymer, which comprises the following preparation steps:

(1)TiO₂surface benzylation of nanowires

Adding TiO into the mixture₂Dispersing the nano-wires in toluene, stirring, adding 4- (chloromethyl) phenyltrichlorosilane after stirring is finished, continuously stirring, adding triethylamine after stirring is finished, stirring and reacting under the protection of nitrogen, and performing suction filtration after the reaction is finished to obtain the TiO with the benzylated surface₂A nanowire;

(2)TiO₂nanowire synthesis RAFT chain transfer reagent

Adding phenyl magnesium bromide into tetrahydrofuran, stirring uniformly, heating, and adding carbon disulfide for reaction after heating is finished; after the reaction is finished, adding surface benzylated TiO₂Stirring the nanowires for reaction, washing and drying a product after the reaction to obtain the RAFT chain transfer reagent;

(3) preparation of limited entry media-molecularly imprinted polymer

Dissolving estradiol and methacrylic acid in acetonitrile/toluene, standing, adding trihydroxymethyl acrylate, azodiisobutyronitrile, glycidyl methacrylate and the RAFT chain transfer reagent prepared in the step (2) after standing is finished, reacting under the protection of nitrogen, performing suction filtration after the reaction is finished, washing and drying to obtain a polymerization product, performing Soxhlet extraction on the polymerization product, washing and drying, and grinding to obtain a molecularly imprinted polymer; and adding perchloric acid/water solution into the molecularly imprinted polymer, sealing and stirring, and after stirring is finished, washing and drying to obtain the limited access medium-molecularly imprinted polymer prepared based on the RAFT method.

Preferably, TiO in step (1)₂The weight-volume ratio of the nanowire, toluene, 4- (chloromethyl) phenyltrichlorosilane, and triethylamine is 1(g): 15-20 (ml): 0.8-1.2 (ml): 0.4-0.6 (ml), and more preferably, TiO₂The weight-volume ratio of the nanowire, the toluene, the 4- (chloromethyl) phenyltrichlorosilane and the triethylamine is 1(g), 17-18 (ml), 0.9-1.1 (ml) and 0.45-0.55 (ml).

Preferably, step (1) TiO₂The time for dispersing the nanowires in toluene and stirring is 25-35 min; stirring for 28-32 min after adding 4- (chloromethyl) phenyl trichlorosilane; and after triethylamine is added, stirring and reacting for 18-30 h under the protection of nitrogen.

Preferably, in step (2), tetrahydrofuran, phenylmagnesium bromide, carbon disulfide, and surface-benzylated TiO₂The volume weight ratio of the nano-wire is 6-10 (ml): 2-4 (ml): 4-6 (ml):1(g), and the preferable materials are tetrahydrofuran, phenyl magnesium bromide, carbon disulfide and TiO with benzylation surface₂The volume-to-weight ratio of the nanowires is 7-9 (ml): 2.5-3 (ml): 5-5.5 (ml):1 (g).

Preferably, the heating temperature in the step (2) is 45-55 ℃, and further preferably, the heating temperature is 47-52 ℃; the reaction time after the carbon disulfide is added is 0.5-1.5 h, and preferably 0.8-1.2 h; adding surface-benzylated TiO₂Stirring and reacting for 5-7 h after the nano wire is processed, and preferably, adding surface benzylated TiO₂The stirring reaction time after the nano wires is 5.5-6.5 h.

Preferably, the washing in the step (2) is sequentially washed for 2-3 times by using tetrahydrofuran and then washed for 5-7 times by using ethanol; the drying temperature is 60-80 ℃.

Preferably, in the step (3), the volume weight ratio of the estradiol, the methacrylic acid, the acetonitrile/toluene, the trihydroxymethyl acrylate, the azobisisobutyronitrile, the glycidyl methacrylate and the RAFT chain transfer reagent is 0.25-0.3 (g): 0.4-0.6 (g): 140-160 (ml): 6-7.5 (g): 0.1-0.2 (g): 0.1-0.3 (ml): 1.0 to 2.0(g), preferably estradiol, methacrylic acid, acetonitrile/toluene, trimethylol acrylate, azobisisobutyronitrile, glycidyl methacrylate, and RAFT chain transfer agent in a volume to weight ratio of 0.26 to 0.28(g):0.4.5 to 0.55(g):144 to 151(ml):6.5 to 7.1 (g):0.1.2 to 0.17(g):0.15 to 0.23 (ml): 1.3 to 1.7 (g).

Preferably, the standing temperature in the step (3) is 3-5 ℃, and the time is 30-42 h; the reaction temperature is 65-75 ℃ under the protection of nitrogen, and the reaction time is 12-36 h; the Soxhlet extraction time is 36-60 h.

Preferably, the volume ratio of acetonitrile to toluene in the acetonitrile/toluene solution in step (3) is 3: 1.

Preferably, the first washing in step (3) is: sequentially washing the mixture with methanol/acetic acid, water and methanol for 2-4 times respectively, and drying the washed mixture at the temperature of 65-75 ℃; wherein the volume ratio of methanol to acetic acid in the methanol/acetic acid solution is 9: 1.

Preferably, after the Soxhlet extraction in the step (3) is finished, washing the product to be neutral by using deionized water, and then washing the product for 2-4 times by using methanol; and then drying at 65-75 ℃.

Preferably, the weight-to-volume ratio of the molecularly imprinted polymer and the perchloric acid/aqueous solution in step (3) is 5(g): 90-110 (ml), stirring for 12-36 h, then washing with water, methanol and n-hexane for 2-3 times respectively in sequence, and drying at 65-75 ℃.

The invention also provides a limited entry medium-molecularly imprinted polymer prepared by the preparation method.

The invention also provides application of the limited entry medium-molecularly imprinted polymer in detection of the residual quantity of estradiol in milk powder.

The technical solutions in the embodiments of the present application will be described clearly and completely below, and it should be understood that the described embodiments are only a part of the embodiments of the present application, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.

Example 1

A preparation method of a limited entry medium-molecularly imprinted polymer comprises the following preparation steps:

(1)TiO₂surface benzylation of nanowires

Adding TiO into the mixture₂Dispersing the nano-wires in toluene, stirring for 25min, adding 4- (chloromethyl) phenyltrichlorosilane after stirring, continuing stirring for 28min, adding triethylamine after stirring, stirring and reacting for 18h under the protection of nitrogen, and performing suction filtration after the reaction is finished to obtain the TiO with the surface benzylation₂A nanowire;

(2)TiO₂nanowire synthesis RAFT chain transfer reagent

Adding phenyl magnesium bromide into tetrahydrofuran, uniformly stirring, heating to 45 ℃, and adding carbon disulfide for reaction for 0.5h after heating is finished; after the reaction is finished, adding surface benzylated TiO₂Stirring and reacting the nanowires for 5 hours, washing the product after reaction for 2 times by using tetrahydrofuran, then washing the product for 7 times by using ethanol, and drying the product at the temperature of 60 ℃ to obtain the RAFT chain transfer reagent;

(3) preparation of limited entry media-molecularly imprinted polymer

Dissolving estradiol and methacrylic acid in acetonitrile/toluene, standing for 30 hours at the temperature of 3 ℃, adding trihydroxymethyl acrylate, azodiisobutyronitrile, glycidyl methacrylate and the RAFT chain transfer reagent prepared in the step (2) after standing is finished, reacting for 36 hours at 65 ℃ under the protection of nitrogen, performing suction filtration after the reaction is finished, sequentially washing for 2 times by using methanol/acetic acid, water and methanol respectively, and drying at 65 ℃ to obtain a polymerization product, performing Soxhlet extraction on the polymerization product for 36 hours, washing the polymerization product to be neutral by using deionized water, and washing for 2 times by using methanol; drying at 65 ℃ and grinding to obtain a molecularly imprinted polymer;

in the molecular engram polymer, according to the weight volume ratio of the molecular engram polymer to perchloric acid/water solution of 5(g): adding perchloric acid/water solution with the ratio of 90(ml), sealing and stirring for 12h, washing with water, methanol and n-hexane for 2 times in sequence after stirring is finished, and drying at 65 ℃ to obtain the access limiting medium-molecularly imprinted polymer prepared based on the RAFT method.

In this example, TiO in step (1)₂The weight-volume ratio of the nano wire, the toluene, the 4- (chloromethyl) phenyltrichlorosilane and the triethylamine is 1(g):15(ml):0.8(ml):0.4 (ml).

In this example, step (2), tetrahydrofuran, phenylmagnesium bromide, carbon disulfide, and surface-benzylated TiO₂The volume-to-weight ratio of the nanowires is 6(ml):2(ml):4(ml):1 (g).

In step (3) of this example, the volume to weight ratio of estradiol, methacrylic acid, acetonitrile/toluene, trimethylol acrylate, azobisisobutyronitrile, glycidyl methacrylate, RAFT chain transfer agent was 0.25(g):0.4(g):140(ml):6(g):0.1(g):0.1 (ml):1 (g).

Example 2

A preparation method of a limited entry medium-molecularly imprinted polymer comprises the following preparation steps:

(1)TiO₂surface benzylation of nanowires

Adding TiO into the mixture₂Dispersing the nano-wires in toluene, stirring for 30min, adding 4- (chloromethyl) phenyltrichlorosilane after stirring, continuing stirring for 30min, adding triethylamine after stirring, stirring and reacting for 24h under the protection of nitrogen, and performing suction filtration after the reaction is finished to obtain the TiO with the surface benzylation₂A nanowire;

(2)TiO₂nanowire synthesis RAFT chain transfer reagent

Adding phenyl magnesium bromide into tetrahydrofuran, uniformly stirring, heating to 50 ℃, and adding carbon disulfide for reaction for 1h after heating is finished; after the reaction is finished, adding surface benzylated TiO₂Stirring and reacting the nanowires for 6h, washing the product after reaction for 2 times by using tetrahydrofuran, then washing the product for 6 times by using ethanol, and drying the product at the temperature of 70 ℃ to obtain the RAFT chain transfer reagent;

(3) preparation of limited entry media-molecularly imprinted polymer

Dissolving estradiol and methacrylic acid in acetonitrile/toluene, standing for 36h at 4 ℃, adding trihydroxymethyl acrylate, azobisisobutyronitrile, glycidyl methacrylate and the RAFT chain transfer reagent prepared in the step (2) after standing is finished, reacting for 24h under the protection of nitrogen at 70 ℃, performing suction filtration after the reaction is finished, sequentially washing for 3 times by using methanol/acetic acid, water and methanol respectively, and drying at 70 ℃ to obtain a polymerization product, performing Soxhlet extraction on the polymerization product for 48h, washing with deionized water to be neutral, and washing with methanol for 3 times; drying at 70 ℃, and grinding to obtain a molecularly imprinted polymer;

in the molecular engram polymer, according to the weight volume ratio of the molecular engram polymer to perchloric acid/water solution of 5(g): adding perchloric acid/water solution into 100(ml) of ratio, sealing and stirring for 24h, washing with water, methanol and n-hexane for 2 times in sequence after stirring is finished, and drying at 70 ℃ to obtain the access limiting medium-molecularly imprinted polymer prepared based on the RAFT method.

In this example, TiO in step (1)₂The weight-volume ratio of the nano wire, the toluene, the 4- (chloromethyl) phenyltrichlorosilane and the triethylamine is 1(g):17(ml):1(ml):0.5 (ml).

In this example, step (2), tetrahydrofuran, phenylmagnesium bromide, carbon disulfide, and surface-benzylated TiO₂The volume-to-weight ratio of the nanowires is 8(ml):3(ml):5(ml):1 (g).

In step (3) of this example, the volume to weight ratio of estradiol, methacrylic acid, acetonitrile/toluene, trimethylol acrylate, azobisisobutyronitrile, glycidyl methacrylate, RAFT chain transfer agent was 0.27(g):0.5(g):150(ml):6.76(g):0.15(g):0.2 (ml): 1.5 (g).

Example 3

A preparation method of a limited entry medium-molecularly imprinted polymer comprises the following preparation steps:

(1)TiO₂surface benzylation of nanowires

Adding TiO into the mixture₂Dispersing the nano-wires in toluene, stirring for 35min, adding 4- (chloromethyl) phenyltrichlorosilane after stirring, continuously stirring for 32min, adding triethylamine after stirring, stirring and reacting for 30h under the protection of nitrogen, and performing suction filtration after the reaction is finished to obtain the TiO with the surface benzylation₂A nanowire;

(2)TiO₂nanowire synthesisRAFT chain transfer reagent

Adding phenyl magnesium bromide into tetrahydrofuran, uniformly stirring, heating to 55 ℃, and adding carbon disulfide for reaction for 1.5h after heating is finished; after the reaction is finished, adding surface benzylated TiO₂Stirring and reacting the nanowires for 7h, washing the product after reaction for 3 times by using tetrahydrofuran, then washing the product for 7 times by using ethanol, and drying the product at the temperature of 80 ℃ to obtain the RAFT chain transfer reagent;

(3) preparation of limited entry media-molecularly imprinted polymer

Dissolving estradiol and methacrylic acid in acetonitrile/toluene, standing for 42h at the temperature of 5 ℃, adding trihydroxymethyl acrylate, azodiisobutyronitrile, glycidyl methacrylate and the RAFT chain transfer reagent prepared in the step (2) after standing is finished, reacting for 36h at 75 ℃ under the protection of nitrogen, performing suction filtration after the reaction is finished, sequentially washing for 4 times by using methanol/acetic acid, water and methanol respectively, and drying at 75 ℃ to obtain a polymerization product, performing Soxhlet extraction on the polymerization product for 60h, washing the polymerization product to be neutral by using deionized water, and washing for 4 times by using methanol; drying at 75 ℃ and grinding to obtain a molecularly imprinted polymer;

in the molecular engram polymer, according to the weight volume ratio of the molecular engram polymer to perchloric acid/water solution of 5(g): adding perchloric acid/water solution into the mixture with the ratio of 110(ml), sealing and stirring the mixture for 36h, washing the mixture for 3 times by using water, methanol and n-hexane in sequence after stirring is finished, and drying the mixture at the temperature of 75 ℃ to obtain the access limiting medium-molecularly imprinted polymer prepared based on the RAFT method.

In this example, TiO in step (1)₂The weight-volume ratio of the nano wire, the toluene, the 4- (chloromethyl) phenyltrichlorosilane and the triethylamine is 1(g):20(ml):1.2(ml):0.6 (ml).

In this example, step (2), tetrahydrofuran, phenylmagnesium bromide, carbon disulfide, and surface-benzylated TiO₂The volume-to-weight ratio of the nanowires is 10(ml):4(ml):6(ml):1 (g).

In step (3) of this example, the volume to weight ratio of estradiol, methacrylic acid, acetonitrile/toluene, trimethylol acrylate, azobisisobutyronitrile, glycidyl methacrylate, RAFT chain transfer agent was 0.3(g):0.6(g):160(ml):7.5(g):0.2(g):0.3 (ml):2 (g).

Through detection, the limited entry media-molecularly imprinted polymers prepared in embodiments 1-3 of the present invention have narrow molecular weight distribution and uniform structure, and the limited entry media-molecularly imprinted polymers prepared in embodiment 2 are characterized in detail:

FIG. 3 is an infrared spectrum of the limited entry medium-molecularly imprinted polymer prepared in example 2 of the present invention, and 3478cm as seen in FIG. 3^-12963cm, which is a strong absorption peak of-OH in stretching vibration^-1The characteristic peak is-CH₃1733cm, respectively^-1Is a characteristic absorption peak of Ti-O, 1149cm^-1Is a C-O stretching vibration absorption peak and accords with the characteristic infrared spectrum of the limited dielectric molecularly imprinted material based on the nano titanium dioxide.

Fig. 4 is a distribution diagram of the pore size of the limited entry media-molecularly imprinted polymer prepared in example 2 of the present invention, and it can be seen from fig. 4 that the main pore size of the material is about 40nm, and the limited entry media-molecularly imprinted material belongs to a mesoporous material.

Application example

According to the invention, an addition recovery method is selected for methodological evaluation, estradiol standard solutions with different concentration levels are respectively added into milk powder in a sample without an analyte background, estradiol with different concentration levels (1, 5 and 10 mu g/kg) is respectively added according to the requirement of estradiol residue limit, and each addition level is subjected to 4 parallel experiments.

And (3) putting 1g of MIP into a test tube, adding methanol to remove micro particles, and filling the micro particles into a solid-phase extraction empty column to prepare the molecularly imprinted solid-phase extraction column. Weighing 0.5g of milk powder in a test tube, adding 10ml of water for vortex dissolution, transferring the milk powder to a molecularly imprinted solid phase extraction column (activated by 5 ml), rinsing the solid phase extraction column with 10ml of water, adding 20ml of acetonitrile for elution, collecting all eluent, concentrating the eluent until the eluent is dry, performing derivatization by a derivatization reagent N-methyl-N-trimethylsilyl trifluoroacetamide (MSTFA) -Trimethoxyiodosilane (TMIS) -Dithioerythritol (DTE), and determining by a gas chromatography mass spectrometer. According to the above detection method, estradiol in milk powder was detected using MIPs prepared in examples 1 to 3 respectively corresponding to estradiol at concentration levels of (1, 5, 10 μ g/kg), and the detection results are shown in table 1.

Wherein, the instrument measurement conditions are as follows:

1) chromatographic conditions are as follows:

a) a chromatographic column: 30m x 0.25mm (inside diameter), 0.25 μm thick, Rxi-5ms quartz capillary column, or equivalent;

b) carrier gas: helium with purity not less than 99.999% and flow rate 1.5 ml/min;

c) temperature of the column: maintaining at 100 deg.C for 1min, and heating to 280 deg.C with a program of 20 deg.C/min for 5 min.

d) Sample inlet temperature: 240 ℃;

e) and (3) sample introduction mode: injecting sample without split flow, and opening the valve after 0.75 min;

f) sample introduction amount: 1 mu L of the solution;

2) mass spectrum conditions:

a) an ionization mode: EI;

b) interface temperature of chromatography-mass spectrometry: 260 ℃;

c) ion source temperature: 230 ℃;

d) solvent retardation: 5 min;

e) ion detection mode: ion reaction monitoring (SIM) is selected, and ions and their abundance ratios 416, 285, 232 are monitored.

TABLE 1 results of measurement of estradiol in milk powder by MIP prepared in examples 1 to 3

	Estradiol content (μ g/kg)	Recovery (%)	Relative standard deviation (%)
				Example 1	1.0	87.0	5.4
Example 2	5.0	92.6	5.8
				Example 3	10.0	104.9	2.9

The result shows that the estradiol is added into the milk powder sample by 1-10 mug/kg, the recovery rate ranges from 87-104.9%, and the relative standard deviation ranges from 2.9-5.8%.

Fig. 5 is a sample-selected ion flow graph obtained in example 2, with retention time on the abscissa and response value on the ordinate. Wherein m/z416 is quantitative ion, m/z285, 232 is qualitative ion, retention time of the target is 11.72min, and peaks at other times are impurity peaks. From fig. 5, it can be seen that: the quantitative ion peak and the qualitative ion peak are not interfered by impurity peaks, and the quantitative determination of the estradiol is not influenced, which shows that the purification has better effect.

According to the embodiment and the application example, the limited entry medium, namely the molecularly imprinted polymer, provided by the invention, is a polymer with narrow molecular weight distribution and uniform structure, the main pore diameter of the material is about 40nm, and the material has good adsorption performance, when the material is applied to estradiol test in milk powder, the material can effectively eliminate the interference of macromolecular impurities in the milk powder, has specific adsorption on estradiol, and can accurately detect the residual quantity of the estradiol in the milk powder.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.