CN102382273B - Preparation method of 17 beta-estradiol molecular-imprinted composite microspheres - Google Patents

Abstract

The invention relates to a preparation method of 17 beta-estradiol molecular-imprinted composite microspheres. 1,4-dioxane is used as a solvent to prepare vinyl-bonded monodisperse crosslinked epoxypropyl methacrylate microspheres. The vinyl-bonded monodisperse crosslinked epoxypropyl methacrylate microspheres are used as a carrier to prepare the 17 beta-estradiol molecular-imprinted composite microspheres by a surface imprinting technique. The method has the advantages of mild reaction conditions, high product stability, high recognition capability for 17 beta-estradiol, and high adsorption rate, can be used for establishing simple sample pretreatments (extraction, control and concentration), and evaluating the quality and composition of the estrogen-containing product; the method can be used for high efficiency liquid chromatography subjected to high-flow-rate high-pressure operation, thereby greatly enhancing the column efficiency and reproducibility; and the method can also be used for quick and efficient solid-phase extraction of adsorbent.

Description

Preparation method of 17β-estradiol molecularly imprinted composite microspheres

technical field

The invention belongs to the technical field of materials, and in particular relates to a molecularly imprinted composite microsphere for analyzing and measuring 17β-estradiol in biological samples and food samples.

Background technique

17β-Estradiol (17β-Estradiol) is a kind of synthetic estrogen, it is mainly used in medicine to treat women's climacteric syndrome and other diseases, but long-term intake of 17β-Estradiol will produce some side effects and increase certain In view of these risks, the country strictly prohibits the use of synthetic estrogens such as 17β-estradiol in food. However, there are some criminals who add 17β-estradiol and other synthetic estrogens with estrogen-like effects in order to strengthen their product functions and reduce costs. In addition, the abuse of anabolic hormones will cause direct or potential harm to human and animal health, society and the ecological environment. Therefore, the monitoring of synthetic estrogens such as 17β-estradiol in food and health food is also a necessary measure to ensure the hygiene and safety of such food.

17β-estradiol can enter the environment through the metabolism of living organisms, or enter the environment as the parent compound produced by estrogen drugs along with industrial wastewater, so that it remains in aquatic organisms and enters the human body through the food chain, and its metabolites are related to human cancer. have a close relationship. Therefore, the food nutrition and safety problems caused by it cannot be ignored, and the research on the existence level and biological activity of this kind of substances has important practical significance. Due to the low concentration and large fluctuation range of the residual drug to be tested in food, the complex sample matrix, many interfering substances, and the uncertainty of the sample matrix and the components to be tested, it is necessary to remove the interfering impurities in the sample and enrich the trace amount before testing. The target component to be tested.

Molecularly imprinted polymers (MIPs) have specific recognition, can be used for sample pretreatment of complex matrices, and are easy to prepare, stable and reusable, and have been used in separation science, biomedicine, sensor technology and control of chemical reactions. Has been widely used. However, the existing methods for preparing MPIMs have obvious limitations: the polymer obtained by bulk polymerization is in the form of a block, which must be ground and sieved before use. The irregular size and shape make sample processing difficult, cumbersome and time-consuming. And the yield is low (generally <50%), and the application efficiency is greatly reduced; the precipitation polymerization method has high requirements for solvents, and is difficult to be widely used; the MPIMs obtained by the emulsion polymerization method have a small particle size (<1 μm), which is not suitable for liquid chromatography. It is not easy to separate after adsorption; when using spherical silica gel surface imprinting or sacrificial silica gel method, the hydroxyl groups on the silica gel surface will affect the force between functional monomers and template molecules, so that the recognition ability of MPIMs is weak; although the suspension polymerization method has a simple preparation process , but the obtained MPIMs have a wide particle size distribution and can only be used after tedious classification treatment; although the multi-step swelling polymerization method can produce micron-sized and uniform particle size MPIMs, it can be used after simple washing, but it needs to be processed Multi-step swelling, the process is more cumbersome, the preparation cycle is longer, and it is difficult to realize industrialization. In contrast, molecularly imprinted polymer microspheres have better performance, are more convenient to prepare and use, and are especially suitable for analytical detection, sensor arrays and separation components. Therefore, the preparation and application of molecularly imprinted polymer microspheres has become one of the research hotspots of molecularly imprinted technology. The binding site of the surface imprint is located on the surface of the polymer, and the template molecule is easy to elute, which can increase the binding capacity; the target molecule does not need to enter its rigid structure when recombining, which greatly speeds up the kinetic process of binding. However, the matrix currently used in this method is mainly limited to silica gel. When using particulate silica gel, special attention should be paid to its pH range of 2 to 7.5. If it is too alkaline (pH>7.5), the silica gel will be crushed or dissolved; if it is too acidic (pH< 2), the chemical bonds of the bonded phase will be broken. After the reaction, it is troublesome to remove the matrix. Generally, hydrofluoric acid corrosion is used, but it has a certain adverse effect on the skeleton structure of the polymer.

Invention of internal dissolution

The technical problem to be solved by the present invention is to overcome the disadvantages of the above-mentioned preparation methods, and provide a preparation method of 17β-estradiol molecularly imprinted composite microspheres with mild reaction conditions, good product stability and strong recognition ability.

The technical solution adopted to solve the above technical problems consists of the following steps:

1. Synthesis of monodisperse polystyrene dispersion

Add styrene, azobisisobutyronitrile, and polyvinylpyrrolidone into absolute ethanol, and the mass ratio of styrene to azobisisobutyronitrile, polyvinylpyrrolidone, and absolute ethanol is 1:0.01～0.04:0.1～0.3 : 7.5 ~ 9, ultrasonic dispersion, nitrogen deoxygenation, stirring, 70 ° C polymerization reaction for 24 hours, prepared into polystyrene; the polystyrene is ultrasonically dispersed with a mass fraction of 0.2% sodium lauryl sulfate aqueous solution to obtain Monodisperse polystyrene dispersion.

The above-mentioned polyvinylpyrrolidone has a molecular weight of 10,000-70,000 and is provided by Paini Chemical Reagent Factory, Zhengzhou, China.

2. Preparation of monodisperse cross-linked glycidyl methacrylate microspheres

Benzoyl peroxide, glycidyl methacrylate, ethylene glycol dimethacrylate, dispersant aqueous solution are added in the mixed solvent of cyclohexanol and toluene, toluene and cyclohexanol, benzoyl peroxide, The mass ratio of glycidyl methacrylate, ethylene glycol dimethacrylate, and dispersant aqueous solution is 1:2:0.15:2:3:60, and the cell disruptor is used to ultrasonicate at a power of 300-500W. Ultrasonic 1 time at intervals of 10-20 seconds, 10-20 seconds each time, ultrasonically emulsify until the upper layer has no oil droplets, add to the monodisperse polystyrene dispersion obtained in step 1, glycidyl methacrylate and monodisperse The mass ratio of polystyrene in the polystyrene dispersion is 1:0.1, stirred and swelled at 30°C for 10 hours, purged with nitrogen to remove oxygen, polymerized at 70°C for 24 hours, filtered, washed with methanol and acetone in sequence, and vacuum-dried at 60°C for 4 Hours, monodisperse cross-linked glycidyl methacrylate microspheres were prepared.

The dispersant in the above-mentioned dispersant aqueous solution is sodium lauryl sulfate and polyvinyl alcohol, and the mass fraction of sodium lauryl sulfate is 0.2%, and the mass fraction of polyvinyl alcohol is 2%, and the mass fraction of described polyvinyl alcohol The degree of polymerization is 1700, and the degree of alcoholysis is 88%, provided by Tianjin Tianda Chemical Experimental Factory.

3. Extraction of monodisperse cross-linked glycidyl methacrylate microspheres

Extract the monodisperse crosslinked glycidyl methacrylate microspheres prepared in step 2 with toluene in a Soxhlet extractor at 140°C for 48 hours, wash with absolute ethanol and acetone in turn, and dry to obtain monodisperse porous crosslinked microspheres Glycidyl methacrylate microspheres.

4. Monodisperse cross-linked glycidyl methacrylate microspheres surface-bonded vinyl

Add the monodisperse porous cross-linked glycidyl methacrylate microspheres prepared in step 3 into 1,4-dioxane, and add 3 to 10 g of monodisperse porous cross-linked microspheres per 100 mL of 1,4-dioxane Glycidyl methacrylate microspheres, stirred and swelled at room temperature for 4 to 12 hours, deoxidized by nitrogen gas, added with hydroxyethyl methacrylate, 1,4-dioxane, and boron trifluoride ether at a mass ratio of 1 : 2～5: 4～6 mixed solution, the mass ratio of monodisperse porous cross-linked glycidyl methacrylate microspheres to hydroxyethyl methacrylate is 1: 0.1～2, stirring at 30～60°C for 8～ After 15 hours, filter, and wash the product with 1,4-dioxane, methanol, distilled water, and acetone in sequence, and vacuum-dry to obtain monodisperse cross-linked glycidyl methacrylate microspheres bonded to vinyl groups on the surface.

5. Synthesis of 17β-estradiol molecularly imprinted composite microspheres

Using acetonitrile as a solvent, ultrasonically disperse 17β-estradiol, methacrylic acid, and surface-bonded vinyl monodisperse crosslinked glycidyl methacrylate microspheres, stir and swell at room temperature for 4 to 12 hours, add ethylene glycol di The mass ratio of methacrylate, azobisisobutyronitrile, 17β-estradiol and surface-bonded vinyl monodisperse cross-linked glycidyl methacrylate microspheres to propyl ester microspheres is 1:20-120 , the molar ratio of 17β-estradiol to methacrylic acid, ethylene glycol dimethacrylate, and azobisisobutyronitrile is 1:2～12:8～20:1～2, nitrogen deoxygenation, 60 Polymerization at ~70°C for 16 to 48 hours, the product was eluted with an eluent with a volume ratio of methanol to acetic acid of 8:2 to remove 17β-estradiol, washed with distilled water until neutral, then washed with methanol, vacuum dried to prepare 17β-estradiol molecularly imprinted composite microspheres.

The best quality of monodisperse porous crosslinked glycidyl methacrylate microspheres and hydroxyethyl methacrylate in step 4 of monodisperse crosslinked glycidyl methacrylate microsphere surface bonded vinyl of the present invention The ratio is 1:1, and the optimum mass ratio of hydroxyethyl methacrylate to 1,4-dioxane and boron trifluoride ether is 1:2.6:5.

In step 4 of the monodisperse crosslinked glycidyl methacrylate microsphere surface-bonded vinyl group of the present invention, it is best to add the monodisperse porous crosslinked glycidyl methacrylate microsphere prepared in step 3 into 1,4 - Dioxane, stirred and swelled at room temperature for 6 hours, purged with nitrogen to remove oxygen, added a mixture of 1,4-dioxane, hydroxyethyl methacrylate and boron trifluoride ether, stirred at 45°C for 12 hours, Monodisperse cross-linked glycidyl methacrylate microspheres with vinyl groups bonded to the surface were obtained.

In step 5 of synthesizing 17β-estradiol molecularly imprinted composite microspheres of the present invention, the optimal mass ratio of 17β-estradiol to monodisperse cross-linked glycidyl methacrylate microspheres bound to vinyl on the surface is 1 : 40, the optimal molar ratio of 17β-estradiol to methacrylic acid, ethylene glycol dimethacrylate and azobisisobutyronitrile is 1: 4: 10: 1.2.

In step 5 of the synthesis of 17β-estradiol molecularly imprinted composite microspheres of the present invention, it is best to dissolve 17β-estradiol and methacrylic acid in acetonitrile, and add the monodisperse cross-linking of surface-bonded vinyl groups obtained in step 4 Glycidyl methacrylate microspheres were ultrasonically dispersed, stirred and swelled at room temperature for 6 hours, ethylene glycol dimethacrylate and azobisisobutyronitrile were added, oxygen was removed by nitrogen gas, and polymerization was carried out at 70°C for 24 hours.

The invention uses 1,4-dioxane as a solvent to prepare monodisperse crosslinked glycidyl methacrylate microspheres with vinyl groups bonded on the surface, and monodisperse crosslinked methacrylic acid rings with vinyl groups bonded to the surface Oxypropyl ester microspheres are used as the carrier, and 17β-estradiol molecularly imprinted composite microspheres are prepared by surface imprinting technology. This method has mild reaction conditions, and the product has good stability. Fast, can be used to establish simple extraction, chemical control and concentrated sample pretreatment, to evaluate the quality and composition of estrogen-containing products, and can be used in high-performance liquid chromatography with high flow rate and high pressure operation, greatly improving column efficiency and gravity Presentity. In addition, since the imprinted binding site is located on the surface of the polymer, the template molecule is easy to elute, and the target molecule does not need to enter its rigid structure during binding, which greatly speeds up the kinetic process of adsorption and desorption, and can be used as a fast and efficient Solid Phase Extraction Sorbent.

Description of drawings

Figure 1 is a chromatographic separation diagram of 17β-estradiol, resveratrol and capsaicin on an imprinted column.

Fig. 2 is a chromatographic separation diagram of 17β-estradiol, resveratrol and capsaicin on a non-imprinted column.

Fig. 3 is a chromatographic separation diagram of 17β-estradiol and estrone on an imprinted column.

Fig. 4 is a chromatographic separation diagram of 17β-estradiol and estrone on a non-imprinted column.

Detailed ways

The present invention will be described in further detail below in conjunction with the accompanying drawings and embodiments, but the present invention is not limited to these embodiments.

Example 1

1. Synthesis of monodisperse polystyrene dispersion

Add 10.908g of styrene, 0.2406g of azobisisobutyronitrile, and 1.5006g of polyvinylpyrrolidone with a molecular weight of 10,000 to 70,000 into a 250mL single-necked flask filled with 96g of absolute ethanol. The mass ratio of vinylpyrrolidone and absolute ethanol is 1:0.02:014:8.8, use an ultrasonic cleaner to disperse ultrasonically for 20 minutes at a power of 80W, blow nitrogen for 15 minutes to remove oxygen, stir, and polymerize at 70°C for 24 hours, and the reaction is over Finally, centrifuge to remove the solvent, wash with absolute ethanol, and prepare polystyrene; 0.2g polystyrene is ultrasonically dispersed in 10mL of 0.2% sodium lauryl sulfate aqueous solution with an ultrasonic cleaner at a power of 80W , a monodisperse polystyrene dispersion was obtained.

2. Preparation of monodisperse cross-linked glycidyl methacrylate microspheres

A mixture of benzoyl peroxide 0.1667g, glycidyl methacrylate 2.064g, ethylene glycol dimethacrylate 3.03g, sodium lauryl sulfate and polyvinyl alcohol (88% degree of alcoholysis) Add 62.35g of aqueous solution into a 250mL beaker containing 1.92g cyclohexanol and 1.0392g toluene, toluene and cyclohexanol, benzoyl peroxide, glycidyl methacrylate, ethylene glycol dimethacrylate, ten The mass ratio of the mixture aqueous solution of dialkyl sodium sulfate and polyvinyl alcohol is 1:2:0.15:2:3:60, sonicate with a cell disruptor at a power of 400W, and sonicate once every 15 seconds. 15 seconds, ultrasonic emulsification to the upper layer without oil droplets, join in the monodisperse polystyrene dispersion obtained in step 1, the mass ratio of glycidyl methacrylate and polystyrene in the monodisperse polystyrene dispersion is 1 : 0.1, stirred at 120 rpm, swelled at 30°C for 10 hours, purged with nitrogen for 20 minutes to remove oxygen, polymerized at 70°C for 24 hours, filtered with glass sand funnel, washed with distilled water at 70°C, and then washed with methanol and acetone in sequence. Vacuum drying at 60°C for 4 hours to prepare monodisperse crosslinked glycidyl methacrylate microspheres.

In the above-mentioned mixture aqueous solution of sodium lauryl sulfate and polyvinyl alcohol, the mass fraction of sodium lauryl sulfate is 0.2%, the mass fraction of polyvinyl alcohol is 2%, the polymerization degree of polyvinyl alcohol is 1700, alcohol The resolution is 88%.

3. Extraction of monodisperse cross-linked glycidyl methacrylate microspheres

Put the monodisperse cross-linked glycidyl methacrylate microspheres prepared in step 2 in a Soxhlet extractor, add 50 mL of toluene, extract at 140°C for 48 hours, wash with absolute ethanol and acetone in turn, Dry at 60° C. for 3 hours under 0.06 MPa to obtain monodisperse porous crosslinked glycidyl methacrylate microspheres.

4. Monodisperse cross-linked glycidyl methacrylate microspheres surface-bonded vinyl

Add 2.0 g of the monodisperse porous cross-linked glycidyl methacrylate microspheres prepared in step 3 into 50 mL of 1,4-dioxane, stir at 200 rpm, swell at room temperature for 6 hours, and blow nitrogen for 20 minutes to remove oxygen , add 2.0g hydroxyethyl methacrylate, 5.2g 1,4-dioxane, 10.0g mixture of boron trifluoride ether, hydroxyethyl methacrylate and 1,4-dioxane, three The mass ratio of boron fluoride ether is 1:2.6:5, the mass ratio of monodisperse porous cross-linked glycidyl methacrylate microspheres to hydroxyethyl methacrylate is 1:1, stir at 45°C for 12 hours, filter , the product was washed successively with 1,4-dioxane, methanol, distilled water, and acetone, and dried in vacuum at 30°C for 2 hours to obtain monodisperse crosslinked glycidyl methacrylate microspheres bonded to vinyl groups on the surface.

5. Synthesis of 17β-estradiol molecularly imprinted composite microspheres

Dissolve 20 mg (0.078 mmol) of 17β-estradiol and 27 μL (0.32 mmol) of methacrylic acid in 15 mL of acetonitrile, and add the monodisperse cross-linked glycidyl methacrylate microspheres bound to vinyl groups on the surface obtained in step 4 0.8g, ultrasonically disperse with an ultrasonic cleaner at a power of 80W for 5 minutes, stir at 200 rpm, swell at room temperature for 6 hours, add ethylene glycol dimethacrylate 147μL (0.774mmol), azobisisobutyronitrile 15mg (0.094mmol), the mass ratio of 17β-estradiol to vinyl monodisperse cross-linked glycidyl methacrylate microspheres bound to the surface is 1:40, 17β-estradiol to methacrylic acid, ethylene glycol The molar ratio of alcohol dimethacrylate to azobisisobutyronitrile is 1:4:10:1.2, nitrogen gas is passed for 20 minutes to remove oxygen, and the polymerization reaction is carried out at 70°C for 24 hours. The product is first washed with distilled water and methanol in sequence, and then washed with The volume ratio of methanol to acetic acid was 8:2 eluent and eluted for 24 hours to remove 17β-estradiol, then washed with distilled water until neutral, washed with methanol, and dried in vacuum at 30°C for 2 hours to prepare 17β-estradiol Estradiol Molecularly Imprinted Composite Microspheres.

Comparative Example 1

According to the method of Steps 1-4 of Example 1, monodisperse cross-linked glycidyl methacrylate microspheres with vinyl groups bonded on the surface were prepared. Without adding the template molecule 17β-estradiol, directly dissolve 27 μL (0.32 mmol) of methacrylic acid in 15 mL of acetonitrile, add 0.8 g of monodisperse cross-linked glycidyl methacrylate microspheres with vinyl groups on the surface, and use Ultrasonic dispersion with an ultrasonic cleaner at a power of 80W for 5 minutes, stirring at 200 rpm, swelling at room temperature for 6 hours, adding 147 μL (0.774 mmol) of ethylene glycol dimethacrylate and 15 mg (0.094 mmol) of azobisisobutyronitrile , deoxygenated by nitrogen gas for 20 minutes, polymerized at 70°C for 24 hours, washed with distilled water and methanol in turn, and vacuum-dried at 30°C for 2 hours to prepare non-molecularly imprinted composite microspheres.

Example 2

In step 4 of the monodisperse crosslinked glycidyl methacrylate microsphere surface-bonded vinyl group in Example 1, 1.6 g of the monodisperse porous crosslinked glycidyl methacrylate microsphere prepared in step 3 was added to 53.2 In mL 1,4-dioxane, stir at 200 rpm, swell at room temperature for 6 hours, blow nitrogen for 20 minutes to remove oxygen, add 0.16g hydroxyethyl methacrylate, 0.32g 1,4-dioxane, A mixture of 0.64g boron trifluoride ether, the mass ratio of hydroxyethyl methacrylate to 1,4-dioxane and boron trifluoride ether is 1:2:4, monodisperse porous cross-linked methacrylic acid The mass ratio of glycidyl ester microspheres to hydroxyethyl methacrylate is 1:0.1, and other steps of this step are the same as in Example 1. In step 5 of the synthesis of 17β-estradiol molecularly imprinted composite microspheres, 40 mg (0.156 mmol) of 17β-estradiol and 26 μL (0.312 mmol) of methacrylic acid were dissolved in 15 mL of acetonitrile, and the surface-bonded Vinyl monodisperse cross-linked glycidyl methacrylate microspheres 0.8g, use an ultrasonic cleaner to disperse ultrasonically for 5 minutes at a power of 80W, 200 rpm, swell at room temperature for 6 hours, add ethylene glycol dimethyl 237 μL (1.248 mmol) of acrylate, 26 mg (0.156 mmol) of azobisisobutyronitrile, and the mass ratio of 17β-estradiol to monodisperse cross-linked glycidyl methacrylate microspheres bound to vinyl on the surface is 1 : 20, the molar ratio of 17β-estradiol to methacrylic acid, ethylene glycol dimethacrylate, and azobisisobutyronitrile is 1: 2: 8: 1, and other steps of this step are the same as in Example 1 . The other steps were the same as in Example 1 to prepare 17β-estradiol molecularly imprinted composite microspheres.

Example 3

In step 4 of the monodisperse crosslinked glycidyl methacrylate microsphere surface-bonded vinyl group in Example 1, 1.6 g of the monodisperse porous crosslinked glycidyl methacrylate microsphere prepared in step 3 was added to 16 mL In 1,4-dioxane, stir at 200 rpm, swell at room temperature for 6 hours, blow nitrogen for 20 minutes to remove oxygen, add 3.2g hydroxyethyl methacrylate, 16g 1,4-dioxane, 19.2g The mixture of boron trifluoride ether, the mass ratio of hydroxyethyl methacrylate to 1,4-dioxane and boron trifluoride ether is 1:5:6, monodisperse porous cross-linked methacrylic epoxy The mass ratio of propyl ester microspheres to hydroxyethyl methacrylate is 1:2, and other steps of this step are the same as in Example 1. In step 5 of the synthesis of 17β-estradiol molecularly imprinted composite microspheres, 6.7 mg (0.026 mmol) of 17β-estradiol and 25 μL (0.31 mmol) of methacrylic acid were dissolved in 15 mL of acetonitrile, and the surface bond obtained in step 4 was added. Vinyl-based monodisperse cross-linked glycidyl methacrylate microspheres 0.8g, use an ultrasonic cleaner to disperse ultrasonically for 5 minutes at a power of 80W, 200 rpm, swell at room temperature for 6 hours, add ethylene glycol dimethyl Acrylate 100μL (0.52mmol), azobisisobutyronitrile 9.3mg (0.056mmol), the mass ratio of 17β-estradiol to monodisperse cross-linked glycidyl methacrylate microspheres bound to vinyl on the surface 1:120, the molar ratio of 17β-estradiol to methacrylic acid, ethylene glycol dimethacrylate, and azobisisobutyronitrile is 1:12:20:2. Other steps and examples of this step 1 is the same. The other steps were the same as in Example 1 to prepare 17β-estradiol molecularly imprinted composite microspheres.

Example 4

In the step 4 of monodisperse cross-linked glycidyl methacrylate microspheres surface-bonded vinyl group in Examples 1-3, monodisperse porous cross-linked glycidyl methacrylate microspheres were added to 1,4-bis In oxygen hexane, stir at 200 rpm, swell at room temperature for 4 hours, deoxygenate with nitrogen for 20 minutes, add a mixture of hydroxyethyl methacrylate, 1,4-dioxane, and boron trifluoride ether, 30 °C for 15 hours, and other steps in this step are the same as those in the corresponding examples. The other steps were the same as those in the corresponding examples to prepare 17β-estradiol molecularly imprinted composite microspheres.

Example 5

In the step 4 of monodisperse cross-linked glycidyl methacrylate microspheres surface-bonded vinyl group in Examples 1-3, monodisperse porous cross-linked glycidyl methacrylate microspheres were added to 1,4-bis In oxygen hexane, stir at 200 rpm, swell at room temperature for 12 hours, deoxygenate with nitrogen for 20 minutes, add a mixture of hydroxyethyl methacrylate, 1,4-dioxane, and boron trifluoride ether, 60 °C for 8 hours, and the other steps of this step are the same as those in the corresponding examples. The other steps were the same as those in the corresponding examples to prepare 17β-estradiol molecularly imprinted composite microspheres.

Example 6

In Step 5 of the synthesis of 17β-estradiol molecularly imprinted composite microspheres in Examples 1-5, 17β-estradiol, methacrylic acid, and monodisperse cross-linked glycidyl methacrylate with vinyl groups bound to the surface Ultrasonic dispersion of microspheres in acetonitrile, stirring at 200 rpm, swelling at room temperature for 4 hours, adding ethylene glycol dimethacrylate and azobisisobutyronitrile, nitrogen gas for 20 minutes to remove oxygen, and polymerization at 60°C for 48 hours. Other steps of this step are the same as those in the corresponding embodiment. The other steps were the same as those in the corresponding examples to prepare 17β-estradiol molecularly imprinted composite microspheres.

Example 7

In Step 5 of the synthesis of 17β-estradiol molecularly imprinted composite microspheres in Examples 1-5, 17β-estradiol, methacrylic acid, and monodisperse cross-linked glycidyl methacrylate with vinyl groups bound to the surface Ultrasonic dispersion of microspheres in acetonitrile, stirring at 200 rpm, swelling at room temperature for 12 hours, adding ethylene glycol dimethacrylate and azobisisobutyronitrile, nitrogen gas for 20 minutes to remove oxygen, and polymerization at 70°C for 16 hours. Other steps of this step are the same as those in the corresponding embodiment. The other steps were the same as those in the corresponding examples to prepare 17β-estradiol molecularly imprinted composite microspheres.

In order to verify the beneficial effects of the present invention, the inventors carried out various tests using the 17β-estradiol molecularly imprinted composite microspheres prepared in Example 1 of the present invention and the non-molecularly imprinted composite microspheres prepared in Comparative Example 1. The specific test conditions as follows:

Experimental reagents: benzoyl peroxide (BPO, A.R. Tianjin Fuchen Chemical Reagent Factory); glycidyl methacrylate (GMA, Sigma-Aldrich); ethylene glycol dimethacrylate (EDMA, Sigma-Aldrich ); Methacrylic acid (MAA, Sigma-Aldrich); Hydroxyethyl methacrylate (HEMA, Sigma-Aldrich); 1,4-dioxane (A.R. Tianjin Fuchen Chemical Reagent Factory); N, N- Dimethylformamide (DMF, Tianjin Fuchen Chemical Reagent Factory); 17β-estradiol, estriol and estrone were purchased from Shanghai Jingchun Industrial Co., Ltd.

1. Chromatography experiment

(1) Weigh 0.8 g of 17β-estradiol molecularly imprinted composite microspheres (MIP) and non-molecularly imprinted composite microspheres (NIP) in 30 mL of isopropanol, homogenize by ultrasonic for 10 minutes, under 20 MPa pressure, Acetonitrile was used as the displacement liquid, and the column (50mm×4.6mm) was wet-packed, and acetonitrile was used as the mobile phase until a stable baseline was obtained. A chromatographic column filled with 17β-estradiol molecularly imprinted composite microspheres is called an imprinted column, and a chromatographic column filled with non-molecularly imprinted composite microspheres is called a non-imprinted column.

Chromatographic conditions: 36% acetonitrile-water solution as mobile phase; flow rate 0.5mL/min; injection volume 5.0μL, sample 20μg·mL ^-1 acetonitrile-water solution; detection wavelength 280nm.

Determination data: capacity factor k'=(t _R -t ₀ )/t ₀ , where t _R is the retention time of the analyte, t ₀ is the dead time (determined by NaNO ₂ ); imprinting factor IF=k' _MIP /k ′ _NIP , where k′ _MIP and k′ _NIP are the capacity factors of 17β-estradiol molecularly imprinted composite microspheres and non-molecularly imprinted composite microspheres; separation factor α=k ₁ ′/k ₂ ′, where k ₁ ′ and k ₂ ' are the capacity factors of 17β-estradiol and estriol on the blot column, respectively.

(2) Retention of 17β-estradiol on imprinted and non-imprinted columns

Taking 17β-estradiol and resveratrol and capsaicin, which have large structural differences, as the research objects, the selectivity of 17β-estradiol molecularly imprinted composite microspheres and non-molecularly imprinted composite microspheres was studied. The results are shown in Figure 1 and Figure 2 shows. It can be seen from Figure 1 that under the same separation conditions, the retention of 17β-estradiol on the imprinted column is stronger than that of resveratrol and capsaicin, indicating that the 17β-estradiol molecularly imprinted composite microspheres have a strong effect on the retention of 17β-estradiol. It has a certain adsorption selectivity; as shown in Figure 2, under the same separation conditions, the retention times of the three substances on the non-imprinted column are very close, which proves that the molecularly imprinted composite microspheres have specific selection for template molecules. This is due to the formation of imprinted holes and binding sites that can match the spatial structure of the template molecule 17β-estradiol during the preparation of the 17β-estradiol molecularly imprinted composite microspheres, which have specific recognition capabilities for the template molecule; The structures of resveratrol and capsaicin are different from those of 17β-estradiol, and it is difficult to match with the imprinted holes left by template molecules. Therefore, 17β-estradiol molecularly imprinted composite microspheres do not have the ability to recognize them . There is no such specific binding site on non-molecularly imprinted composite microspheres, and the retention times of the three substances on non-imprinted columns are very close.

In addition, estrone, a structural analogue of 17β-estradiol, was selected as the research object to study the recognition effect of molecularly imprinted composite microspheres on similar substances. It can be seen from Figure 3 that the retention times of estradiol and estrone on the imprinted column are very close, indicating that the 17β-estradiol molecularly imprinted composite microspheres have similar retention of structural analogues. From the comparison of Figures 3 and 4, it can be seen that the retention time of 17β-estradiol and its structural analogue estrone on the non-imprinted column is shorter than that on the imprinted column, which further proves that the molecularly imprinted composite microspheres have a positive effect on the template. Molecules have specific recognition.

(3) Effect of pH value on separation

Respectively with acetonitrile and pH 3, 5, 7, 9, 11 PBS buffer (25mmol/L phosphate buffer) in a volume ratio of 36:64 as the mobile phase, investigate the pH of the mobile phase on the 17β - Effect of estradiol and estriol retention on the blot column. The results are shown in Table 1.

Influence of the pH value of the phosphate hydrochloric acid wash solution in the mobile phase on the separation in Table 1

pH k'(17β-estradiol) k'(estriol) α k′(non) IF 3 6.24 1.45 4.30 4.23 1.48 5 6.48 1.45 4.47 4.33 1.50 7 6.50 1.43 4.55 4.41 1.47 9 6.30 1.47 4.29 3.96 1.59 11 6.18 1.43 4.32 3.78 1.63

It can be seen from Table 1 that as the pH increases, the retention behavior of 17β-estradiol on the imprinted column and non-imprinted column becomes slightly smaller, but the change is not obvious, indicating that the 17β-estradiol molecularly imprinted composite microspheres have Strong acid and alkali resistance and good stability; the recognition ability of 17β-estradiol molecularly imprinted composite microspheres is produced by the imprinting process and is slightly affected by pH.

(4) Effect of column temperature on separation

With 36% acetonitrile aqueous solution as the mobile phase and a flow rate of 0.5mL/min, the effects of different temperatures (20, 30, 40, 50, 60° C.) on 17β-estradiol and estriol on the imprinted column were investigated. Influence. The experimental results are shown in Table 2.

Table 2 The influence of column temperature on separation

Temperature (°C) k'(17β-estradiol) k'(estriol) α k′(non) IF 20 6.50 1.6 4.06 4.41 1.47 30 5.50 1.43 3.85 3.88 1.42 40 5.21 1.3 4.01 3.65 1.43 50 4.49 1.16 3.87 3.08 1.46 60 3.84 1.04 3.69 2.51 1.53

It can be seen from Table 2 that the separation factor reaches the highest value of 4.06 at 20°C. As the temperature increases, the capacity factors of 17β-estradiol and estriol decrease, but the change is not large, while the imprinting factor changes little. Obviously, it shows that thermodynamic factors have almost no influence on the stability of the imprinted and non-imprinted microspheres, which further proves that the thermal stability of the 17β-estradiol molecularly imprinted composite microspheres prepared by the present invention is very good.

The experimental results of comprehensive experiments (2) to (4) show that the 17β-estradiol molecularly imprinted composite microspheres prepared by the present invention have strong specific recognition ability to 17β-estradiol and its structural analogues, and are resistant to acid and alkali. The pH range is wider than that of silica gel 2-7.5. It has good thermal stability and its performance is almost not affected by temperature. It can be used in many fields.

Claims (7)

1. A preparation method of 17β-estradiol molecularly imprinted composite microspheres, consisting of the following steps: (1) Synthesis of monodisperse polystyrene dispersion Add styrene, azobisisobutyronitrile, and polyvinylpyrrolidone into absolute ethanol, and the mass ratio of styrene to azobisisobutyronitrile, polyvinylpyrrolidone, and absolute ethanol is 1:0.01～0.04:0.1～0.3 : 7.5 ~ 9, ultrasonic dispersion, nitrogen deoxygenation, stirring, 70 ° C polymerization reaction for 24 hours, prepared into polystyrene; the polystyrene is ultrasonically dispersed with a mass fraction of 0.2% sodium lauryl sulfate aqueous solution to obtain Monodisperse polystyrene dispersion; The molecular weight of the above-mentioned polyvinylpyrrolidone is 10000～70000; (2) Preparation of monodisperse crosslinked glycidyl methacrylate microspheres Benzoyl peroxide, glycidyl methacrylate, ethylene glycol dimethacrylate, dispersant aqueous solution are added in the mixed solvent of cyclohexanol and toluene, toluene and cyclohexanol, benzoyl peroxide, The mass ratio of glycidyl methacrylate, ethylene glycol dimethacrylate, and dispersant aqueous solution is 1:2:0.15:2:3:60, and the cell disruptor is used to ultrasonicate at a power of 300-500W. Ultrasonic 1 time at intervals of 10-20 seconds, 10-20 seconds each time, ultrasonic emulsification until the upper layer is free of oil droplets, and added to the monodisperse polystyrene dispersion obtained in step (1), glycidyl methacrylate and The mass ratio of polystyrene in the monodisperse polystyrene dispersion is 1:0.1, stirred and swelled at 30°C for 10 hours, deoxidized by nitrogen gas, and polymerized at 70°C for 24 hours to prepare a monodisperse cross-linked glycidyl methacrylate Ester microspheres; The dispersant in the above-mentioned dispersant aqueous solution is sodium lauryl sulfate and polyvinyl alcohol, and the mass fraction of sodium lauryl sulfate is 0.2%, and the mass fraction of polyvinyl alcohol is 2%, and the mass fraction of described polyvinyl alcohol The degree of polymerization is 1700, and the degree of alcoholysis is 88%; (3) Extraction of monodisperse cross-linked glycidyl methacrylate microspheres Extract the monodisperse crosslinked glycidyl methacrylate microspheres prepared in step (2) with toluene in a Soxhlet extractor at 140°C for 48 hours to obtain monodisperse porous crosslinked glycidyl methacrylate microspheres ; (4) Monodisperse cross-linked glycidyl methacrylate microspheres with vinyl groups bonded to the surface Add the monodisperse porous crosslinked glycidyl methacrylate microspheres prepared in step (3) into 1,4-dioxane, and add 3 to 10 g of monodisperse porous Cross-linked glycidyl methacrylate microspheres, stirred and swelled at room temperature for 4 to 12 hours, passed nitrogen gas to remove oxygen, and added the mass ratio of hydroxyethyl methacrylate to 1,4-dioxane and boron trifluoride ether It is a mixed solution of 1:2~5:4~6, the mass ratio of monodisperse porous crosslinked glycidyl methacrylate microspheres to hydroxyethyl methacrylate is 1:0.1~2, stirred at 30~60°C After 8 to 15 hours, monodisperse crosslinked glycidyl methacrylate microspheres with vinyl groups bonded to the surface are obtained; (5) Synthesis of 17β-estradiol molecularly imprinted composite microspheres Using acetonitrile as a solvent, ultrasonically disperse 17β-estradiol, methacrylic acid, and surface-bonded vinyl monodisperse crosslinked glycidyl methacrylate microspheres, stir and swell at room temperature for 4 to 12 hours, add ethylene glycol di The mass ratio of methacrylate, azobisisobutyronitrile, 17β-estradiol and monodisperse cross-linked glycidyl methacrylate microspheres bonded to vinyl on the surface is 1:20-120, 17β-estradiol The molar ratio of diol to methacrylic acid, ethylene glycol dimethacrylate, and azobisisobutyronitrile is 1:2~12:8~20:1~2, nitrogen gas is used to remove oxygen, and polymerization is carried out at 60~70°C After reacting for 16-48 hours, the 17β-estradiol is eluted, and the 17β-estradiol molecularly imprinted composite microspheres are prepared. 2. The preparation method of 17β-estradiol molecularly imprinted composite microspheres according to claim 1, characterized in that: in the monodisperse crosslinked glycidyl methacrylate microsphere surface bonding vinyl step (4) Among them, the mass ratio of the monodisperse porous crosslinked glycidyl methacrylate microspheres to hydroxyethyl methacrylate is 1:1. 3. The preparation method of 17β-estradiol molecularly imprinted composite microspheres according to claim 1, characterized in that: in the monodisperse crosslinked glycidyl methacrylate microsphere surface bonding vinyl step (4) Among them, the mass ratio of hydroxyethyl methacrylate to 1,4-dioxane and boron trifluoride ether is 1:2.6:5. 4. The preparation method of 17β-estradiol molecularly imprinted composite microspheres according to claim 1, characterized in that: the step of bonding vinyl on the surface of monodisperse crosslinked glycidyl methacrylate microspheres (4) , the monodisperse porous cross-linked glycidyl methacrylate microspheres prepared in step (3) were added to 1,4-dioxane, stirred and swelled at room temperature for 6 hours, deoxygenated by nitrogen, and 1,4- A mixture of dioxane, hydroxyethyl methacrylate, and boron trifluoride ether was stirred at 45° C. for 12 hours to obtain monodisperse crosslinked glycidyl methacrylate microspheres with vinyl groups bonded to the surface. 5. The preparation method of 17β-estradiol molecularly imprinted composite microspheres according to claim 1, characterized in that: in the step (5) of synthesizing 17β-estradiol molecularly imprinted composite microspheres, the 17β- The mass ratio of estradiol to surface-bonded monodisperse cross-linked glycidyl methacrylate microspheres with vinyl groups was 1:40. 6. The preparation method of 17β-estradiol molecularly imprinted composite microspheres according to claim 1, characterized in that: in the step (5) of synthesizing 17β-estradiol molecularly imprinted composite microspheres, the 17β- The molar ratio of estradiol to methacrylic acid, ethylene glycol dimethacrylate and azobisisobutyronitrile is 1:4:10:1.2. 7. The preparation method of 17β-estradiol molecularly imprinted composite microspheres according to claim 1, characterized in that: in the step (5) of synthesizing 17β-estradiol molecularly imprinted composite microspheres, 17β-estradiol Dissolve alcohol and methacrylic acid in acetonitrile, add monodisperse cross-linked glycidyl methacrylate microspheres with vinyl groups on the surface obtained in step (4), ultrasonically disperse, stir and swell at room temperature for 6 hours, add ethylene glycol Dimethacrylate, azobisisobutyronitrile, deoxygenated by nitrogen gas, polymerized at 70°C for 24 hours.

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