CN102382273B - Preparation method of 17 beta-estradiol molecular-imprinted composite microspheres - Google Patents
Preparation method of 17 beta-estradiol molecular-imprinted composite microspheres Download PDFInfo
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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
Technical field
The invention belongs to the material technology field, be specifically related to the molecular imprinting complex microsphere of 17 beta estradiols in a kind of assay determination biological sample and the food samples.
Background technology
(17 β-Estradiol) are that synthetic is estrogenic a kind of to 17 beta estradiols; It is mainly used in diseases such as medically treating climacteric syndrome; But take in 17 beta estradiols for a long time and can produce some spinoffs; And increase the initiation potential of some disease, in view of having these risks, so country forbids in food, to use synthetic oestrogenic hormon such as 17 beta estradiols.But be no lack of some lawless persons for strengthening its product function, reduce cost and add the synthetic oestrogenic hormon of the estrogen-like effects that 17 beta estradiols etc. have.In addition, the abuse anabolic hormone, healthy to humans and animals, all can cause directly or potential harm social and ecotope.Therefore the oestrogenic hormon monitoring to synthetic such as 17 beta estradiols in food, the protective foods also is the requisite measure that guarantees this type of food sanitation safe.
17 beta estradiols can get into environment through the metabolism of life entity; Or get into environment with trade effluent as the parent compound of female hormone class drug manufacture; So that residual in aquatic organism, get into human body by food chain, and its meta-bolites there is confidential relation with human cancer.Therefore, food nutrition and safety-problems that it causes can not be ignored, and the research that has level and BA of this type material is had important practical significance.Because the drug level residual to be measured in the food is low and fluctuation range is big, sample substrate is complicated, interfering substance is many, the characteristics such as uncertainty of sample substrate and component to be measured, needs before detecting disturb impurity, enrichment tracer level purpose component to be measured in the removal sample.
Molecularly imprinted polymer (MIPs) has specific recognition; The sample pre-treatments that can be used for complex matrices; And preparation is simple, stable in properties and reusable, has all obtained at the aspects such as control of separation science, biomedicine, sensor technology and chemical reaction using widely.But all there is tangible limitation in existing preparation MPIMs method: the polymkeric substance that mass polymerization obtains is for block; Must be before using through processing such as grinding, screenings; The irregular sample preparation difficulty that makes of size and dimension; It is loaded down with trivial details, time-consuming to work, and productive rate low (general＜50%), and application efficiency significantly reduces; Precipitation polymerization method is high to the requirement of solvent, is difficult to be used widely; The MPIMs particle diameter that emulsion polymerization makes little (＜1 μ m), be not suitable in liquid chromatography using and absorption after not easily separated; Utilize spherical silica gel surface imprinted or when sacrificing the silica gel method, the hydroxyl on silica gel surface can influence the reactive force between function monomer and template molecule, thus the MPIMs recognition capability a little less than; Though suspension polymerization preparation technology is simple, the MPIMs size distribution that obtains is wide, needs just can use through loaded down with trivial details stage treatment; Though multistep swollen-state polymerization method can make micron order and the uniform MPIMs of particle diameter, can use through simple washing, needs through the multistep swelling, process is comparatively loaded down with trivial details, preparation cycle is longer, is difficult to realize industriallization.By contrast, molecular blotting polymer microsphere better performances, preparation and use are more convenient, are specially adapted to analyzing and testing, sensor array and separation assembly.Therefore, the preparation of molecular blotting polymer microsphere and applied research become one of research focus of molecular imprinting.Surface imprinted binding site is positioned at the surface of polymkeric substance, and template molecule is easy to wash-out, can improve binding capacity; Target molecule need not to get into its rigid structure when combining again, has accelerated the bonded dynamic process greatly.But the matrix that this method is used at present mainly is confined to silica gel, and the use pH scope of using silica particles will pay special attention to it is 2～7.5, if cross alkali (pH＞7.5), silica gel can be pulverized or dissolve; If peracid (pH＜2), the chemical bond of bonding phase can rupture.It is cumbersome that reaction finishes back removal matrix, generally adopts the hydrofluoric acid corroding method, but the skeleton structure of polymkeric substance is had certain detrimentally affect.
Dissolve in the invention
Technical problem to be solved by this invention is to overcome above-mentioned preparing method's shortcoming, provides a kind of reaction conditions gentle, the preparation method of the 17 beta-estradiol molecular engram complex microspheres that product stability is good, recognition capability is strong.
Solving the problems of the technologies described above the technical scheme that is adopted is made up of following step:
1, synthetic monodisperse polystyrene dispersion liquid
Vinylbenzene, Diisopropyl azodicarboxylate, Vinylpyrrolidone polymer are added in the absolute ethyl alcohol; The mass ratio of vinylbenzene and Diisopropyl azodicarboxylate, Vinylpyrrolidone polymer, absolute ethyl alcohol is 1: 0.01～0.04: 0.1～0.3: 7.5～9; Ultra-sonic dispersion, logical nitrogen deoxygenation is stirred; 70 ℃ of polyreactions 24 hours are prepared into PS; It is 0.2% lauryl sodium sulfate aqueous solution ultra-sonic dispersion that PS is used massfraction, obtains the monodisperse polystyrene dispersion liquid.
The molecular weight of above-mentioned Vinylpyrrolidone polymer is 10000～70000, sends Buddhist nun's chemical reagent factory Zhengzhou to provide by China.
2, the monodisperse cross-linked GMA microballoon of preparation
Lucidol, GMA, ethylene glycol dimethacrylate, aqueous dispersant are added in the mixed solvent of hexalin and toluene, and the mass ratio of toluene and hexalin, Lucidol, GMA, ethylene glycol dimethacrylate, aqueous dispersant is 1: 2: 0.15: 2: 3: 60, using the cytoclasis appearance was ultrasonic under 300～500W at power; Every interval 10～20 seconds ultrasonic 1 time; Each ultrasonic 10～20 seconds, ultrasonic emulsification to upper strata did not have oil droplet, joined in the monodisperse polystyrene dispersion liquid that step 1 obtains; The mass ratio of PS is 1: 0.1 in GMA and the monodisperse polystyrene dispersion liquid; 30 ℃ were stirred swelling 10 hours, logical nitrogen deoxygenation, 70 ℃ of polyreactions 24 hours; Filter; Use methyl alcohol, washing with acetone successively, 60 ℃ of vacuum-drying 4 hours is prepared into monodisperse cross-linked GMA microballoon.
Dispersion agent in the above-mentioned aqueous dispersant is sodium lauryl sulphate and Z 150PH; The massfraction of sodium lauryl sulphate is 0.2%, and the massfraction of Z 150PH is 2%, and the polymerization degree of described Z 150PH is 1700; Alcoholysis degree is 88%, and huge chemical experimental factory provides by Tianjin.
3, the monodisperse cross-linked GMA microballoon of extracting
The monodisperse cross-linked GMA microballoon of step 2 preparation with toluene 140 ℃ of extractings 48 hours in cable type extractor according, is used absolute ethyl alcohol, washing with acetone successively, and drying obtains the sour epoxy propyl ester of monodisperse porous crosslinked methacrylic microballoon.
4, monodisperse cross-linked GMA microsphere surface bonding ethylene base
The monodisperse porous crosslinked methacrylic acid epoxy propyl ester microballoon of step 3 preparation is added 1, in the 4-dioxane, every 100mL 1; Add the monodisperse porous crosslinked methacrylic acid of 3～10g epoxy propyl ester microballoon in the 4-dioxane, stirring at room swelling 4～12 hours, logical nitrogen deoxygenation; Add Rocryl 400 and 1, the mass ratio of 4-dioxane, BFEE is 1: 2～5: 4～6 mixed solution, and the mass ratio of monodisperse porous crosslinked methacrylic acid epoxy propyl ester microballoon and Rocryl 400 is 1: 0.1～2; 30～60 ℃ were stirred 8～15 hours; Filter, product uses 1 successively, 4-dioxane, methyl alcohol, zero(ppm) water, washing with acetone; Vacuum-drying obtains the monodisperse cross-linked GMA microballoon of surface bond vinyl.
5, synthetic 17 beta-estradiol molecular engram complex microspheres
With the acetonitrile is solvent; The monodisperse cross-linked GMA microballoon of ultra-sonic dispersion 17 beta estradiols, methylacrylic acid, surface bond vinyl, stirring at room swelling 4～12 hours adds ethylene glycol dimethacrylate, Diisopropyl azodicarboxylate; The mass ratio of the monodisperse cross-linked GMA microballoon propyl ester microballoon of 17 beta estradiols and surface bond vinyl is 1: 20～120; The mol ratio of 17 beta estradiols and methylacrylic acid, ethylene glycol dimethacrylate, Diisopropyl azodicarboxylate is 1: 2～12: 8～20: 1～2, logical nitrogen deoxygenation, 60～70 ℃ of polyreactions 16～48 hours; The volume ratio of product use methyl alcohol and acetate is 8: 2 an elutriant wash-out; Remove 17 beta estradiols, be washed till neutrality with zero(ppm) water earlier, use methanol wash again; Vacuum-drying is prepared into 17 beta-estradiol molecular engram complex microspheres.
In the monodisperse cross-linked GMA microsphere surface bonding ethylene base step 4 of the present invention; The optimum quality ratio of monodisperse porous crosslinked methacrylic acid epoxy propyl ester microballoon and Rocryl 400 is 1: 1; Rocryl 400 and 1, the optimum quality ratio of 4-dioxane, BFEE are 1: 2.6: 5.
In the monodisperse cross-linked GMA microsphere surface bonding ethylene base step 4 of the present invention, best monodisperse porous crosslinked methacrylic acid epoxy propyl ester microballoon with step 3 preparation adds 1, in the 4-dioxane; Stirring at room swelling 6 hours; Logical nitrogen deoxygenation adds 1, the mixed solution of 4-dioxane and Rocryl 400, BFEE; 45 ℃ were stirred 12 hours, obtained the monodisperse cross-linked GMA microballoon of surface bond vinyl.
In the synthetic 17 beta-estradiol molecular engram complex microsphere steps 5 of the present invention; The optimum quality ratio of the monodisperse cross-linked GMA microballoon of 17 beta estradiols and surface bond vinyl is 1: 40, and the optimum mole ratio of 17 beta estradiols and methylacrylic acid, ethylene glycol dimethacrylate, Diisopropyl azodicarboxylate is 1: 4: 10: 1.2.
In the synthetic 17 beta-estradiol molecular engram complex microsphere steps 5 of the present invention; The best is dissolved in 17 beta estradiols, methylacrylic acid in the acetonitrile, the monodisperse cross-linked GMA microballoon of the surface bond vinyl that adding step 4 obtains, ultra-sonic dispersion; Stirring at room swelling 6 hours; Add ethylene glycol dimethacrylate, Diisopropyl azodicarboxylate, logical nitrogen deoxygenation, 70 ℃ of polyreactions 24 hours.
The present invention is with 1; The 4-dioxane is a solvent, and the monodisperse cross-linked GMA microballoon of preparation surface bond vinyl is a carrier with the monodisperse cross-linked GMA microballoon of surface bond vinyl; Adopt surface imprinted technology to be prepared into 17 beta-estradiol molecular engram complex microspheres; This method reaction conditions is gentle, and the products therefrom good stability is strong to the recognition capability of 17 beta estradiols, rate of adsorption is fast; Can be used for setting up convenient extraction, controlization and concentrating sample pre-treatment; Quality and composition to containing estrogen product are assessed, and can be used for standing the performance liquid chromatography of high flow rate high pressure operation simultaneously, improve post greatly and imitate and circulation ratio.In addition; Because the binding site of trace is positioned at the surface of polymkeric substance, template molecule is easy to wash-out, in conjunction with the time target molecule need not to get into its rigid structure; Accelerate the dynamic process of adsorption and desorption greatly, can it be used as solid phase extraction adsorbents fast and efficiently.
Description of drawings
Fig. 1 is 17 beta estradiols, trans-resveratrol and the capsicine chromatographic fractionation figure on the trace post.
Fig. 2 is 17 beta estradiols, trans-resveratrol and the capsicine chromatographic fractionation figure on non-trace post.
Fig. 3 is 17 beta estradiols and the chromatographic fractionation figure of oestrone on the trace post.
Fig. 4 is 17 beta estradiols and the chromatographic fractionation figure of oestrone on non-trace post.
Embodiment
To further explain of the present invention, but the invention is not restricted to these embodiment below in conjunction with accompanying drawing and embodiment.
Embodiment 1
1, synthetic monodisperse polystyrene dispersion liquid
With vinylbenzene 10.908g, Diisopropyl azodicarboxylate 0.2406g, molecular weight is that 10000～70000 Vinylpyrrolidone polymer 1.5006g adds and fills in the 250mL single port flask of 96g absolute ethyl alcohol; The mass ratio of vinylbenzene and Diisopropyl azodicarboxylate, Vinylpyrrolidone polymer, absolute ethyl alcohol is 1: 0.02: 014: 8.8, and use ultrasonic cleaner to be ultra-sonic dispersion under the 80W 20 minutes, logical nitrogen deoxygenation in 15 minutes at power; Stir; 70 ℃ of polyreactions 24 hours, after reaction finished, centrifugal removing desolvated; Use absolute ethanol washing, be prepared into PS; With the 0.2g PS use ultrasonic cleaner power as ultra-sonic dispersion under the 80W in the 10mL massfraction is 0.2% lauryl sodium sulfate aqueous solution, obtain the monodisperse polystyrene dispersion liquid.
2, the monodisperse cross-linked GMA microballoon of preparation
The mixture aqueous solution 62.35g of Lucidol 0.1667g, GMA 2.064g, ethylene glycol dimethacrylate 3.03g, sodium lauryl sulphate and Z 150PH (alcoholysis degree is 88%) added fill in the 250mL beaker of 1.92g hexalin and 1.0392g toluene, the mass ratio of the mixture aqueous solution of toluene and hexalin, Lucidol, GMA, ethylene glycol dimethacrylate, sodium lauryl sulphate and Z 150PH is 1: 2: 0.15: 2: 3: 60, use the cytoclasis appearance at power as ultrasonic under the 400W; Every interval 15 seconds ultrasonic 1 time; Each ultrasonic 15 seconds, ultrasonic emulsification to upper strata did not have oil droplet, joined in the monodisperse polystyrene dispersion liquid that step 1 obtains; The mass ratio of PS is 1: 0.1 in GMA and the monodisperse polystyrene dispersion liquid; 120 rev/mins of stirrings, 30 ℃ of swellings 10 hours, logical nitrogen deoxygenation in 20 minutes; 70 ℃ of polyreactions 24 hours; Use the glass sand hourglass suction filtration,, use methyl alcohol, washing with acetone more successively with 70 ℃ of distilled water washs; 60 ℃ of vacuum-drying 4 hours is prepared into monodisperse cross-linked GMA microballoon.
In the above-mentioned sodium lauryl sulphate and the mixture aqueous solution of Z 150PH, the massfraction of sodium lauryl sulphate is 0.2%, and the massfraction of Z 150PH is 2%, and the polymerization degree of Z 150PH is 1700, and alcoholysis degree is 88%.
3, the monodisperse cross-linked GMA microballoon of extracting
The monodisperse cross-linked GMA microballoon of step 2 preparation is placed cable type extractor according; Add 50mL toluene; 140 ℃ of extractings 48 hours; Using absolute ethyl alcohol, washing with acetone successively, is 60 ℃ of dryings 3 hours under the 0.06MPa in vacuum tightness, obtains monodisperse porous crosslinked methacrylic acid epoxy propyl ester microballoon.
4, monodisperse cross-linked GMA microsphere surface bonding ethylene base
The monodisperse porous crosslinked methacrylic acid epoxy propyl ester microballoon 2.0g of step 3 preparation is added 50mL 1, in the 4-dioxane, 200 rev/mins of stirrings; Room temperature swelling 6 hours, logical nitrogen deoxygenation in 20 minutes adds 2.0g Rocryl 400,5.2g 1; The mixed solution of 4-dioxane, 10.0g BFEE, Rocryl 400 and 1, the mass ratio of 4-dioxane, BFEE are 1: 2.6: 5; The mass ratio of monodisperse porous crosslinked methacrylic acid epoxy propyl ester microballoon and Rocryl 400 is 1: 1, and 45 ℃ were stirred 12 hours, filtered; Product uses 1 successively; 4-dioxane, methyl alcohol, zero(ppm) water, washing with acetone, 30 ℃ of vacuum-drying 2 hours obtains the monodisperse cross-linked GMA microballoon of surface bond vinyl.
5, synthetic 17 beta-estradiol molecular engram complex microspheres
17 beta estradiol 20mg (0.078mmol), methylacrylic acid 27 μ L (0.32mmol) are dissolved in the 15mL acetonitrile, add the monodisperse cross-linked GMA microballoon 0.8g of the surface bond vinyl that step 4 obtains, use ultrasonic cleaner to be ultra-sonic dispersion under the 80W 5 minutes at power; 200 rev/mins of stirrings; Room temperature swelling 6 hours adds ethylene glycol dimethacrylate 147 μ L (0.774mmol), Diisopropyl azodicarboxylate 15mg (0.094mmol), and the mass ratio of the monodisperse cross-linked GMA microballoon of 17 beta estradiols and surface bond vinyl is 1: 40; The mol ratio of 17 beta estradiols and methylacrylic acid, ethylene glycol dimethacrylate, Diisopropyl azodicarboxylate is 1: 4: 10: 1.2; Logical nitrogen deoxygenation in 20 minutes, 70 ℃ of polyreactions 24 hours, product is used earlier zero(ppm) water, methanol wash successively; The volume ratio of using methyl alcohol and acetate again is 8: 2 an elutriant wash-out 24 hours; Remove 17 beta estradiols, be washed till neutrality with zero(ppm) water then, use methanol wash again; 30 ℃ of vacuum-drying 2 hours is prepared into 17 beta-estradiol molecular engram complex microspheres.
The comparative example 1
According to the method for embodiment 1 step 1～4, be prepared into the monodisperse cross-linked GMA microballoon of surface bond vinyl.Do not add template molecule 17 beta estradiols, directly methylacrylic acid 27 μ L (0.32mmol) are dissolved in the 15mL acetonitrile, add the monodisperse cross-linked GMA microballoon of 0.8g surface bond vinyl; Use ultrasonic cleaner to be ultra-sonic dispersion under the 80W 5 minutes, 200 rev/mins of stirrings, room temperature swelling 6 hours at power; Add ethylene glycol dimethacrylate 147 μ L (0.774mmol), Diisopropyl azodicarboxylate 15mg (0.094mmol); Logical nitrogen deoxygenation in 20 minutes, 70 ℃ of polyreactions 24 hours, product is used zero(ppm) water, methanol wash successively; 30 ℃ of vacuum-drying 2 hours is prepared into non-molecular imprinting complex microsphere.
In the monodisperse cross-linked GMA microsphere surface bonding ethylene base step 4 of embodiment 1; The monodisperse porous crosslinked methacrylic acid epoxy propyl ester microballoon 1.6g of step 3 preparation is added 53.2mL 1, in the 4-dioxane, 200 rev/mins of stirrings; Room temperature swelling 6 hours; Logical nitrogen deoxygenation in 20 minutes adds 0.16g Rocryl 400,0.32g 1, the mixed solution of 4-dioxane, 0.64g BFEE; Rocryl 400 and 1; The mass ratio of 4-dioxane, BFEE is 1: 2: 4, and the mass ratio of monodisperse porous crosslinked methacrylic acid epoxy propyl ester microballoon and Rocryl 400 is 1: 0.1, and other steps of this step are identical with embodiment 1.In synthetic 17 beta-estradiol molecular engram complex microsphere steps 5; 17 beta estradiol 40mg (0.156mmol), methylacrylic acid 26 μ L (0.312mmol) are dissolved in the 15mL acetonitrile; The monodisperse cross-linked GMA microballoon 0.8g of the surface bond vinyl that adding step 4 obtains; Use ultrasonic cleaner to be ultra-sonic dispersion under the 80W 5 minutes at power; 200 rev/mins; Room temperature swelling 6 hours adds ethylene glycol dimethacrylate 237 μ L (1.248mmol), Diisopropyl azodicarboxylate 26mg (0.156mmol), and the mass ratio of the monodisperse cross-linked GMA microballoon of 17 beta estradiols and surface bond vinyl is 1: 20; The mol ratio of 17 beta estradiols and methylacrylic acid, ethylene glycol dimethacrylate, Diisopropyl azodicarboxylate is 1: 2: 8: 1, and other steps of this step are identical with embodiment 1.Other steps are identical with embodiment 1, are prepared into 17 beta-estradiol molecular engram complex microspheres.
In the monodisperse cross-linked GMA microsphere surface bonding ethylene base step 4 of embodiment 1; The monodisperse porous crosslinked methacrylic acid epoxy propyl ester microballoon 1.6g of step 3 preparation is added 16mL 1, in the 4-dioxane, 200 rev/mins of stirrings; Room temperature swelling 6 hours; Logical nitrogen deoxygenation in 20 minutes adds 3.2g Rocryl 400,16g 1, the mixed solution of 4-dioxane, 19.2g BFEE; Rocryl 400 and 1; The mass ratio of 4-dioxane, BFEE is 1: 5: 6, and the mass ratio of monodisperse porous crosslinked methacrylic acid epoxy propyl ester microballoon and Rocryl 400 is 1: 2, and other steps of this step are identical with embodiment 1.In synthetic 17 beta-estradiol molecular engram complex microsphere steps 5; 17 beta estradiol 6.7mg (0.026mmol), methylacrylic acid 25 μ L (0.31mmol) are dissolved in the 15mL acetonitrile; The monodisperse cross-linked GMA microballoon 0.8g of the surface bond vinyl that adding step 4 obtains; Use ultrasonic cleaner to be ultra-sonic dispersion under the 80W 5 minutes at power; 200 rev/mins; Room temperature swelling 6 hours adds ethylene glycol dimethacrylate 100 μ L (0.52mmol), Diisopropyl azodicarboxylate 9.3mg (0.056mmol), and the mass ratio of the monodisperse cross-linked GMA microballoon of 17 beta estradiols and surface bond vinyl is 1: 120; The mol ratio of 17 beta estradiols and methylacrylic acid, ethylene glycol dimethacrylate, Diisopropyl azodicarboxylate is 1: 12: 20: 2, and other steps of this step are identical with embodiment 1.Other steps are identical with embodiment 1, are prepared into 17 beta-estradiol molecular engram complex microspheres.
In the monodisperse cross-linked GMA microsphere surface bonding ethylene base step 4 of embodiment 1～3, monodisperse porous crosslinked methacrylic acid epoxy propyl ester microballoon is added 1, in the 4-dioxane; 200 rev/mins of stirrings, room temperature swelling 4 hours, logical nitrogen deoxygenation in 20 minutes; Add Rocryl 400 and 1; The mixed solution of 4-dioxane, BFEE, 30 ℃ were stirred 15 hours, and other steps of this step are identical with corresponding embodiment.Other steps are identical with corresponding embodiment, are prepared into 17 beta-estradiol molecular engram complex microspheres.
Embodiment 5
In the monodisperse cross-linked GMA microsphere surface bonding ethylene base step 4 of embodiment 1～3, monodisperse porous crosslinked methacrylic acid epoxy propyl ester microballoon is added 1, in the 4-dioxane; 200 rev/mins of stirrings, room temperature swelling 12 hours, logical nitrogen deoxygenation in 20 minutes; Add Rocryl 400 and 1; The mixed solution of 4-dioxane, BFEE, 60 ℃ were stirred 8 hours, and other steps of this step are identical with corresponding embodiment.Other steps are identical with corresponding embodiment, are prepared into 17 beta-estradiol molecular engram complex microspheres.
Embodiment 6
In the synthetic 17 beta-estradiol molecular engram complex microsphere steps 5 of embodiment 1～5; With the monodisperse cross-linked GMA microballoon ultra-sonic dispersion of 17 beta estradiols, methylacrylic acid, surface bond vinyl in acetonitrile, 200 rev/mins of stirrings, room temperature swelling 4 hours; Add ethylene glycol dimethacrylate, Diisopropyl azodicarboxylate; Logical nitrogen deoxygenation in 20 minutes, 60 ℃ of polyreactions 48 hours, other steps of this step are identical with corresponding embodiment.Other steps are identical with corresponding embodiment, are prepared into 17 beta-estradiol molecular engram complex microspheres.
Embodiment 7
In the synthetic 17 beta-estradiol molecular engram complex microsphere steps 5 of embodiment 1～5; With the monodisperse cross-linked GMA microballoon ultra-sonic dispersion of 17 beta estradiols, methylacrylic acid, surface bond vinyl in acetonitrile, 200 rev/mins of stirrings, room temperature swelling 12 hours; Add ethylene glycol dimethacrylate, Diisopropyl azodicarboxylate; Logical nitrogen deoxygenation in 20 minutes, 70 ℃ of polyreactions 16 hours, other steps of this step are identical with corresponding embodiment.Other steps are identical with corresponding embodiment, are prepared into 17 beta-estradiol molecular engram complex microspheres.
In order to verify beneficial effect of the present invention, the contriver adopts the 17 beta-estradiol molecular engram complex microspheres of the embodiment of the invention 1 preparation and the non-molecular imprinting complex microsphere of comparative example's 1 preparation to carry out various tests, and concrete test situation is following:
Experiment reagent: Lucidol (BPO, A.R. Tianjin good fortune chemical reagent in morning factory); GMA (GMA, Sigma-Aldrich); Ethylene glycol dimethacrylate (EDMA, Sigma-Aldrich); Methylacrylic acid (MAA, Sigma-Aldrich); Rocryl 400 (HEMA, Sigma-Aldrich); 1,4-dioxane (A.R. Tianjin good fortune chemical reagent in morning factory); N, dinethylformamide (DMF, Tianjin good fortune chemical reagent in morning factory); 17 beta estradiols, trihydroxy-oestrin and oestrone are all purchased the brilliant pure Industrial Co., Ltd. from Shanghai.
1, chromatography experiment
(1) take by weighing 17 beta-estradiol molecular engram complex microspheres (MIP), each 0.8g of non-molecular imprinting complex microsphere (NIP) places the 30mL Virahol; Ultrasonic homogenate 10 minutes; Under 20MPa pressure, with acetonitrile as displacing liquid, wet method dress post (50mm * 4.6mm); With the acetonitrile is moving phase, up to obtaining stable baseline.The chromatographic column of filling with 17 beta-estradiol molecular engram complex microspheres is called the trace post, and the chromatographic column of filling with non-molecular imprinting complex microsphere is called non-trace post.
Chromatographic condition: with volume(tric)fraction is that 36% acetonitrile-aqueous solution is a moving phase; Flow velocity is 0.5mL/ minute; Sample size is 5.0 μ L, and sample is 20 μ gmL
-1Acetonitrile solution; The detection wavelength is 280nm.
Determination data: capacity factor k '=(t
R-t
0)/t
0, t wherein
RBe the RT of analyte, t
0Be that the dead time is (with NaNO
2Measure); Trace factor IF=k '
MIP/ k '
NIP, k ' wherein
MIPAnd k '
NIPBe respectively the capacity factor of 17 beta-estradiol molecular engram complex microspheres and non-molecular imprinting complex microsphere; Separation factor alpha=k
1'/k
2', k wherein
1' and k
2' be respectively 17 beta estradiols and the capacity factor of trihydroxy-oestrin on the trace post.
The reservation situation of (2) 17 beta estradiols on trace post and non-trace post
With 17 beta estradiols and with the bigger trans-resveratrol of its textural difference and capsicine as research object, study the selectivity of 17 beta-estradiol molecular engram complex microspheres and non-molecular imprinting complex microsphere, the result is as depicted in figs. 1 and 2.Visible by Fig. 1, under identical separation condition, the reservation of 17 beta estradiols on the trace post is stronger than trans-resveratrol, capsicine, shows that 17 beta-estradiol molecular engram complex microspheres have certain adsorption selectivity to 17 beta estradiols; As shown in Figure 2, under the identical separation condition, the RT of three kinds of materials on non-trace post is very approaching, proves that the molecular imprinting complex microsphere has specificity to template molecule and selects.This is that it has the specific recognition ability to template molecule owing in the preparation process of 17 beta-estradiol molecular engram complex microspheres, formed the trace hole and the binding site that can be complementary with template molecule 17 beta estradiol space structures; The structure of the structure of trans-resveratrol and capsicine and 17 beta estradiols is inequality, and the trace hole that is difficult to stay with template molecule is complementary, and therefore, 17 beta-estradiol molecular engram complex microspheres do not possess recognition capability to it.But not having such specific binding site on the molecular imprinting complex microsphere, the RT of three kinds of materials on non-trace post is very approaching.
In addition, the analog oestrone of selecting 17 beta estradiols is as the recognition reaction of research object research molecular imprinting complex microsphere to allied substances.Can find out that by Fig. 3 Theelin,dihydro-and the oestrone RT on the trace post is very approaching, explain that 17 beta-estradiol molecular engram complex microspheres have similar reservation situation to analog.Can find out that by Fig. 3,4 contrasts 17 beta estradiols and the RT of its analog oestrone on non-trace post further prove thus that all less than the RT on the trace post molecular imprinting complex microsphere has the specific recognition effect to template molecule.
(3) the pH value is to isolating influence
Be that the volume ratio of 3,5,7,9,11 PBS damping fluid (phosphate buffered saline buffer of 25mmol/L) is that 36: 64 mixed solution is a moving phase with acetonitrile and pH respectively, the pH that investigates moving phase keeps the influence of situation on the trace post to 17 beta estradiols and trihydroxy-oestrin.The result is as shown in table 1.
In table 1 moving phase phosphoric acid hydrochloric acid towards the pH value of liquid to isolating influence
| pH | K ' (17 beta estradiol) | K ' (trihydroxy-oestrin) | 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 |
Visible by table 1, along with the increase of pH, the retention behavior of 17 beta estradiols on trace post and non-trace post has slightly and diminishes, but changes not obviously, explains that 17 beta-estradiol molecular engram complex microspheres have stronger resistance to acids and bases, good stability; The recognition capability of 17 beta-estradiol molecular engram complex microspheres is that produce and influence that receive pH is very little by the trace process.
(4) column temperature is to isolating influence
With volume(tric)fraction is that 36% acetonitrile solution is a moving phase, and flow velocity is 0.5mL/ minute, investigates differing temps (20,30,40,50,60 ℃) to 17 beta estradiols and the influence of trihydroxy-oestrin on the trace post.Experimental result is seen table 2.
Table 2 column temperature is to isolating influence
| Temperature (℃) | K ' (17 beta estradiol) | K ' (trihydroxy-oestrin) | 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 |
Visible by table 2; In the time of 20 ℃, separate the factor and reach mxm. 4.06, along with the rising of temperature, the capacity factor of 17 beta estradiols and trihydroxy-oestrin all decreases; But change little; And the trace factor variations is not obvious, and the stability almost not influence of thermodynamics factor to trace and non-trace microballoon is described, has proved that further the thermostability of the 17 beta-estradiol molecular engram complex microspheres that the present invention prepares is fine.
The experimental result of Comprehensive Experiment (2)～(4); Explain the present invention preparation 17 beta-estradiol molecular engram complex microspheres 17 beta estradiols and analog thereof are had strong specific recognition ability; Anti acid alkali performance, the pH use range is wider than 2～7.5 of silica gel, Heat stability is good; Its performance receives Influence of Temperature hardly, can in multi-field, use.
Claims (7)
1. the preparation method of a beta-estradiol molecular engram complex microsphere, be made up of following step:
(1) synthetic monodisperse polystyrene dispersion liquid
Vinylbenzene, Diisopropyl azodicarboxylate, Vinylpyrrolidone polymer are added in the absolute ethyl alcohol; The mass ratio of vinylbenzene and Diisopropyl azodicarboxylate, Vinylpyrrolidone polymer, absolute ethyl alcohol is 1: 0.01～0.04: 0.1～0.3: 7.5～9; Ultra-sonic dispersion, logical nitrogen deoxygenation is stirred; 70 ℃ of polyreactions 24 hours are prepared into PS; It is 0.2% lauryl sodium sulfate aqueous solution ultra-sonic dispersion that PS is used massfraction, obtains the monodisperse polystyrene dispersion liquid;
The molecular weight of above-mentioned Vinylpyrrolidone polymer is 10000～70000;
(2) the monodisperse cross-linked GMA microballoon of preparation
Lucidol, GMA, ethylene glycol dimethacrylate, aqueous dispersant are added in the mixed solvent of hexalin and toluene; The mass ratio of toluene and hexalin, Lucidol, GMA, ethylene glycol dimethacrylate, aqueous dispersant is 1: 2: 0.15: 2: 3: 60; Using the cytoclasis appearance is ultrasonic under 300～500W at power; Every interval 10～20 seconds ultrasonic 1 time, each ultrasonic 10～20 seconds, ultrasonic emulsification to upper strata did not have oil droplet; Join in the monodisperse polystyrene dispersion liquid that step (1) obtains; The mass ratio of PS is 1: 0.1 in GMA and the monodisperse polystyrene dispersion liquid, and 30 ℃ were stirred swelling 10 hours, logical nitrogen deoxygenation; 70 ℃ of polyreactions 24 hours are prepared into monodisperse cross-linked GMA microballoon;
Dispersion agent in the above-mentioned aqueous dispersant is sodium lauryl sulphate and Z 150PH, and the massfraction of sodium lauryl sulphate is 0.2%, and the massfraction of Z 150PH is 2%, and the polymerization degree of described Z 150PH is 1700, and alcoholysis degree is 88%;
(3) the monodisperse cross-linked GMA microballoon of extracting
The monodisperse cross-linked GMA microballoon of step (2) preparation with toluene 140 ℃ of extractings 48 hours in cable type extractor according, is obtained the sour epoxy propyl ester of monodisperse porous crosslinked methacrylic microballoon;
(4) monodisperse cross-linked GMA microsphere surface bonding ethylene base
The monodisperse porous crosslinked methacrylic acid epoxy propyl ester microballoon of step (3) preparation is added 1; In the 4-dioxane; Every 100mL 1 adds the monodisperse porous crosslinked methacrylic acid of 3～10g epoxy propyl ester microballoon, stirring at room swelling 4～12 hours in the 4-dioxane; Logical nitrogen deoxygenation; Add Rocryl 400 and 1, the mass ratio of 4-dioxane, BFEE is 1: 2～5: 4～6 mixed solution, and the mass ratio of monodisperse porous crosslinked methacrylic acid epoxy propyl ester microballoon and Rocryl 400 is 1: 0.1～2; 30～60 ℃ were stirred 8～15 hours, obtained the monodisperse cross-linked GMA microballoon of surface bond vinyl;
(5) synthetic 17 beta-estradiol molecular engram complex microspheres
With the acetonitrile is solvent; The monodisperse cross-linked GMA microballoon of ultra-sonic dispersion 17 beta estradiols, methylacrylic acid, surface bond vinyl; Stirring at room swelling 4～12 hours; Add ethylene glycol dimethacrylate, Diisopropyl azodicarboxylate, the mass ratio of the monodisperse cross-linked GMA microballoon of 17 beta estradiols and surface bond vinyl is that the mol ratio of 1: 20～120,17 beta estradiols and methylacrylic acid, ethylene glycol dimethacrylate, Diisopropyl azodicarboxylate is 1: 2～12: 8～20: 1～2; Logical nitrogen deoxygenation; 60～70 ℃ of polyreactions 16～48 hours, wash-out 17 beta estradiols are prepared into 17 beta-estradiol molecular engram complex microspheres.
2. the preparation method of 17 beta-estradiol molecular engram complex microspheres according to claim 1; It is characterized in that: in monodisperse cross-linked GMA microsphere surface bonding ethylene base step (4), the mass ratio of described monodisperse porous crosslinked methacrylic acid epoxy propyl ester microballoon and Rocryl 400 is 1: 1.
3. the preparation method of 17 beta-estradiol molecular engram complex microspheres according to claim 1; It is characterized in that: in monodisperse cross-linked GMA microsphere surface bonding ethylene base step (4); Described Rocryl 400 and 1, the mass ratio of 4-dioxane, BFEE are 1: 2.6: 5.
4. the preparation method of 17 beta-estradiol molecular engram complex microspheres according to claim 1; It is characterized in that: in monodisperse cross-linked GMA microsphere surface bonding ethylene base step (4), with the monodisperse porous crosslinked methacrylic acid epoxy propyl ester microballoon adding 1 of step (3) preparation, in the 4-dioxane; Stirring at room swelling 6 hours; Logical nitrogen deoxygenation adds 1, the mixed solution of 4-dioxane and Rocryl 400, BFEE; 45 ℃ were stirred 12 hours, obtained the monodisperse cross-linked GMA microballoon of surface bond vinyl.
5. the preparation method of 17 beta-estradiol molecular engram complex microspheres according to claim 1; It is characterized in that: in synthetic 17 beta-estradiol molecular engram complex microsphere steps (5), the mass ratio of the monodisperse cross-linked GMA microballoon of described 17 beta estradiols and surface bond vinyl is 1: 40.
6. the preparation method of 17 beta-estradiol molecular engram complex microspheres according to claim 1; It is characterized in that: in synthetic 17 beta-estradiol molecular engram complex microsphere steps (5), the mol ratio of described 17 beta estradiols and methylacrylic acid, ethylene glycol dimethacrylate, Diisopropyl azodicarboxylate is 1: 4: 10: 1.2.
7. the preparation method of 17 beta-estradiol molecular engram complex microspheres according to claim 1; It is characterized in that: in synthetic 17 beta-estradiol molecular engram complex microsphere steps (5); 17 beta estradiols, methylacrylic acid are dissolved in the acetonitrile the monodisperse cross-linked GMA microballoon of the surface bond vinyl that adding step (4) obtains, ultra-sonic dispersion; Stirring at room swelling 6 hours; Add ethylene glycol dimethacrylate, Diisopropyl azodicarboxylate, logical nitrogen deoxygenation, 70 ℃ of polyreactions 24 hours.
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