CN111116823B - Preparation method and application of material for screening thyroxin interferon - Google Patents
Preparation method and application of material for screening thyroxin interferon Download PDFInfo
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- CN111116823B CN111116823B CN201811282189.2A CN201811282189A CN111116823B CN 111116823 B CN111116823 B CN 111116823B CN 201811282189 A CN201811282189 A CN 201811282189A CN 111116823 B CN111116823 B CN 111116823B
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F222/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by a carboxyl radical and containing at least one other carboxyl radical in the molecule; Salts, anhydrides, esters, amides, imides, or nitriles thereof
- C08F222/36—Amides or imides
- C08F222/38—Amides
- C08F222/385—Monomers containing two or more (meth)acrylamide groups, e.g. N,N'-methylenebisacrylamide
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
- B01D15/20—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to the conditioning of the sorbent material
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F2/00—Processes of polymerisation
- C08F2/44—Polymerisation in the presence of compounding ingredients, e.g. plasticisers, dyestuffs, fillers
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
- C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
- C08F220/52—Amides or imides
- C08F220/54—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide
- C08F220/60—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide containing nitrogen in addition to the carbonamido nitrogen
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J9/00—Working-up of macromolecular substances to porous or cellular articles or materials; After-treatment thereof
- C08J9/28—Working-up of macromolecular substances to porous or cellular articles or materials; After-treatment thereof by elimination of a liquid phase from a macromolecular composition or article, e.g. drying of coagulum
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J2335/00—Characterised by the use of homopolymers or copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by a carboxyl radical, and containing at least one other carboxyl radical in the molecule, or of salts, anhydrides, esters, amides, imides or nitriles thereof; Derivatives of such polymers
Abstract
The invention discloses a preparation method and application of a material for screening thyroxine interferon, which comprises the preparation of a thyroxine imprinted material and the screening of thyroxine interferon. The prepared thyroxine imprinted material can specifically adsorb thyroxine interferon, and shows different retention times on a chromatographic column, thereby achieving the purpose of separating and screening the environment thyroxine interferon. Compared with the traditional screening method of the thyroxine interferon, the method does not need to use a biological effect original piece, can realize effect identification and component separation in one step through chromatographic evaluation, has the advantages of rapidness, simplicity, convenience, low cost and the like, and is suitable for screening the environment thyroxine interferon.
Description
Technical Field
The invention relates to the field of new materials, in particular to a preparation method and application of a material for screening thyroxine interferon.
Background
Thyroxine disruptors are a class of environmental endocrine disruptors that affect the synthesis, secretion, transport and metabolism of the body's thyroxine, and have become another class of important environmental endocrine disruptors following environmental estrogens. It has been shown that transthyretin interferes with the levels of thyroxine in the body, causing hormonal disturbances and affecting the function of various organs in the body (Miller M D, et al. environmental Health Perspectives,2009,117(7): 1033-.
The current screening methods of the thyroxine interferon mainly comprise thyroxine transporter competitive binding experiments, thyroxine receptor competitive binding experiments and the like (Ren X M, et al. Toxicology,2016, 366-. And when the thyroxine interferon in an actual environment sample is screened, in order to obtain a relatively single component, a plurality of times of cyclic processes of chromatographic separation and effect screening are often required, the process is complicated, and the efficiency is low. Therefore, if the evaluation of the thyroxine interference effect and the chromatographic separation can be combined together, the screening steps of the thyroxine interference can be greatly simplified, and the time and the money cost can be saved. Affinity chromatography can simultaneously realize the identification and separation of specific targets, but affinity chromatography columns prepared by protein, receptor and other equivalent elements have poor stability and are difficult to tolerate organic solvents, so the affinity chromatography columns are limited in the identification and separation application of pollutants.
Most of the thyroxine interferors are capable of producing a thyroxine interference effect because they have a structure similar to that of thyroxine, and thus the development of artificial receptors that specifically recognize thyroxine can be used to identify the thyroxine interferors. The molecular imprinting technology is a technology for forming a specific recognition site by memorizing a binding site and a spatial structure of a target molecule, has the characteristics of an artificial antibody, has good stability, can tolerate an organic solvent, and has high mechanical strength. Therefore, the thyroxine imprinted material can specifically recognize thyroxine interferents; further, the thyroxine imprinted material is filled in a chromatographic column, so that separation and screening of thyroxine interferents can be realized.
Disclosure of Invention
The invention aims to provide a preparation method and application of a material for screening thyroxine interferon by using a bionic recognition means.
The purpose of the invention is realized by the following technical scheme:
a preparation method and application of a material for screening thyroxine interferon comprise the preparation of a thyroxine imprinted material and the screening of thyroxine interferon. The method comprises the following specific steps:
1. preparation of thyroxine imprinted material
Dissolving a certain mass of thyroxine in dimethyl sulfoxide, adding a functional monomer and a cross-linking agent, adding particles modified by a metal catalyst, a ligand and an initiator, and stirring at the temperature of 60-80 ℃ for 12-24 hours; oscillating the polymerized particles in the eluent for 1-2 hours, and repeating for 6-8 times;
preferably, the concentration of the thyroxine is 5-10% of the dimethyl sulfoxide in terms of mass (g)/volume (ml) percentage concentration (W/V).
Preferably, the molar ratio of the thyroxine to the functional monomer is 1: 1.8-1: 4.2, and the functional monomer is one of 4-vinylpyridine, dimethylaminopropyl methacrylamide and 2- (3-indole) ethyl methacrylamide.
Preferably, the molar ratio of the functional monomer to the cross-linking agent is 1: 5-1: 10, and the cross-linking agent is one of ethylene glycol dimethacrylate, trimethylpropane trimethacrylate, divinylbenzene and methylene bisacrylamide.
Preferably, the metal catalyst is cuprous bromide or cuprous chloride, the ligand is 2, 2-bipyridyl, pentamethyldiethylenetriamine, tris [2- (methylamino) ethyl ] amine or tris (2-pyridylmethyl) amine, and the molar ratio of the metal catalyst to the ligand is 1: 1-1: 3.
Preferably, the particles modified by the initiator are 2-bromoisobutyryl or 2-bromopropionyl modified silica or polystyrene particles, and the concentration of the particles is 2-5% by mass (g)/volume (ml) percentage concentration (W/V).
Preferably, the eluent is acetic acid or methanol or ethanol solution of ammonia water, and the concentration of the eluent is 5-10% in terms of mass (g)/volume (ml) percentage concentration (W/V).
2. Screening for thyroxine interferons
The thyroxine imprinted material prepared by the method provided by the invention is filled in a chromatographic column, and the size of the chromatographic column is 4.6 x 100-200 mm. The mobile phase is 0.02-0.05% acetic acid methanol solution, the flow rate is 0.5-1 mL/min, the column temperature is 25-35 ℃, and the detection wavelength is 230-290 nm. The contaminants evaluated were phthalates, UV absorbers, parabens, halogenated phenols, bisphenols, halogenated bisphenols, and the like.
The invention has the following outstanding advantages:
the thyroxine imprinted material prepared by the molecular imprinting technology has the specific recognition capability on thyroxine interferents, and has the advantages of low cost, high stability and organic solvent tolerance compared with conventional biological effect elements such as thyroxine transporters, thyroxine binding globulin and the like. In addition, the identification and separation of the thyroxine interferon can be realized simultaneously by a chromatographic separation mode, and the screening step of the thyroxine interferon in an actual sample is greatly simplified.
Drawings
FIG. 1 is an electron micrograph of a thyroxine imprinted material.
FIG. 2 shows adsorption capacity and imprinted factors of thyroxine imprinted material on thyroxine and its structural analogs.
Figure 3 is a spectrum of various contaminants on a thyroxine blotting column.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
Example 1 preparation of thyroxine imprinted material
(1) Synthesis of functional monomer 2- (3-indole) ethyl methacrylamide
2g tryptamine was added to 50mL tetrahydrofuran and mixed by sonication, and 3.46mL triethylamine was added. 2.42mL of methacryloyl chloride was added dropwise with stirring and stirred at room temperature for 6 h. After centrifugation, the supernatant yellow liquid was distilled under reduced pressure to 2mL, purified by silica gel column chromatography (dichloromethane: methanol 99:1, Rf 0.25), and evaporated to dryness to obtain a yellow oily liquid.
(2) Preparation of thyroxine imprinted silica gel
Weighing 570mg of 2- (3-indole) ethyl methacrylamide and 973mg of thyroxine, dissolving in 125mL of dimethyl sulfoxide, performing ultrasonic treatment for 10min, adding 3850mg of methylene bisacrylamide, and uniformly stirring; adding 70mg of cuprous bromide and 75mg of 2, 2-bipyridine, and uniformly stirring; adding 2.5g of 2-bromoisobutyryl modified silicon dioxide, introducing nitrogen for 5min, and stirring at 70 ℃ for reacting for 18 h.
The preparation process of the non-thyroxine imprinted silica gel is the same as that described above, except that thyroxine is not added in the preparation process, and a thyroxine specific recognition site cannot be formed and is used for comparison.
FIG. 1 is a scanning electron microscope image of thyroxine-imprinted silica gel. Compared with unmodified silica gel, a plurality of protrusions are formed on the surface of the thyroxine-imprinted silica gel, which indicates that the molecularly imprinted polymer is successfully prepared on the surface of the silica gel.
Example 2 evaluation of adsorption Performance and Selectivity Performance of thyroxine imprinted Material
And (2) preparing methanol solutions of the thyroxine and the structural analogs thereof with the concentration of 100 mu g/mL respectively, weighing 10mg of the thyroxine imprinted silica gel and the non-thyroxine imprinted silica gel prepared in the example 1, adding the weighed thyroxine imprinted silica gel and the non-thyroxine imprinted silica gel into 1mL of the above solutions respectively, shaking the solutions at room temperature for 6h, measuring the concentration of a supernatant, and calculating the adsorption amount of the thyroxine imprinted material to the thyroxine and the structural analogs thereof. As shown in figure 2, the adsorption amount of the thyroxine imprinted material to thyroxine is 5.4mg/g, and the imprinting factor is 2.0, which are all higher than that of the structural analogues, thus indicating that the thyroxine imprinted material has specific recognition performance to thyroxine.
Example 3 screening for thyroxin interferons
The thyroxine imprinted silica gel particles were packed in a 4.6 x 100mm stainless steel chromatography column to prepare a thyroxine imprinted column. Preparing a pollutant mixed standard solution with the concentration of 1 mu g/mL, and analyzing the peak-off time of different pollutants by using 0.05% methanol as a mobile phase. As shown in figure 3, the peak time of the thyroxine interferon such as halogenated phenol, tetrabromobisphenol A and the like is higher than that of other pollutants, and the thyroxine imprinted column is proved to have the capability of screening the thyroxine interferon.
It will be understood by those skilled in the art that the foregoing is only a preferred embodiment of the present invention, and is not intended to limit the invention, and that any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (6)
1. A chromatographic column for screening a thyroxine interferon is characterized by being filled with a thyroxine interferon screening material prepared by the following method, and comprising the following steps:
dissolving thyroxine in dimethyl sulfoxide, adding a functional monomer and a cross-linking agent, adding a metal catalyst, a ligand and particles modified by an initiator, wherein the particles modified by the initiator are 2-bromoisobutyryl modified silicon dioxide particles, and stirring at the temperature of 60-80 ℃ for 12-24 hours; and oscillating the polymerized particles in the eluent for 1-2 hours, and repeating for 6-8 times.
2. The chromatographic column for screening the thyroxine interferon according to claim 1, wherein the mole ratio of the thyroxine to the functional monomer is 1:1.8 to 1:4.2, and the mole ratio of the functional monomer to the cross-linking agent is 1:5 to 1: 10.
3. The chromatography column for screening for a thyroxin interferon according to claim 1 or 2, wherein: the functional monomer is one of 4-vinylpyridine, dimethylaminopropyl methacrylamide and 2- (3-indole) ethyl methacrylamide; the cross-linking agent is one of ethylene glycol dimethacrylate, trimethylpropane trimethacrylate, divinylbenzene and methylene bisacrylamide.
4. The chromatographic column for screening the thyroxine interferon according to claim 1, wherein the metal catalyst is cuprous bromide or cuprous chloride, and the ligand is 2, 2-bipyridyl, pentamethyldiethylenetriamine, tris [2- (methylamino) ethyl ] amine or tris (2-pyridylmethyl) amine, in a molar ratio of 1:1 to 1: 3.
5. The chromatographic column for screening the thyroxine interferon according to claim 1, wherein the eluent is a methanol or ethanol solution of acetic acid or ammonia, and the concentration of the eluent is 5-10% by mass/volume percentage (W/V).
6. The method for screening the thyroxine interferon is characterized in that the chromatographic column of claim 1 is adopted, a methanol solution with 0.02-0.05% of acetic acid in volume percentage is used as a mobile phase, and screening is realized by a chromatographic separation method.
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