CN109946409B - Solid-phase extraction affinity column for estrogen interferent and preparation method and application thereof - Google Patents

Solid-phase extraction affinity column for estrogen interferent and preparation method and application thereof Download PDF

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CN109946409B
CN109946409B CN201910219012.6A CN201910219012A CN109946409B CN 109946409 B CN109946409 B CN 109946409B CN 201910219012 A CN201910219012 A CN 201910219012A CN 109946409 B CN109946409 B CN 109946409B
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silica gel
estrogen
phase extraction
affinity column
estrogen receptor
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CN109946409A (en
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张鸿雁
张景华
臧利国
王晓文
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Shandong Normal University
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Abstract

The invention relates to a preparation method and application of a solid-phase extraction affinity column for detecting estrogen interferent, which utilizes an estrogen receptor binding element to improve enrichment and purification efficiency, and belongs to the technical field of sample pretreatment of substance detection, and the steps are as follows: acidifying pretreatment of silica gel, reacting the pretreated silica gel with sulfonyl chloride to obtain chlorinated silica gel, reacting the chlorinated silica gel with adipic acid to obtain carboxylated silica gel, combining the carboxylated silica gel with an estrogen receptor binding element, and filling the combined product into a solid-phase extraction hollow column tube to obtain a solid-phase extraction affinity column; the estrogen receptor binding element is a DNA molecule that binds to an estrogen receptor. The natural activity of the estrogen receptor is kept by utilizing the estrogen receptor binding element, the enrichment and purification efficiency of the solid phase extraction method can be obviously improved, and the wide application of the detection method combining the estrogen receptor binding element with the high performance liquid chromatography is facilitated.

Description

Solid-phase extraction affinity column for estrogen interferent and preparation method and application thereof
Technical Field
The disclosure belongs to the technical field of sample pretreatment of substance detection, and particularly relates to a preparation method and application of an estrogen interferent solid-phase extraction affinity column for improving enrichment and purification efficiency by using an estrogen receptor binding element.
Background
The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
Estrogen disruptors are a natural or synthetic class of exogenous compounds that exist in the environment and interfere in some way with endocrine function. National standards for food safety strictly regulate the limited standards of various estrogen disruptors, and the detection of the estrogen disruptors has important significance for food safety supervision. At present, the detection methods of estrogen disruptors are mainly divided into biological detection methods and chemical detection methods. Biological detection methods are roughly classified into three categories according to the difference of tested materials: animal in vivo experiments, cell experiments and non-cell experiments are mainly used for preliminary screening.
The existing chemical detection methods mainly comprise high performance liquid chromatography, gas chromatography, liquid chromatography tandem mass spectrometry, gas chromatography tandem mass spectrometry and other instrument analysis methods. The method can accurately determine the content of the estrogen with low concentration, but because the matrix of the food sample is complex, a large amount of interferents existing in the matrix can influence the accuracy of the detection result. Therefore, in practical application, the sample pretreatment technologies, such as liquid-liquid extraction, solid-phase extraction, etc., need to be combined at the same time. The sample pretreatment technology can realize the extraction and purification of the object to be detected, and effectively remove the interference of the object not to be detected in the food sample. Particularly, the method combining solid phase extraction and liquid chromatography or liquid chromatography-mass spectrometry is widely used for detecting various substances, and the method is adopted in the national standard of food safety for determining various food hazards. At present, the commonly used solid phase extraction method generally adopts silica gel and the like as a filler, the silica gel and the like have nonspecific adsorption with the target substance to be detected, meanwhile, other interferents with similar properties can be retained to a certain extent, and the specific enrichment and purification of the target substance to be detected cannot be realized. The specific binding of the target substance to be detected can be realized by using the antibody, the molecularly imprinted polymer and the like as the recognition molecules of the solid phase extraction column packing, but the preparation principle of the recognition molecules determines that the specific recognition can only be limited to a limited number of estrogen interferents.
The estrogen interferent is mostly combined with the estrogen receptor to play an interference role in an endocrine system, so that the estrogen receptor is adopted as a recognition material of the solid-phase extraction column packing, so that the nonspecific combination with other impurities in the matrix can be effectively avoided, and the family specificity recognition of all the estrogen interferents can be realized. However, estrogen receptors are very unstable and very easy to inactivate, and when the estrogen receptors are used for preparing a solid-phase extraction column, most of activity is lost if the estrogen receptors are chemically crosslinked on a solid-phase extraction column filler, so that the estrogen receptors have poor enrichment and purification effects on estrogen interferents.
BRIEF SUMMARY OF THE PRESENT DISCLOSURE
In view of the problems in the prior art, one aspect of the present disclosure is to provide a method for preparing an estrogen receptor binding element-enhanced solid phase extraction affinity column for enriching and purifying estrogen receptor.
In order to solve the above technical problem, the technical scheme of the present disclosure is:
a preparation method of an estrogen interferent solid-phase extraction affinity column for improving enrichment and purification efficiency by utilizing an estrogen receptor binding element comprises the following steps: acidifying pretreatment of silica gel, reacting the pretreated silica gel with sulfonyl chloride to obtain chlorinated silica gel, reacting the chlorinated silica gel with adipic acid to obtain carboxylated silica gel, coupling an estrogen receptor binding element on the carboxylated silica gel, and filling the conjugated estrogen receptor binding element on the silica gel into a solid-phase extraction hollow column tube after the conjugated estrogen receptor binding element is combined with an estrogen receptor to obtain a solid-phase extraction affinity column;
the estrogen receptor binding element is a DNA molecule that binds to an estrogen receptor.
Preferably, the sequence of the DNA molecule is 5'-GTCAGGTCACAGTGACCTGATCAAAGTTAATG-3'.
According to the method, an estrogen receptor binding element is coupled on a carboxylated silica gel filler and then is specifically bound with an estrogen receptor, the whole solid-phase extraction affinity column is made, and the estrogen receptor bound on the solid-phase extraction affinity column filler can be specifically bound with an estrogen interferent, so that the estrogen interferent is effectively enriched and purified. The enrichment purification method of the solid-phase extraction affinity column is used as a sample pretreatment step and can be combined with high performance liquid chromatography to establish a sensitive and accurate estrogen interferent detection method. The present disclosure is designed based on the principle that estrogen receptor can recognize estrogen interferent, and the estrogen receptor is fixed by using an estrogen receptor binding element to maintain the natural activity of the estrogen receptor as much as possible and improve the enrichment purification efficiency of the solid phase extraction affinity column.
A preparation method of a solid-phase extraction affinity column for detecting estrogen interferent by utilizing an estrogen receptor binding element to improve enrichment and purification efficiency comprises the following specific steps:
1) mixing the chromatographic column silica gel with hydrochloric acid, heating, refluxing, filtering, washing and drying to obtain pretreated silica gel;
2) dissolving the pretreated silica gel in anhydrous pyridine, adding sulfonyl chloride for mixing, washing and drying the obtained product to obtain chlorinated silica gel;
3) mixing the chlorinated silica gel obtained in the step 2) with adipic acid, toluene and pyridine, heating for reaction, and filtering and washing the obtained product to obtain carboxylated silica gel;
4) activating the carboxylated silica gel, mixing the activated carboxylated silica gel with the estrogen receptor binding element for reaction, and washing a product by using ultrapure water and a sealant A to obtain silica gel coupled with the estrogen receptor binding element;
5) and mixing and incubating silica gel coupled with the estrogen receptor binding element and the estrogen receptor, washing the product with ultrapure water and a sealant B, washing with the ultrapure water, and filling the washed product into a solid-phase extraction hollow column tube to obtain the solid-phase extraction affinity column.
Preferably, the mass percent of the hydrochloric acid in the step 1) is 8-12%; preferably, the volume of hydrochloric acid corresponding to 1g of silica gel is 8-15 mL; preferably, the reflux temperature is 100-110 ℃; preferably, the refluxing time is 6-10 h; preferably, the drying temperature is 100-120 ℃, and the drying time is 22-26 h.
The silica gel is firstly treated by refluxing acid: the main purpose of acidification is to form free silicon hydroxyl on the surface of the silica gel so as to increase the number of the silicon hydroxyl participating in bonding reaction on the surface of the silica gel and remove metal ions on the surface of the silica gel so as to purify the silica gel. Because the boiling point of the hydrochloric acid is low and volatile, the rapid volatilization of the hydrochloric acid can be avoided by selecting condensation reflux, so that the silica gel is fully contacted with the hydrochloric acid to be fully purified. When the concentration of the hydrochloric acid is too low, metal ions on the surface of the silica gel cannot be sufficiently removed, and the concentration of the hydrochloric acid is too high, the hydrochloric acid is extremely easy to volatilize in the heating and stirring processes, so that 10% of hydrochloric acid is selected.
Preferably, the volume of the anhydrous pyridine and the sulfonyl chloride corresponding to 1g of silica gel in the step 2) is 5-7 mL and 1.5-2.5 mL; preferably, the detergent for washing the product in the step 2) is a mixture of toluene and anhydrous sodium sulfate, methanol and acetone in sequence; preferably, the drying temperature in the step 2) is 100-120 ℃, and the drying time is 22-26 h.
Function of preparing the chlorinated silica gel: the silica gel bonding stationary phase has two main ways, firstly, the silicon hydroxyl on the surface of the silica gel is converted into various active reactive groups, and then the reactive groups on the silicon hydroxyl and organic compounds are subjected to further derivatization reaction to generate a silica gel bonding phase; secondly, firstly, the organic compound is converted into a compound with a specific functional group which is easy to react with the silicon hydroxyl, and then the compound is bonded to the surface of the silica gel to obtain a silica gel bonding phase. The first method is selected by the present disclosure. Sulfonyl chloride is a common chlorinating agent in experiments, is commonly used for chlorination of aromatic compounds, carboxylic acids and other various organic and inorganic compounds, and has a chlorination effect inferior to that of sulfonyl chloride.
Preferably, the mass ratio of the chlorinated silica gel to the adipic acid and the toluene in the step 3) is 1:1: 3-6; preferably, the volume of pyridine corresponding to 1g of chlorinated silica gel is 5-7 mL; preferably, the heating reaction in the step 3) is carried out at the temperature of 100-120 ℃ for 10-14 h.
The carboxylation of the silicon hydroxyl group on the surface of the silica gel can enable the silica gel to be stably connected with an estrogen receptor binding element to be used as a substrate for fixing and binding estrogen receptors.
The effect of the pyridine addition here: pyridine is used as an acid-binding agent, is alkaline, has a catalytic effect on the silica gel bonded carboxyl, and can better promote the silica gel bonded carboxyl.
Preferably, the activating agent for activating the carboxylated silica gel in step 4) is EDC/NHS; preferably, the activation time in the step 4) is 1.5-2.5 h.
Preferably, the estrogen receptor binding element in step 4) is estrogen receptor binding DNA having the sequence 5'-GTCAGGTCACAGTGACCTGATCAAAGTTAATG-3'; preferably, the incubation time in the step 4) is 1.5-2.5 h; preferably, the sealant A in the step 4) is bovine serum albumin or ovalbumin, and further preferably, the mass percentage of the bovine serum albumin is 2-4%; preferably, 1mg of silica gel coupled with an estrogen receptor binding element corresponds to 28-32 mu L of the sealant A; preferably, the reaction time of the silica gel coupled with the estrogen receptor binding element and the sealant A is 1.5-2.5 h; preferably, the mass of the estrogen receptor binding element corresponding to 1mg of the carboxylated silica gel in the step 4) is 6-7 mu g.
The DNA molecules are combined with the estrogen receptor, the combination of the silica gel and the estrogen receptor is tighter through the DNA molecules, a stable solid phase extraction affinity column is favorably formed, a sealing agent A is used for sealing macromolecular sites of the silica gel, and the nonspecific adsorption of the silica gel to the estrogen receptor is eliminated.
Preferably, the concentration of the estrogen receptor in the step 5) is 4-6 g/L; preferably, the mass ratio of the silica gel coupled with the estrogen receptor binding element in the step 5) to the estrogen receptor is 40: 1-2; preferably, the incubation time in the step 5) is 0.5-4 h; preferably, the blocking agent B in the step 5) is triphenylmethylamine or phenylmethylamine; preferably, the concentration of the sealant B is 0.02-0.1 g/L; preferably, the volume of the sealant B corresponding to the mass of 1mg of the silica gel coupled with the estrogen receptor binding element is 28-32 muL.
The combination of estrogen concentration and estrogen receptor binding element has saturation, 1 mug of estrogen receptor binding element can be combined with 5 mug of estrogen receptor, when the concentration of estrogen receptor is too high, the estrogen receptor can not be fully combined, and when the concentration of estrogen receptor is too low, the estrogen receptor binding element can not be fully reacted.
And (3) blocking the micromolecule binding site of the silica gel by using a blocking agent B, and eliminating non-specific adsorption of other micromolecule interferents in the sample matrix so as to ensure that the estrogen receptor can be specifically bound with the estrogen interferents.
The second aspect of the present disclosure is to provide a solid phase extraction affinity column prepared by the above preparation method.
The third aspect of the present disclosure provides an application of the above estrogen interferent solid-phase extraction affinity column in detecting estrogen interferent.
Preferably, the estrogen disruptor is 17 β -estradiol (17 β -E) 2 ) Small molecule substances with estrogenic properties in bisphenol a (bpa), Diethylstilbestrol (DES) or veterinary residues.
The method for detecting the estrogen interferent by using the solid-phase extraction affinity column comprises the following specific steps:
1) adding the estrogen interferent into the solid-phase extraction affinity column, washing the solid-phase extraction affinity column with ultrapure water and an eluant in sequence, and collecting eluent;
2) detecting the eluent obtained in the step 1) by utilizing high performance liquid chromatography.
Preferably, the eluent in the step 1) is an aqueous solution of methanol, and the mass fraction of the methanol is 35-45%.
Preferably, the detection conditions of the high performance liquid chromatography in the step 2) are that the mobile phase is 40-50% acetonitrile, the column temperature is 25-35 ℃, the flow rate of the mobile phase is 0.7-1.3 mL/min, and the sample injection volume is 18-22 muL.
The beneficial effect of this disclosure:
the method utilizes an estrogen receptor binding element to maintain the biological activity of an estrogen receptor, and establishes a sensitive, simple and convenient estrogen interferent detection method according to the principle of solid-phase extraction and high performance liquid chromatography combination. The method not only can detect the known estrogen interferent in the sample, but also can enrich and purify the unknown estrogen interferent in the sample.
As for a common silica gel matrix solid phase extraction column, the purification effect is not ideal because the column can carry out nonspecific adsorption on estrogen interferents. The specific binding of the target substance to be detected can be realized by using the antibody, the molecularly imprinted polymer and the like as the recognition molecules of the solid phase extraction column packing, but the preparation principle of the recognition molecules determines that the specific recognition can only be limited to a limited number of estrogen interferents. The estrogen receptor is used as the identification material of the solid phase extraction column packing, so that non-specific combination with other impurities in the matrix can be effectively avoided, and group specific identification of all estrogen interferents can be realized. However, estrogen receptors are very unstable and very easy to inactivate, and when the estrogen receptors are used for preparing a solid-phase extraction column, most of activity is lost if the estrogen receptors are chemically crosslinked on a solid-phase extraction filler, and the enrichment and purification effects on estrogen interferents are poor.
The method comprises the steps of coupling an estrogen receptor binding element on a carboxylated silica gel filler, then specifically binding the estrogen receptor, and integrally preparing a solid-phase extraction affinity column which reacts with an estrogen interferent. The estrogen receptor generates various physiological effects in vivo and is realized by the combination of the estrogen receptor and the estrogen receptor binding element, so that the estrogen receptor binding element is used as a connecting unit between the estrogen receptor and the filler, the inactivation problem of the estrogen receptor can be effectively avoided, and the enrichment and purification effects of the solid-phase extraction affinity column are obviously improved. The kit is combined with high performance liquid for detection, a standard curve is established, and sensitive and accurate quantitative detection of estrogen interferents is realized.
Drawings
The accompanying drawings, which are included to provide a further understanding of the disclosure, illustrate embodiments of the disclosure and together with the description serve to explain the disclosure and are not to be construed as limiting the disclosure.
FIG. 1 is a schematic representation of the estrogen receptor binding element, estrogen receptor and estrogen interferent binding;
FIG. 2 is a schematic illustration of a process for solid phase extraction of estrogen interferents based on estrogen receptor binding elements;
FIG. 3 is a graph comparing the effect of BSA blocking the macromolecular sites of carboxylated silica gels;
FIG. 4 is a graph of the optimization of estrogen receptor binding element binding time to estrogen receptor;
FIG. 5 is a graph showing the effect of triphenylmethylamine blocking on samples from different solid phase extraction columns;
FIG. 6 is a graph of the effect of different concentrations of triphenylmethylamine;
FIG. 7 is a graph showing the comparative effect of adsorption of BPA of different concentrations by different solid phase extraction columns;
FIG. 8 is a graph showing the effect of different solid phase extraction columns on the adsorption of BPA;
FIG. 9 is a graph showing the effect of different solid phase extraction columns on the adsorption of DES;
FIG. 10 shows a 17 beta-E pair of different solid phase extraction columns 2 A comparative effect graph of adsorption conditions of (1);
FIG. 11 shows a 17 beta-E pair of different solid phase extraction columns 2 And the adsorption comparison effect graph of the BPA and DES mixed standard substance.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present disclosure. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
The disclosure is further illustrated with reference to the following examples
The sequences of the DNA molecules of the estrogen receptor binding elements in the following examples are:
5’-GTCAGGTCACAGTGACCTGATCAAAGTTAATG-3’。
the silica gel may be a chromatography column silica gel or a gel.
Example 1
Weighing 20g of chromatographic column silica gel into a three-neck flask, adding 200mL of 10% HCl, stirring, heating to 105 ℃, performing reflux acidification treatment for 8 hours, filtering, washing to be neutral by deionized water, placing in an oven, drying for 24 hours at 110 ℃, and placing in a dryer for storage for later use.
5g of the pretreated silica gel is taken, 30mL of anhydrous pyridine is added into a dry three-neck flask, and about 10mL of sulfonyl chloride is slowly dripped under the condition of uniform stirring. The reaction was vigorous, producing a large amount of heat, so the three-neck flask was placed in a water bath to aid in heat dissipation. After the reaction is finished, filtering the product, washing the product for a plurality of times by using toluene (anhydrous sodium sulfate treatment), methanol and acetone in sequence, putting the product into an oven, drying the product at 110 ℃, and putting the dried product into a dryer for storage for later use.
5g of chlorinated silica gel is taken and added with 5g of adipic acid, 1:5 of toluene and pyridine (acid-binding agent) (30mL), the mixture is stirred at a constant speed and at 110 ℃ for 12h, after the reaction is finished, the product is filtered, washed for a plurality of times by toluene, methanol and acetone in sequence, and then the carboxylated silica gel is obtained after drying.
10mg of carboxylated silica gel is taken, activated by EDC/NHS for 2h, combined with 66 mu g of estrogen receptor binding element (fully dissolved by 66 mu L of ultrapure water), incubated with shaking for 2h, washed by ultrapure water after the reaction is finished, and the unbound estrogen receptor binding element is washed away to obtain the silica gel coupled with the estrogen receptor binding element.
The silica gel coupled with the estrogen receptor binding element was blocked with 300. mu.L of 3% BSA (bovine serum albumin) for 2h to eliminate the non-specific adsorption of the silica gel to the estrogen receptor as much as possible. After the reaction was completed, the reaction mixture was washed three times with ultrapure water to remove unbound BSA.
And (3) shaking and incubating 50 mu L of estrogen receptor with the concentration of 5mg/mL and silica gel coupled with an estrogen receptor binding element for 2h, and measuring the binding condition of the estrogen receptor by using a Coomassie brilliant blue method. The time period 2h for the estrogen receptor binding element to bind to the estrogen receptor allows the estrogen receptor binding element to adsorb as much of the estrogen receptor as possible. After the reaction is finished, washing with ultrapure water for three times to wash away the unbound estrogen receptor.
And (2) sealing the silica gel coupled with the estrogen receptor and the estrogen receptor binding element by using 300 mu L of triphenylmethylamine with the concentration of 0.03mg/mL, and sealing unreacted carboxyl of the silica gel as much as possible so as to eliminate non-specific adsorption of the silica gel on the estrogen interferent and ensure that the estrogen interferent can be specifically bound with the estrogen receptor. After the reaction is finished, washing the reaction product for three times by using ultrapure water, and washing out the unbound triphenylmethylamine. The concentration of the triphenylmethylamine is 0.02mg/mL, so that the triphenylmethylamine can block the non-specific binding sites of the estrogen interferent as much as possible.
FIG. 2 shows the preparation process and detection process of the extraction affinity column, silica gel molecules are firstly combined with an Estrogen Receptor Element (ERE), Bovine Serum Albumin (BSA) seals the macromolecular sites of the silica gel coupled with the estrogen receptor binding element, and then is combined with an estrogen receptor to obtain the silica gel coupled with the estrogen receptor and the estrogen receptor binding element, triphenylmethylamine seals the small molecular sites of the silica gel coupled with the estrogen receptor and the estrogen receptor binding element, the obtained product is loaded into a solid-phase extraction empty column tube to obtain a solid-phase extraction affinity column, an estrogen interferent is injected into the solid-phase extraction affinity column for extraction, and the eluent is detected through washing and elution.
Test example 1
Adsorption of estrogen receptors.
Samples 1 to 4 are silica gels coupled to estrogen receptor binding elements, sample 5 is silica gel coupled to estrogen receptor binding elements with macromolecular sites blocked by BSA in example 1, samples 1 to 5 are shown in fig. 3, wherein 1 represents comparative example 1, 2 represents comparative example 2, 3 represents comparative example 3, 4 represents comparative example 4, and 5 represents example 1. As can be seen from fig. 3, sample 1 did not absorb estrogen receptor, sample 2 had the most amount of adsorption of estrogen receptor due to the silica gel not having a blocked site, sample 3 had a much reduced amount of adsorption of estrogen receptor due to the blocked macromolecular site, sample 4 had a smaller amount of adsorption of estrogen receptor than silica gel alone due to the binding of a portion of the sites to the estrogen receptor binding member, and sample 5 had a smaller amount of adsorption of estrogen receptor than the silica gel not having a coupled estrogen receptor binding member due to the blocked macromolecular sites of the silica gel coupled to the estrogen receptor binding member.
Sample 1 only estrogen receptors in the extraction affinity column.
Sample 2 silica gel was not bound to estrogen receptor elements, and was not blocked by the macromolecular sites of silica gel with BSA, and the silica gel directly adsorbed estrogen receptor to give extraction affinity columns.
Sample 3 silica gel was not bound to estrogen receptor element, and was blocked at silica gel macromolecular sites by BSA and adsorbed to estrogen receptor to obtain extraction affinity column.
Sample 4 silica gel was bound to estrogen receptor element without blocking the macromolecular sites of silica gel with BSA, and the silica gel coupled to estrogen receptor binding element was bound to estrogen receptor to obtain an extraction affinity column.
The extraction affinity columns obtained in comparative example 1, comparative example 2, comparative example 3, comparative example 4 and example 1 are schematically shown in table 1.
TABLE 1 different extraction affinity columns
Figure BDA0002002982100000071
Figure BDA0002002982100000081
Example 2
Weighing 20g of silica gel in a three-neck flask, adding 200mL of 10% HCl, stirring, heating to 110 ℃, carrying out reflux acidification treatment for 8h, filtering, washing to be neutral by deionized water, placing in an oven, drying for 24h at 110 ℃, and storing in a dryer for later use.
5g of the pretreated silica gel is taken, 30mL of anhydrous pyridine is added into a dry three-neck flask, and about 10mL of sulfonyl chloride is slowly dropped under the condition of uniform stirring. The reaction was vigorous, producing a large amount of heat, so the three-neck flask was placed in a water bath to aid in heat dissipation. After the reaction is finished, filtering the product, washing the product for a plurality of times by using toluene (anhydrous sodium sulfate treatment), methanol and acetone in sequence, putting the product into an oven, drying the product at 110 ℃, and storing the product in a dryer for later use.
Adding 5g of silica gel chloride into 25mL of adipic acid, 25mL of toluene and 30mL of pyridine (acid binding agent), uniformly stirring, stirring at 120 ℃ for 12h, filtering the product after the reaction is finished, washing the product for several times by using toluene, methanol and acetone in sequence, and drying to obtain the product.
And (3) taking 10mg of carboxylated silica gel, activating the carboxylated silica gel by EDC/NHS for 2h, then combining the activated silica gel with 66 mu g of estrogen receptor binding element, oscillating and incubating for 2h, washing the activated silica gel by ultrapure water after the reaction is finished, and washing away the unbound estrogen receptor binding element to obtain the silica gel coupled with the estrogen receptor binding element.
The silica gel coupled with the estrogen receptor binding element was blocked with 300. mu.L of 3% BSA (bovine serum albumin) for 2h to eliminate the non-specific adsorption of the silica gel to the estrogen receptor as much as possible. After the reaction was completed, the reaction mixture was washed three times with ultrapure water to remove unbound BSA.
50 mu.L of estrogen receptor with the concentration of 5mg/mL and silica gel coupled with an estrogen receptor binding element are incubated for 2h with shaking, and the binding condition of the estrogen receptor is measured by a Coomassie brilliant blue method. The time 2h for the estrogen receptor binding element to bind to the estrogen receptor allows the estrogen receptor binding element to adsorb as much estrogen receptor as possible. After the reaction is finished, washing the mixture for three times by using ultrapure water to wash away the unbound estrogen receptor.
And (2) sealing the silica gel coupled with the estrogen receptor and the estrogen receptor binding element by using 300 mu L of triphenylmethylamine with the concentration of 0.03mg/mL, and sealing unreacted carboxyl of the silica gel as much as possible so as to eliminate non-specific adsorption of the silica gel on the estrogen interferent and ensure that the estrogen interferent can be specifically bound with the estrogen receptor. After the reaction is finished, washing the reaction product for three times by using ultrapure water, and washing out the unbound triphenylmethylamine. The concentration of the triphenylmethylamine is 0.02mg/mL, so that the triphenylmethylamine can block the non-specific binding sites of the estrogen interferent as much as possible.
The process of detecting estrogen interferent by solid phase extraction affinity column in the examples is as follows:
transferring the sealed silica gel coupled with an estrogen receptor and estrogen receptor binding element into a solid-phase extraction hollow column tube, injecting standard liquid of an estrogen interferent into a solid-phase extraction column by using a constant flow pump, and binding the estrogen receptor and the estrogen interferent. Washing with ultrapure water for three times, washing away the unconjugated estrogen interferent, and eluting the estrogen interferent adsorbed by the estrogen receptor by using 40% methanol-water solution as an eluent for detection.
Detecting the estrogen interferent by using high performance liquid chromatography, wherein the mobile phase is 45% acetonitrile, the column temperature is 30 ℃, the flow rate of the mobile phase is 1mL/min, the sample injection volume is 20 mu L, and the ultraviolet detection wavelength is 280nm (adjusted according to the maximum absorption wavelength of different estrogen interferents). And establishing a standard curve, and quantitatively detecting the estrogen interferent.
Example 3
Unlike example 1, the estrogen receptor binding element was incubated for binding to estrogen receptor for 0.5 h.
Example 4
The difference from example 1 is that the estrogen receptor binding element is incubated for binding to the estrogen receptor for 1 h.
Example 5
In contrast to example 1, the estrogen receptor binding element was incubated for binding to estrogen receptor for a period of 3 h.
Example 6
Unlike example 1, the estrogen receptor binding element binds to the estrogen receptor for a period of 4 hours.
The solid phase extraction columns obtained in example 1, example 3, example 4, example 5 and example 6 extract the estrogen disrupter, and the extraction process is as described above, and the result is shown in fig. 4, and it can be seen from fig. 4 that the binding capacity of the estrogen receptor and the estrogen receptor binding element is not changed after incubation for 2h, which indicates that the two elements are fully bound and the estrogen receptor is easily inactivated in vitro for a longer time.
Example 7
The difference from example 1 is that the concentration of triphenylmethylamine is 0.04 mg/mL.
Example 8
Except for example 1 that the concentration of triphenylmethylamine was 0.06 mg/mL.
Example 9
The difference from example 1 is that the concentration of triphenylmethylamine is 0.08 mg/mL.
Example 10
Except for example 1 that the concentration of triphenylmethylamine was 0.1 mg/mL.
Test example 2
Influence of the blocking agent B (triphenylmethylamine) on the coupling estrogen receptor binding element and the estrogen receptor silica gel on the adsorption amount of the estrogen interferent.
The sample 1 is the coupling estrogen receptor binding element and the estrogen receptor silica gel of the small molecular site blocked by the triphenylmethylamine in the example 1, the difference between the sample 2 and the sample 1 is that the sample is not the coupling estrogen receptor binding element and the estrogen receptor silica gel of the small molecular site blocked by the triphenylmethylamine, the sample 1 and the sample 2 respectively extract the bisphenol a, the extraction process is as shown above, the obtained result is as shown in fig. 5, the figure is a comparison graph of the blocking effect of the triphenylmethylamine, 1 is the adsorption amount of the silica gel to the bisphenol a when the triphenylmethylamine is not blocked, 2 is the adsorption amount of the silica gel to the bisphenol a after the triphenylmethylamine is blocked, and the adsorption amount of the bisphenol a in the sample 2 is obviously reduced, which indicates that the blocking effect of the triphenylmethylamine is obvious, and the non-specific adsorption of the silica gel to the bisphenol a can be reduced.
The extraction effects obtained in examples 1, 7, 8, 9, and 10 are shown in fig. 6, and it can be seen from fig. 6 that the effect of the concentration of tritylamine on the blocking result is small, the effect of adsorbing estrogen-interfering substances is the best when the concentration of tritylamine is 20mg/mL, and the blocking effect of tritylamine is stable.
Test example 3
The adsorption effect of silica gel without estrogen receptor binding versus estrogen receptor binding elements coupled to the estrogen receptor on bisphenol a was compared.
Sample 1 is the solid phase extraction affinity column prepared in example 1, sample 2 is different from sample 1 in that the sample is silica gel of a coupled estrogen receptor binding element which is not bound to an estrogen receptor, the preparation process of sample 2 is the process of removing the binding with the estrogen receptor in the preparation process of example 1, wherein the process comprises the process of blocking a small molecule site by tritylamine, and sample 1 and sample 2 are respectively subjected to the same extraction process of an estrogen interferent, and the obtained result is shown in fig. 7, wherein a is sample 2, b is sample 1, and the abscissa in fig. 7 is the concentration of bisphenol a, and the adsorption effect of BPA on BPA by the silica gel of the coupled estrogen receptor and the estrogen receptor binding element is much higher than that of the silica gel of the coupled estrogen receptor binding element which is not bound to the estrogen receptor.
Test example 4
The comparison of bisphenol A adsorption in different solid-phase extraction column preparation processes is shown above, and the process of detecting estrogen interferent by using the solid-phase extraction affinity column is shown above, wherein the ultraviolet wavelength of the high performance liquid chromatography is 278 nm.
Sample 3 is the solid phase extraction affinity column obtained in example 1, sample 1 is different from sample 3 in that silica gel is not coupled with an estrogen receptor binding element and does not adsorb an estrogen receptor during the preparation process, then silica gel passes through a solid phase extraction affinity column obtained by BSA, triphenylmethylamine blocking large molecules and small molecule sites, sample 2 is different from sample 3 in that silica gel is not coupled with an estrogen receptor binding element during the preparation process, then silica gel passes through a solid phase extraction affinity column obtained by BSA, triphenylmethylamine blocking large molecules and small molecule sites, sample 4 is different from sample 3 in that silica gel is coupled with an estrogen receptor binding element during the preparation process, silica gel passes through a BSA blocking large molecule site and does not adsorb an estrogen receptor, and triphenylmethylamine blocks small molecule sites to obtain a solid phase extraction affinity column, the extraction conditions of the estrogen interferent bisphenol A by sample 1, sample 2, sample 3 and sample 4 are shown in FIG. 8, where 1 represents sample 1, 2 represents sample 2, 3 represents sample 3, and 4 represents sample 4. As can be seen from fig. 8, after the macromolecular sites and the small molecular sites of the silica gel are blocked, only the silica gel that binds to the estrogen receptor has a good effect of adsorbing the estrogen interferent, but if the silica gel does not bind to the estrogen receptor binding element first, the binding strength between the silica gel and the estrogen receptor is not strong, and the effect of adsorbing the obtained BPA is not good.
Test example 5
The comparison of the preparation processes of different solid-phase extraction columns on diethylstilbestrol adsorption is shown above, the process of detecting estrogen interferent by the solid-phase extraction affinity column is carried out, and the ultraviolet wavelength of the corresponding high performance liquid chromatography is 254 nm.
Sample 3 is the solid phase extraction affinity column obtained in example 1, sample 1 is different from sample 3 in that silica gel is not coupled to an estrogen receptor binding member and does not adsorb an estrogen receptor during the preparation process, then silica gel is passed through a solid phase extraction affinity column obtained by BSA, tritylamine blocking large molecule and small molecule sites, sample 2 is different from sample 3 in that silica gel is not coupled to an estrogen receptor binding member during the preparation process, then silica gel is passed through a solid phase extraction affinity column obtained by BSA, tritylamine blocking large molecule and small molecule sites, sample 4 is different from sample 3 in that silica gel is coupled to an estrogen receptor binding member during the preparation process, silica gel is passed through BSA blocking large molecule sites and does not adsorb an estrogen receptor, and tritylamine blocks small molecule sites, the extraction of sample 1, sample 2, sample 3, and sample 4 to an estrogen interference substance, diethylstilbestrol is shown in fig. 9, where 1 denotes sample 1, 2 denotes sample 2, 3 denotes sample 3, and 4 denotes sample 4. As can be seen from fig. 9, after the macromolecular sites and the small molecular sites of the silica gel are blocked, only the silica gel that binds to the estrogen receptor has a good effect of adsorbing the estrogen interferent, but if the silica gel is not bound to the estrogen receptor binding element first, the binding strength between the silica gel and the estrogen receptor is not strong, and the effect of adsorbing the obtained DES is not good. The negative adsorption amount is due to some error in the experiment process, and the adsorption amount is almost 0.
Test example 6
The comparison of the preparation processes of different solid-phase extraction columns on 17 beta-estradiol adsorption is shown in the specification, the process of detecting estrogen interferents by using the solid-phase extraction affinity column is shown in the specification, and the ultraviolet wavelength of the corresponding high performance liquid chromatography is 280 nm.
Sample 3 is the solid phase extraction affinity column obtained in example 1, and sample 1 is different from sample 3 in that silica gel is not coupled to an estrogen receptor binding element and does not adsorb estrogen receptor during the preparation process, then silica gel passes through a solid phase extraction affinity column obtained by BSA and triphenylmethylamine blocking large molecular and small molecular sites, the difference between the sample 2 and the sample 3 is that the silica gel is not coupled with an estrogen receptor binding element in the preparation process, and then silica gel passes through a solid phase extraction affinity column obtained by blocking large molecular and small molecular sites with BSA and tritylamine, a sample 4 is a solid phase extraction affinity column which is different from the sample 3 in that the silica gel is coupled with an estrogen receptor binding element in the preparation process, the silica gel passes through the BSA to block the large molecular sites and does not adsorb estrogen receptors, and the tritylamine blocks the small molecular sites, and the sample 1, the sample 2, the sample 3 and the sample 4 are 17 beta-E of estrogen interferents. 2 The extraction is shown in FIG. 10, where 1 denotes sample 1, 2 denotes sample 2, 3 denotes sample 3, and 4 denotes sample 4. As can be seen from fig. 10, after the macromolecular sites and the small molecular sites of the silica gel are blocked, only the silica gel which binds to the estrogen receptor has a good adsorption effect on the estrogen interferent, but if the silica gel is not bound to the estrogen receptor binding element first, the binding strength between the silica gel and the estrogen receptor is not strong, and the 17 β -E obtained by adsorption is not strong 2 The effect of (2) is not good. The negative adsorption was due to the experimental processThere was a certain error, and the amount of adsorption was almost 0.
Test example 7
The comparison of the mixed adsorption of 17 beta-estradiol, bisphenol A and diethylstilbestrol in the preparation process of different solid-phase extraction columns, the process of detecting estrogen interferents by using a solid-phase extraction affinity column is shown as above, and the ultraviolet wavelength of the corresponding high performance liquid chromatography is (280, 278 and 254) nm. The liquid phase can be set to four different wavelengths simultaneously.
Sample 3 is the solid phase extraction affinity column obtained in example 1, sample 1 is different from sample 3 in that silica gel is not coupled with an estrogen receptor binding element and does not adsorb an estrogen receptor during the preparation process, then silica gel passes through a solid phase extraction affinity column obtained by BSA, triphenylmethylamine blocking large molecules and small molecule sites, sample 2 is different from sample 3 in that silica gel is not coupled with an estrogen receptor binding element during the preparation process, then silica gel passes through a solid phase extraction affinity column obtained by BSA, triphenylmethylamine blocking large molecules and small molecule sites, sample 4 is different from sample 3 in that silica gel is coupled with an estrogen receptor binding element during the preparation process, silica gel passes through a BSA blocking large molecule site and does not adsorb an estrogen receptor, and triphenylmethylamine blocks small molecule sites to obtain a solid phase extraction affinity column, the extraction conditions of sample 1, sample 2, sample 3 and sample 4 on the mixed estrogen interferent are shown in FIG. 11, wherein 1 represents sample 1, 2 represents sample 2, 3 represents sample 3, 4 represents sample 4, a is BPA and b is 17. beta. -E 2 And c is DES. As can be seen from fig. 11, after the macromolecular sites and the small molecular sites of the silica gel are blocked, only the silica gel that binds to the estrogen receptor has a good effect of adsorbing the estrogen interferent, but if the silica gel does not bind to the estrogen receptor binding element first, the binding strength between the silica gel and the estrogen receptor is not strong, and the effect of adsorbing the obtained mixed estrogen interferent is not good. And from this figure it can be seen that the estrogen receptor has an ability to adsorb estrogen interferents with DES > 17 β -E 2 BPA, which is consistent with the affinity of estrogen receptors for the three. The negative adsorption amount is due to some error in the experiment process, and the adsorption amount is almost 0.
As can be seen from the accompanying figure 11, the solid-phase extraction column based on the estrogen receptor binding element disclosed by the invention has a good detection effect on estrogen interferents, namely 17 beta-estradiol, bisphenol A and diethylstilbestrol.
The above description is only a preferred embodiment of the present disclosure and is not intended to limit the present disclosure, and various modifications and changes may be made to the present disclosure by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present disclosure should be included in the protection scope of the present disclosure.
SEQUENCE LISTING
<110> university of eastern Shandong teacher
<120> estrogen interferent solid-phase extraction affinity column, preparation method and application thereof
<130>
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 32
<212> DNA
<213> Artificial sequence
<400> 1
gtcaggtcac agtgacctga tcaaagttaa tg 32

Claims (28)

1. The method for detecting the estrogen interferent by utilizing the solid-phase extraction affinity column is characterized by comprising the following steps:
the method comprises the following specific steps: injecting the estrogen interferent into the solid phase extraction affinity column, washing the solid phase extraction affinity column by ultrapure water and eluent in sequence, and collecting eluent; detecting the obtained eluent by using high performance liquid chromatography; the eluent is a water solution of methanol, and the mass fraction of the methanol is 35-45%;
the preparation method of the solid phase extraction affinity column comprises the following steps: acidifying pretreatment of silica gel, reacting the pretreated silica gel with sulfonyl chloride to obtain chlorinated silica gel, reacting the chlorinated silica gel with adipic acid to obtain carboxylated silica gel, combining the carboxylated silica gel with an estrogen receptor binding element, and filling the combined element into a solid-phase extraction hollow column tube to obtain a solid-phase extraction affinity column;
the estrogen receptor binding element is a DNA molecule which is combined with an estrogen receptor;
the sequence of the DNA molecule is: 5'-GTCAGGTCACAGTGACCTGATCAAAGTTAATG-3';
the preparation method of the solid-phase extraction affinity column comprises the following specific steps:
1) mixing silica gel with hydrochloric acid, heating and refluxing, filtering, washing and drying to obtain pretreated silica gel;
2) dissolving the pretreated silica gel in anhydrous pyridine, adding sulfonyl chloride, mixing, washing and drying the obtained product to obtain chlorinated silica gel;
3) mixing the chlorinated silica gel obtained in the step 2) with adipic acid, toluene and pyridine, heating for reaction, and filtering and washing the obtained product to obtain carboxylated silica gel;
4) activating the carboxylated silica gel, mixing the activated carboxylated silica gel with the estrogen receptor binding element for reaction, and washing a product with ultrapure water and a sealant A to obtain a carboxylated silica gel-coupled estrogen receptor binding element;
5) mixing and incubating the carboxylated silica gel coupled estrogen receptor binding element and an estrogen receptor, washing a product by using ultrapure water and a sealant B, washing the product by using the ultrapure water, and filling the washed product into a solid-phase extraction hollow column tube to obtain the solid-phase extraction affinity column.
2. The method for detecting estrogen disruptors using a solid phase extraction affinity column of claim 1, wherein: the mass percentage of the hydrochloric acid in the step 1) is 8-12%.
3. The method for detecting estrogen disruptors using a solid phase extraction affinity column according to claim 1, wherein: the volume of hydrochloric acid corresponding to 1g of silica gel is 8-15 mL.
4. The method for detecting estrogen disruptors using a solid phase extraction affinity column according to claim 1, wherein: the reflux temperature is 100-110 ℃.
5. The method for detecting estrogen disruptors using a solid phase extraction affinity column according to claim 1, wherein: the refluxing time is 6-10 h.
6. The method for detecting estrogen disruptors using a solid phase extraction affinity column according to claim 1, wherein: the drying temperature is 100-120 ℃, and the drying time is 22-26 h.
7. The method for detecting estrogen disruptors using a solid phase extraction affinity column of claim 1, wherein: in the step 2), the volume of the anhydrous pyridine and the volume of the sulfonyl chloride corresponding to 1g of silica gel are 5-7 mL and 1.5-2.5 mL.
8. The method for detecting estrogen disruptors using a solid phase extraction affinity column of claim 1, wherein: the detergent for washing the product in the step 2) is a mixture of toluene and anhydrous sodium sulfate, methanol and acetone in turn.
9. The method for detecting estrogen disruptors using a solid phase extraction affinity column according to claim 1, wherein: the drying temperature in the step 2) is 100-120 ℃, and the drying time is 22-26 h.
10. The method for detecting estrogen disruptors using a solid phase extraction affinity column of claim 1, wherein: in the step 3), the mass ratio of the chlorinated silica gel to the adipic acid, the toluene and the pyridine is 1:1:5: 6.
11. The method for detecting estrogen disruptors using a solid phase extraction affinity column of claim 1, wherein: the heating reaction in the step 3) is carried out at the temperature of 100-120 ℃ for 10-14 h.
12. The method for detecting estrogen disruptors using a solid phase extraction affinity column according to claim 1, wherein: the activating agent for activating the carboxylated silica gel in the step 4) is EDC/NHS.
13. The method for detecting estrogen disruptors using a solid phase extraction affinity column according to claim 1, wherein: the activation time in the step 4) is 1.5-2.5 h.
14. The method for detecting estrogen disruptors using a solid phase extraction affinity column of claim 1, wherein: the mass of the estrogen receptor binding element corresponding to 1mg of the carboxylated silica gel is 6-7 mug.
15. The method for detecting estrogen disruptors using a solid phase extraction affinity column according to claim 1, wherein: the incubation time in the step 4) is 1.5-2.5 h.
16. The method for detecting estrogen disruptors using a solid phase extraction affinity column of claim 1, wherein: in the step 4), the sealant A is beef serum protein or ovalbumin.
17. The method for detecting estrogen disruptors using a solid phase extraction affinity column of claim 16, wherein: the mass percentage of the beef serum protein is 2-4%.
18. The method for detecting estrogen disruptors using a solid phase extraction affinity column of claim 1, wherein: the volume of the 1mg carboxylated silica gel coupled estrogen receptor binding element corresponding to the sealant A is 28-32 mu L.
19. The method for detecting estrogen disruptors using a solid phase extraction affinity column of claim 1, wherein: the reaction time of the silica gel coupled with the estrogen receptor binding element and the sealant A is 1.5-2.5 h.
20. The method for detecting estrogen disruptors using a solid phase extraction affinity column of claim 1, wherein: in the step 5), the concentration of the estrogen receptor is 4-6 mg/mL.
21. The method for detecting estrogen disruptors using a solid phase extraction affinity column of claim 1, wherein: the incubation time in the step 5) is 0.5-4 h.
22. The method for detecting estrogen disruptors using a solid phase extraction affinity column according to claim 1, wherein: in the step 5), the mass ratio of the carboxylated silica gel coupled estrogen receptor binding element to the estrogen receptor is 40: 1-2.
23. The method for detecting estrogen disruptors using a solid phase extraction affinity column according to claim 1, wherein: in the step 5), the sealant B is triphenylmethylamine.
24. The method for detecting estrogen disruptors using a solid phase extraction affinity column of claim 1, wherein: the concentration of the sealant B is 0.02-0.1 mg/mL.
25. The method for detecting estrogen disruptors using a solid phase extraction affinity column of claim 1, wherein: the volume of the sealant B corresponding to the mass of the 1mg carboxylated silica gel coupled estrogen receptor binding element is 28-32 mu L.
26. The method for detecting estrogen disruptors using a solid phase extraction affinity column according to claim 1, wherein: the estrogen interferent is estradiol, bisphenol A or diethylstilbestrol.
27. The method for detecting estrogen disruptors using a solid phase extraction affinity column of claim 1, wherein: the estrogen interferent is a small molecule substance with estrogenic properties in the residue of the veterinary drug.
28. The method for detecting estrogen disruptors using a solid phase extraction affinity column of claim 1, wherein: the detection conditions of the high performance liquid chromatography are that a mobile phase is 40-50% acetonitrile, the column temperature is 25-35 ℃, the flow rate of the mobile phase is 0.7-1.3 mL/min, and the sample injection volume is 18-22 mu L.
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