阅读说明：本技术 一种黄曲霉毒素分子印迹聚合物及其制备方法和应用 (Aflatoxin molecularly imprinted polymer and preparation method and application thereof ) 是由 潘浣钰 黄寿辉 闵曼 孙蕙 何建雄 李丰 郝燕娟 陈晓强 赖飞龙 毓志超 于 2021-07-14 设计创作，主要内容包括：一种黄曲霉毒素分子印迹聚合物及其制备方法和应用。本发明公开了一种黄曲霉毒素分子印迹聚合物的制备方法,包括以下：步骤1,将苏木精作为模板分子与功能单体、乙腈混合,超声处理,加入交联剂和引发剂,得到第一混合物,模板分子：功能单体：交联剂的摩尔质量为1:6～8:30,对第一混合物进行氮气吹扫,进行聚合反应,离心分离得到产物；步骤2,用甲醇洗涤产物,除去未反应的功能单体,用洗涤剂除去苏木精,得到黄曲霉毒素分子印迹聚合物。本发明的制备方法得到的分子印迹聚合物具有易制备、性质稳定、成本低、效率高和对黄曲霉毒素特异性识别能力的优点,有利于推广应用于对中药、食品中黄曲霉毒素含量的检测,提高对中药、食品的食用安全保障。(An aflatoxin molecularly imprinted polymer and a preparation method and application thereof. The invention discloses a preparation method of an aflatoxin molecularly imprinted polymer, which comprises the following steps: step 1, taking hematoxylin as a template molecule, mixing the hematoxylin with a functional monomer and acetonitrile, carrying out ultrasonic treatment, and adding a cross-linking agent and an initiator to obtain a first mixture, wherein the template molecule: functional monomer: the molar mass of the cross-linking agent is 1: 6-8: 30, nitrogen purging is carried out on the first mixture, polymerization reaction is carried out, and centrifugal separation is carried out to obtain a product; and 2, washing the product with methanol to remove unreacted functional monomers, and removing hematoxylin with a detergent to obtain the aflatoxin molecularly imprinted polymer. The molecularly imprinted polymer obtained by the preparation method has the advantages of easiness in preparation, stable property, low cost, high efficiency and capability of specifically identifying aflatoxin, is favorable for popularization and application in detection of aflatoxin content in traditional Chinese medicines and foods, and improves edible safety guarantee of the traditional Chinese medicines and the foods.)

1. The preparation method of the aflatoxin molecularly imprinted polymer is characterized by comprising the following steps of:

step 1, mixing hematoxylin serving as a template molecule with a functional monomer and acetonitrile, carrying out ultrasonic treatment, and adding a cross-linking agent and an initiator to obtain a first mixture, wherein the template molecule: functional monomer: the molar mass of the cross-linking agent is 1: 6-8: 30, nitrogen purging is carried out on the first mixture, polymerization reaction is carried out at a certain temperature, and after the reaction is finished, centrifugal separation is carried out to obtain a product;

and 2, washing the product with methanol to remove unreacted functional monomers, and then removing hematoxylin with a detergent to obtain the aflatoxin molecularly imprinted polymer, wherein the detergent is a mixed solution of methanol and acetic acid.

2. The preparation method of the aflatoxin molecularly imprinted polymer according to claim 1, which comprises the following steps:

step 1, mixing hematoxylin serving as a template molecule with a functional monomer and 30-50ml of acetonitrile, carrying out ultrasonic treatment for 10-20min, and adding a cross-linking agent and an initiator to obtain a first mixture, wherein the template molecule: functional monomer: the molar ratio of the cross-linking agent is 1:8:30, nitrogen purging is carried out on the first mixture for 10-20min, polymerization reaction is carried out for 24 hours at a certain temperature, after the reaction is finished, centrifugal separation is carried out to obtain a product, the functional monomer is one of methacrylic acid or acrylamide, and the initiator is one of azobisisobutyronitrile or azobisisovaleronitrile;

and 2, washing the product with 20-40ml of methanol to remove unreacted functional monomers, and then removing hematoxylin with a detergent to obtain the aflatoxin molecularly imprinted polymer, wherein the detergent is a mixed solution of methanol and acetic acid.

3. The preparation method of the aflatoxin molecularly imprinted polymer as claimed in claim 2, wherein the functional monomer is methacrylic acid, the cross-linking agent is ethylene glycol dimethacrylate, and the initiator is azobisisobutyronitrile.

4. The preparation method of the aflatoxin molecularly imprinted polymer as claimed in claim 2, wherein the volume ratio of methanol to acetic acid in the detergent is 8: 2.

5. the application of the aflatoxin molecularly imprinted polymer prepared by the preparation method according to any one of claims 1-4, wherein the aflatoxin molecularly imprinted polymer is used for measuring the aflatoxin content in traditional Chinese medicines and foods.

6. The application of the aflatoxin molecularly imprinted polymer of claim 5, wherein the traditional Chinese medicine comprises medicinal materials and traditional Chinese medicine preparations.

7. The application of the aflatoxin molecularly imprinted polymer according to claim 5, wherein the detection method for determining the aflatoxin content in traditional Chinese medicines and foods by using the aflatoxin molecularly imprinted polymer comprises the following steps:

step a, preparing a molecular imprinting-solid phase extraction column:

taking a clean SPE small column, placing a polytetrafluoroethylene gasket at the bottom of the clean SPE small column, adding the aflatoxin molecularly imprinted polymer as a filler, uniformly distributing the aflatoxin molecularly imprinted polymer in the column, pressing the polytetrafluoroethylene gasket at the top of the filler again for fixing the filler, and obtaining a molecularly imprinted-solid phase extraction column;

step b, preparing a sample solution to be detected:

crushing and sieving a sample to be detected, taking 15g of sample powder to be detected, placing the sample powder into a homogenizing bottle, adding 75mL of first extracting agent, carrying out ultrasonic oscillation extraction for 30 minutes, filtering by using qualitative filter paper, collecting filtrate, taking 15mL of filtrate, placing the filtrate into a 50mL volumetric flask, adding 2.5mL of water, adding 60% methanol solution in volume fraction, fixing the volume to a scale, and shaking up to obtain a sample extracting solution, wherein the volume ratio of methanol to water in the sample extracting solution is 3:2, and the first extracting agent is 70% methanol solution in volume fraction;

connecting the molecular imprinting-solid phase extraction column to a solid phase extraction device, adding an activating agent into the column, soaking and washing the small column, and activating a filler; after filler activation, taking a certain amount of sample extracting solution to enable the sample extracting solution to slowly pass through a molecular imprinting-solid phase extraction column at the speed of 1.0ml/min, enabling air to pass through the column, adding a proper amount of water to drip wash the column, enabling the air to pass through the column, adding 1.0ml of eluent to carry out elution for three times, incubating for 30 seconds during each elution, and collecting the eluent to obtain a sample solution to be detected;

step c, preparing a reference substance gradient solution to be detected:

measuring a proper amount of aflatoxin B1 standard substance solution with the concentration of 1.0 mu g/ml, and diluting with 70% methanol solution to obtain a reference substance gradient solution with the concentration of aflatoxin B1 of 0.12-10.0 ng/ml;

respectively taking 3.0ml of the control gradient solution with each concentration, slowly passing through a molecular imprinting-solid phase extraction column at the speed of 1.0ml/min, allowing air to pass through the column, adding a proper amount of water to elute the column, allowing air to pass through the column, adding 1.0ml of eluent to elute for three times, incubating for 30 seconds during each elution, and collecting the eluent to obtain a control gradient solution to be detected;

and d, carrying out high performance liquid chromatography-tandem mass spectrometry detection:

sucking 5 mul of each reference substance gradient solution to be measured, injecting the solution into a high performance liquid chromatography-tandem mass spectrometer, measuring the peak area, and drawing a standard curve by taking the peak area as a vertical coordinate and the content concentration of the aflatoxin B1 as a horizontal coordinate; and (3) additionally sucking 5 mu l of the sample liquid to be detected, injecting the sample liquid into a high performance liquid chromatography-tandem mass spectrometer, measuring the peak area, calculating to obtain the concentration equivalent to the aflatoxin B1 in the sample extracting solution according to a standard curve, and then calculating to obtain the content of the aflatoxin in the sample to be detected.

8. The use according to claim 7, wherein the liquid chromatography conditions in step d are: a chromatographic column: a chromatographic column with octadecylsilane chemically bonded silica as a filler, and a mobile phase: phase A: 10mmol/L ammonium acetate solution; phase B: methanol, column temperature: 25 ℃, flow rate: 0.3ml/min, gradient elution, in the manner shown in Table 1:

time (minutes) Mobile phase A (%) Mobile phase B (%) 0～4.5 65→15 35→85 4.5～6 15→0 85→100 6～6.5 0→65 100→35 6.5～10 65 35

9. The use according to claim 7, wherein the mass spectrum detection conditions in step d are: the electrospray ion source has a positive ion acquisition mode, and the corresponding retention time, monitored ion pair and voltage parameters of the target are shown in table 2:

10. the use according to claim 7, wherein the activating agent is methanol, the eluting agent is methanol, and the concentration of aflatoxin B1 in the control gradient concentration solution in step c is, in order: 0.12ng/ml, 0.25ng/ml, 1.0ng/ml, 2.0ng/ml, 5.0ng/ml, 10.0 ng/ml.

Technical Field

The invention relates to the technical field of high molecular materials, in particular to an aflatoxin molecularly imprinted polymer and a preparation method and application thereof.

Background

Aflatoxins are a class of difuranocoumarol metabolites with similar chemical structures produced by a certain strains such as aspergillus flavus and aspergillus parasiticus. The substance is found in moldy crops such as corn, peanut, wheat, rice and soybean. More than 30 aflatoxins have been isolated, of which B1, B2, G1, G2 and two metabolites, M1 and M2, are more common. Wherein aflatoxin B1 was listed as the first carcinogen by the International agency for research on cancer (IARC) in 1993. The toxicity of the compound for inducing hepatocellular carcinoma is 10 times that of potassium cyanide and 75 times that of dimethyl nitrosamine. Aflatoxins can pose serious health problems to humans through contamination of various foods and animal feeds. Therefore, the problem of aflatoxin pollution to food is receiving increasing attention.

How to separate aflatoxin from a complex food sample quickly, accurately and effectively has become an important problem of food safety detection. There are several widely used methods for detecting aflatoxin, including Thin Layer Chromatography (TLC), enzyme linked immunosorbent assay (ELISA), high performance liquid chromatography-mass spectrometry (LC-MS), and electrochemical sensors. However, before detection, the target substance is often required to be enriched, and meanwhile, the interference of impurities is eliminated, so that the influence of the existence of the impurities on the detection sensitivity is prevented. The most commonly used method is the pretreatment of the sample with an immunoaffinity column, which includes drug extraction, centrifugation, filtration, and immunoaffinity column chromatography. The process is complex to operate, the immunoaffinity column is unstable, the price is relatively expensive, the adsorption performance is easily influenced by an organic solvent, a pH value and temperature, the immunoaffinity column is basically disposable, and the popularization of aflatoxin detection is greatly limited.

The molecular imprinting technology provides a new way for the rapid extraction and detection of aflatoxin, and the Molecularly Imprinted Polymer (MIP) prepared by the technology is simple to prepare, resistant to high temperature and acid and alkali and reusable, so that the use cost is greatly reduced. When the complex is applied to a solid phase extraction column, the complex can replace an immunoaffinity column and can also be used as an identification element for a biosensor which can be used for on-line detection and repeated use. Because aflatoxin has high toxicity and high price, high experiment cost and potential danger to operators due to the fact that aflatoxin is used as a template molecule, in actual preparation, an imprinted polymer needs to be synthesized by using a structural analogue of aflatoxin as a virtual template, so that the harm to the experimenters is reduced, and meanwhile, the leakage of the template molecule is avoided. A virtual template of aflatoxin commonly used in the prior art is 5, 7-dimethoxycoumarin, but a molecularly imprinted polymer prepared by taking the 5, 7-dimethoxycoumarin as the virtual template has lower adsorption efficiency on the aflatoxin. Therefore, a cheap virtual template molecule needs to be developed to replace aflatoxin with high toxicity and high cost, and a molecularly imprinted compound with good adsorption effect on aflatoxin is prepared and applied to detection of aflatoxin in food.

Disclosure of Invention

The invention aims to provide an aflatoxin molecularly imprinted polymer and a preparation method and application thereof.

According to one aspect of the invention, the preparation method of the aflatoxin molecularly imprinted polymer is provided, and comprises the following steps:

step 1, mixing hematoxylin serving as a template molecule with a functional monomer and acetonitrile, carrying out ultrasonic treatment, and adding a cross-linking agent and an initiator to obtain a first mixture, wherein the template molecule: functional monomer: the molar ratio of the cross-linking agent is 1: 6-8: 30, nitrogen purging is carried out on the first mixture, polymerization reaction is carried out at a certain temperature, and after the reaction is finished, centrifugal separation is carried out to obtain a product;

and 2, washing the product with methanol to remove unreacted functional monomers, and then removing hematoxylin with a detergent to obtain the aflatoxin molecularly imprinted polymer, wherein the detergent is a mixed solution of methanol and acetic acid.

The invention has the beneficial effects that: the invention takes hematoxylin as template molecule, adopts bulk polymerization method, fully mixes the hematoxylin and functional monomer to play a role, then adds cross linker and initiator to initiate polymerization, finally removes unreacted monomer and hematoxylin, prepares molecular engram polymer, the molar ratio of hematoxylin, functional monomer and cross linker directly influences the specificity and adsorption capacity of molecular engram polymer to aflatoxin, the molar ratio of hematoxylin, functional monomer and cross linker is 1: 6-8: 30, the prepared molecularly imprinted polymer has a good specific adsorption effect on aflatoxin B1, so the aflatoxin molecularly imprinted polymer prepared by using hematoxylin to replace aflatoxin B1 as a template molecule and the preparation method thereof solve the problems of high price, high toxicity, high experiment cost, insecurity for operators, easy leakage and difficulty in removal of the template molecule and the like of the aflatoxin as the template molecule, and the prepared molecularly imprinted polymer has the advantages of easiness in preparation, stable property, low cost, high efficiency and capability of specifically identifying the aflatoxin, is favorable for popularization and application in detection of aflatoxin content in traditional Chinese medicines and foods, and improves edible safety guarantee of the traditional Chinese medicines and the foods.

In some embodiments, a method for preparing an aflatoxin molecularly imprinted polymer comprises the following steps:

step 1, mixing hematoxylin serving as a template molecule with a functional monomer and 30-50ml of acetonitrile, carrying out ultrasonic treatment for 10-20min, and adding a cross-linking agent and an initiator to obtain a first mixture, wherein the template molecule: functional monomer: the molar ratio of the cross-linking agent is 1:8:30, purging the first mixture with nitrogen for 10-20min, carrying out polymerization reaction for 24 hours at a certain temperature, after the reaction is finished, carrying out centrifugal separation to obtain a product, wherein the functional monomer is one of methacrylic acid or acrylamide, and the initiator is one of azobisisobutyronitrile or azobisisovaleronitrile;

and 2, washing the product with 20-40ml of methanol to remove unreacted functional monomers, and then removing hematoxylin with a detergent to obtain the aflatoxin molecularly imprinted polymer, wherein the detergent is a mixed solution of methanol and acetic acid. In the preparation method, the mol ratio of the hematoxylin to the functional monomer and the cross-linking agent is preferably 1:8:30, under the condition of the proportion, the prepared molecularly imprinted polymer has the best specific adsorption effect on aflatoxin B1, and the adsorption quantity is the highest.

In some embodiments, the functional monomer is methacrylic acid, the crosslinker is ethylene glycol dimethacrylate, and the initiator is azobisisobutyronitrile. Methacrylic acid has a carboxyl group in addition to a carbon-carbon double bond in a molecule, and can generate hydrogen bond action with hydroxyl of template molecule hematoxylin; the ethylene glycol dimethacrylate has two ethylene groups in a molecule, and can form a three-dimensional network structure in a polymerization reaction, so that a cavity formed by the polymer after the template molecule is removed is fixed in shape, and the specific adsorption of aflatoxin is realized.

In some embodiments, the volume ratio of methanol to acetic acid in the detergent is 8: 2. the detergent can well remove template molecule hematoxylin in the product.

In some embodiments, the prepared aflatoxin molecularly imprinted polymer is used for measuring the aflatoxin content in traditional Chinese medicines and foods. The prepared aflatoxin molecularly imprinted polymer has a high specific adsorption effect on aflatoxin, can be applied to measurement of aflatoxin content in traditional Chinese medicines and foods, and improves edible safety guarantee for the traditional Chinese medicines and the foods.

In some embodiments, the Chinese medicine comprises medicinal materials and Chinese medicine preparations. The aflatoxin molecularly imprinted polymer can be used for measuring the aflatoxin content in medicinal materials and Chinese medicinal preparations.

In some embodiments, the detection method for determining the aflatoxin content in traditional Chinese medicines and foods by using the aflatoxin molecularly imprinted polymer comprises the following steps:

step a, preparing a molecular imprinting-solid phase extraction column:

taking a clean SPE small column, placing a polytetrafluoroethylene gasket at the bottom of the clean SPE small column, adding an aflatoxin molecularly imprinted polymer as a filler, uniformly distributing the aflatoxin molecularly imprinted polymer in the column, pressing the polytetrafluoroethylene gasket at the top of the filler again for fixing the filler, and obtaining a molecularly imprinted-solid phase extraction column;

step b, preparing a sample solution to be detected:

crushing and sieving a sample to be detected, taking 15g of sample powder to be detected, placing the sample powder into a homogenizing bottle, adding 75mL of first extracting agent, carrying out ultrasonic oscillation extraction for 30 minutes, filtering by using qualitative filter paper, collecting filtrate, taking 15mL of filtrate, placing the filtrate into a 50mL volumetric flask, adding 2.5mL of water, adding 60% methanol solution in volume fraction, fixing the volume to a scale, and shaking up to obtain a sample extracting solution, wherein the volume ratio of methanol to water in the sample extracting solution is 3:2, and the first extracting agent is 70% methanol solution in volume fraction;

connecting the molecular imprinting-solid phase extraction column to a solid phase extraction device, adding an activating agent into the column, soaking and washing the small column, and activating a filler; after filler activation, taking a certain amount of sample extracting solution to enable the sample extracting solution to slowly pass through a molecular imprinting-solid phase extraction column at the speed of 1.0ml/min, enabling air to pass through the column, adding a proper amount of water to drip wash the column, enabling the air to pass through the column, adding 1.0ml of eluent to carry out elution for three times, incubating for 30 seconds during each elution, and collecting the eluent to obtain a sample solution to be detected;

step c, preparing a reference substance gradient solution to be detected:

measuring a proper amount of aflatoxin B1 standard substance solution with the concentration of 1.0 mu g/ml, and diluting with 70% methanol solution to obtain a reference substance gradient solution with the concentration of aflatoxin B1 of 0.12-10.0 ng/ml;

respectively taking 3.0ml of the control gradient solution with each concentration, slowly passing through a molecular imprinting-solid phase extraction column at the speed of 1.0ml/min, allowing air to pass through the column, adding a proper amount of water to elute the column, allowing air to pass through the column, adding 1.0ml of eluent to elute for three times, incubating for 30 seconds during each elution, and collecting the eluent to obtain a control gradient solution to be detected;

and d, carrying out high performance liquid chromatography-tandem mass spectrometry detection:

sucking 5 mul of each reference substance gradient solution to be measured, injecting the solution into a high performance liquid chromatography-tandem mass spectrometer, measuring the peak area, and drawing a standard curve by taking the peak area as a vertical coordinate and the content concentration of the aflatoxin B1 as a horizontal coordinate; and (3) additionally sucking 5 mu l of the sample liquid to be detected, injecting the sample liquid into a high performance liquid chromatography-tandem mass spectrometer, measuring the peak area, calculating to obtain the concentration equivalent to the aflatoxin B1 in the sample extracting solution according to a standard curve, and then calculating to obtain the content of the aflatoxin in the sample to be detected. And in the step b, the aflatoxin molecularly imprinted polymer is used as a filler of the solid-phase extraction column, the aflatoxin contained in the sample to be detected is adsorbed and extracted by utilizing the specific adsorption effect of the molecularly imprinted polymer on the aflatoxin, the aflatoxin contained in the sample to be detected is effectively extracted, the accuracy of a detection result is ensured, the method has the advantages of simple preparation, high temperature resistance, acid and alkali resistance, reusability, greatly reduced cost and capability of replacing an immunoaffinity column, and the problems of complicated operation process, unstable immunoaffinity column, high price, influence of an organic solvent on adsorption performance, pH value and temperature and the like in the conventional detection method are solved.

In some embodiments, the liquid chromatography conditions in step d are: a chromatographic column: a chromatographic column with octadecylsilane chemically bonded silica as a filler, and a mobile phase: phase A: 10mmol/L ammonium acetate solution; phase B: methanol, column temperature: 25 ℃, flow rate: 0.3ml/min, gradient elution mode as shown in table 1:

TABLE 1

Time (minutes)	Mobile phase A (%)	Mobile phase B (%)
			0～4.5	65→15	35→85
4.5～6	15→0	85→100
			6～6.5	0→65	100→35
6.5～10	65	35

In some embodiments, the mass spectrometry detection conditions in step d are: the electrospray ion source has a positive ion acquisition mode, and the corresponding retention time, monitored ion pair and voltage parameters of the target are shown in table 2:

TABLE 2

In some embodiments, the activating agent is methanol, the eluent is methanol, and the concentration of aflatoxin B1 in the control gradient solution in step c is, in order: 0.12ng/ml, 0.25ng/ml, 1.0ng/ml, 2.0ng/ml, 5.0ng/ml, 10.0 ng/ml.

Drawings

Fig. 1 is an infrared spectrum of an aflatoxin molecularly imprinted polymer using hematoxylin as a template molecule according to a preparation method of the aflatoxin molecularly imprinted polymer in an embodiment of the invention.

Fig. 2 is an infrared spectrum of a blank molecularly imprinted polymer of a method for preparing an aflatoxin molecularly imprinted polymer according to an embodiment of the invention.

Fig. 3 is a bar graph of adsorption capacity of a series of molecularly imprinted polymers prepared by using hematoxylin as a template molecule and using template molecules, functional monomers and cross-linking agents in molar ratios of 1:8:15, 1:8:20 and 1:8:30 according to an embodiment of the invention on aflatoxin B1.

Fig. 4 is a bar graph of adsorption capacity of a series of molecularly imprinted polymers prepared by using hematoxylin as a template molecule and using template molecules, functional monomers and a cross-linking agent in a molar ratio of 1:6:30, 1:8:30, 1:10:30 and 1:12:30 according to an embodiment of the invention to aflatoxin B1.

Fig. 5 shows a ratio of methanol to water in a volume ratio of 3:2, thermodynamic experiment chart of different aflatoxin molecularly imprinted polymers on aflatoxin B1 under the condition of adsorption solvent.

Fig. 6 is a standard curve diagram of aflatoxin B1 of the detection method for determining aflatoxin content in traditional Chinese medicines and foods by using the aflatoxin molecularly imprinted polymer in the embodiment of the invention.

Detailed Description

The present invention will be described in further detail with reference to examples.

Example 1

In this embodiment, the aflatoxin B1 standard is selected from aflatoxin B1 standard supplied by Qingdao Pop bioengineering Co., Ltd, ammonium acetate is selected from analytically pure ammonium acetate supplied by Anyingji chemical Co., Ltd, methanol is selected from analytically pure methanol supplied by Anyingji chemical Co., Ltd, analytically pure acetic acid supplied by Anyingji chemical Co., Ltd, methacrylic acid is selected from chemically pure methacrylic acid supplied by national drug group chemical reagent Co., Ltd, ethylene glycol dimethacrylate is selected from ethylene glycol dimethacrylate supplied by Shanghai Albandin Biotech, azodiisobutyronitrile is selected from analytically pure azobisisobutyronitrile supplied by national drug group chemical reagent Co., Ltd, azodiisovaleronitrile is selected from 98% azodiisovaleronitrile supplied by national drug group chemical reagent Co., Ltd, acrylamide is selected from chemically pure acrylamide supplied by national drug group chemical reagent Co., Ltd, the acetonitrile is chemically pure acetonitrile supplied by national medicine group chemical reagent company Limited;

the 5, 7-dimethoxycoumarin is prepared by cyclization reaction of 3, 5-dimethoxyphenol and ethyl propiolate under the catalysis of silver tetrafluoroborate, and the reaction formula is as follows:

the reagents of this example 1 were used in the following examples 2 to 8.

Example 2

The invention relates to a preparation method of an aflatoxin molecularly imprinted polymer, which comprises the following steps:

step 1, mixing 0.2mmol of hematoxylin as a template molecule with 1.6mmol of methacrylic acid and 40ml of acetonitrile, carrying out ultrasonic treatment for 15min, and then adding 6mmol of ethylene glycol dimethacrylate and 0.072mmol of azobisisobutyronitrile to obtain a first mixture, wherein the hematoxylin: methacrylic acid: the molar ratio of ethylene glycol dimethacrylate is 1:8:30, purging the first mixture with nitrogen for 15min, carrying out polymerization reaction for 24 hours at the temperature of 65 ℃, and after the reaction is finished, carrying out centrifugal separation to obtain a product;

and 2, washing the product with 30ml of methanol to remove unreacted methacrylic acid, and then removing hematoxylin with a detergent to obtain the aflatoxin molecularly imprinted polymer, wherein the volume ratio of methanol to acetic acid in the detergent is 8: 2. and (3) carrying out Fourier transform infrared spectrometer analysis on the prepared aflatoxin molecularly imprinted polymer to obtain an infrared spectrogram of the aflatoxin molecularly imprinted polymer, which is shown in the attached figure 1 of the specification. Comparing the description with the accompanying drawings 1 and 2, it can be directly seen that the aflatoxin molecularly imprinted polymer prepared by taking the hematoxylin as a template molecule has an obvious vibration peak between 2900-3000 and a strong signal peak between 1700-1800, which shows that after the hematoxylin and methacrylic acid are polymerized by the action of hydrogen bonds, the polymer generates holes after the hematoxylin is eluted, the number of functional groups exposed outside is increased, and the absorption vibration peaks of carbonyl and alkane are enhanced.

The reaction process of the step 1 and the step 2 is as follows:

in the preparation method of the embodiment, hematoxylin is used as a template molecule, a bulk polymerization method is adopted, hematoxylin and a functional monomer methacrylic acid are fully mixed to play a role, a cross-linking agent ethylene glycol dimethacrylate and an initiator azobisisobutyronitrile are added to initiate a polymerization reaction, and finally unreacted functional monomers methacrylic acid and hematoxylin are removed to prepare a molecularly imprinted polymer, wherein the molar ratio of hematoxylin to methacrylic acid and ethylene glycol dimethacrylate directly influences the specificity and the adsorption capacity of the molecularly imprinted polymer to aflatoxin, and the molar ratio of hematoxylin to the functional monomer and the cross-linking agent is 1:8:30 has better specific adsorption effect on aflatoxin B1. In the polymerization reaction, the molecule of the methacrylic acid has a carboxyl group besides a carbon-carbon double bond, and can react with the hydroxyl group of the template molecule hematoxylin to form a hydrogen bond; the ethylene glycol dimethacrylate has two ethylene groups in a molecule, and can form a three-dimensional network structure in a polymerization reaction, so that a cavity formed by the polymer after the hematoxylin is removed has a stable shape, and the aflatoxin can be conveniently adsorbed.

Example 3

The invention relates to a preparation method of an aflatoxin molecularly imprinted polymer, which comprises the following steps:

step 1, mixing 0.2mmol of hematoxylin as a template molecule with 1.2mmol of methacrylic acid and 30ml of acetonitrile, carrying out ultrasonic treatment for 10min, and then adding 6mmol of ethylene glycol dimethacrylate and 0.072mmol of azobisisobutyronitrile to obtain a first mixture, wherein the hematoxylin: methacrylic acid: the molar ratio of ethylene glycol dimethacrylate is 1:6:30, purging the first mixture with nitrogen for 10min, carrying out polymerization reaction for 24 hours at the temperature of 65 ℃, and after the reaction is finished, carrying out centrifugal separation to obtain a product;

and 2, washing the product with 20ml of methanol to remove unreacted methacrylic acid, and then removing hematoxylin with a detergent to obtain the aflatoxin molecularly imprinted polymer, wherein the volume ratio of methanol to acetic acid in the detergent is 8: 2.

example 4

The invention relates to a preparation method of an aflatoxin molecularly imprinted polymer, which comprises the following steps:

step 1, mixing 0.2mmol of hematoxylin as a template molecule with 1.4mmol of methacrylic acid and 50ml of acetonitrile, carrying out ultrasonic treatment for 20min, and then adding 6mmol of ethylene glycol dimethacrylate and 0.072mmol of azobisisobutyronitrile to obtain a first mixture, wherein the hematoxylin: methacrylic acid: the molar ratio of ethylene glycol dimethacrylate is 1: 7: 30, purging the first mixture with nitrogen for 20min, carrying out polymerization reaction for 24 hours at the temperature of 65 ℃, and after the reaction is finished, carrying out centrifugal separation to obtain a product;

and 2, washing the product with 40ml of methanol to remove unreacted methacrylic acid, and then removing hematoxylin with a detergent to obtain the aflatoxin molecularly imprinted polymer, wherein the volume ratio of methanol to acetic acid in the detergent is 8: 2.

example 5

The influence of different molar ratios of the template molecule, the functional monomer and the cross-linking agent on the adsorption performance of the aflatoxin molecularly imprinted polymer is as follows:

in the embodiment, hematoxylin is used as a template molecule, the molar ratio of the template molecule to the functional monomer to the cross-linking agent is 1:6:30, 1:8:30, 1:10:30, 1:12:30, 1:8:15 and 1:8:20, and the preparation method in the embodiment 2 is adopted to synthesize the template molecule, so as to prepare a series of molecularly imprinted polymers using hematoxylin as the template molecule.

Preparing aflatoxin B1 standard substance into standard sample solution with concentration gradient of 100 ppm, 75 ppm, 50ppm, 25 ppm and 12.5ppm by using methanol, and drawing aflatoxin B1 standard concentration curve by using high performance liquid chromatograph-fluorescence detector to obtain 0.016462x +2.23839 standard curve y and 0.989571 standard curve R2.

The volume ratio of methanol to water is 3:2, preparing an aflatoxin B1 solution with the concentration of 50ppm as an adsorption solution, respectively and correspondingly adding 10mg of a series of molecularly imprinted polymers taking hematoxylin as template molecules into the aflatoxin B1 solution with the concentration of 50ppm, after adsorbing for 1 hour, measuring the concentration of aflatoxin B1 in the aflatoxin B1 solution after adsorption by using a high performance liquid chromatograph-fluorescence detector, and calculating the adsorption quantity Q of the series of molecularly imprinted polymers taking hematoxylin as template molecules on aflatoxin B1 according to the change of the concentrations before and after adsorption, wherein the specific adsorption quantity is shown in the following table 3, the accompanying drawing 3 and the accompanying drawing 4 of the specification, and the calculation formula is as follows:

Q＝(C1-C2)V/M

q: adsorption capacity, ug/mg

C1: concentration before adsorption, ug/mL

C2: post-adsorption concentration, ug/mL

M: mass of polymer, mg

TABLE 3 adsorption capacity of a series of molecularly imprinted polymers with hematoxylin as template molecule on aflatoxin B1

From the above table 3, the attached drawings 3 and 4 in the specification, it can be directly obtained that the adsorption capacity of the prepared molecularly imprinted polymer to aflatoxin B1 is higher and better than that of the molecularly imprinted polymer prepared in other molar ratios by using the molar ratio of the template molecule, the functional monomer and the cross-linking agent of 1: 6-8: 30, so that the optimal molar ratio of the template molecule, the functional monomer and the cross-linking agent is 1: 6-8: 30, and the most preferable molar ratio is 1:8:30 in the preparation method of the invention.

Example 6

The adsorption condition test of the aflatoxin molecularly imprinted polymer of different template molecules on aflatoxin B1:

in this example, 5, 7-dimethoxycoumarin and hematoxylin were used as template molecules, respectively, and synthesized by the preparation method in example 2 without adding a template molecule as a blank control, to prepare a molecularly imprinted polymer 7 using 5, 7-dimethoxycoumarin as a template molecule, a molecularly imprinted polymer 8 using hematoxylin as a template molecule, and a blank molecularly imprinted polymer. And (3) carrying out Fourier transform infrared spectrometer analysis on the prepared blank molecularly imprinted polymer to obtain an infrared spectrogram of the blank molecularly imprinted polymer, which is shown in the attached figure 2 of the specification.

Preparing aflatoxin B1 into standard sample solution with concentration gradient of 100, 75, 50, 25 and 12.5ppm by using methanol, and drawing standard concentration curve of aflatoxin B1 by using high performance liquid chromatograph-fluorescence detector to obtain standard curve y of 0.016462x +2.23839, R²＝0.989571。

Methanol was used as adsorption solution 1, with a methanol to water volume ratio of 3:2 as an adsorption solution 2, preparing 20ppm aflatoxin B1 solutions, adding 10mg of molecularly imprinted polymer 7, molecularly imprinted polymer 8 and blank molecularly imprinted polymer into 20ppm aflatoxin B1 solution, adsorbing for 1 hour, detecting the concentration of aflatoxin B1 in the aflatoxin B1 solution after adsorption by using a high performance liquid chromatograph-fluorescence detector, and calculating the adsorption amounts of the molecularly imprinted polymer 7, the molecularly imprinted polymer 8 and the blank molecularly imprinted polymer to aflatoxin B1 according to the change of the concentrations before and after adsorption by referring to the adsorption amount calculation formula in example 5, and the results are shown in table 4 below.

TABLE 4 adsorption capacity of molecularly imprinted polymers prepared from different template molecules on aflatoxin B1 under different adsorption solvent conditions

It can be directly obtained from table 4 that, for the same molecularly imprinted polymer, the ratio of the adsorption solvent can affect the adsorption effect of the molecularly imprinted polymer on aflatoxin. Under the adsorption condition of an adsorption solution 2 (the volume ratio of methanol to water is 3:2), the adsorption amount of the molecularly imprinted polymer 8 taking hematoxylin as a template molecule to the aflatoxin B1 in a 20ppm aflatoxin B1 solution is 0.577 mug/mg which is higher than that under the condition of the adsorption solution 1, and the adsorption amounts of the molecularly imprinted polymer 7 taking 5, 7-dimethoxycoumarin as a template molecule and the blank molecularly imprinted polymer under the condition of the adsorption solution 1 are both smaller than that under the condition of the adsorption solution 2, so that the volume ratio of methanol to water is 3:2 as an adsorption solvent in the application of the invention, so that the adsorption effect of the molecularly imprinted polymer taking hematoxylin as a template molecule on the aflatoxin is improved, and the accuracy of subsequent detection is ensured. Under the same condition of adsorbing the solution 2, the adsorption quantity of the molecularly imprinted polymer 8 is improved by 28.79 percent compared with that of the molecularly imprinted polymer 7 and is improved by 20.71 percent compared with that of a blank molecularly imprinted polymer.

Example 7

Adsorption performance experiment of aflatoxin molecularly imprinted polymers of different template molecules on aflatoxin B1:

in this embodiment, three different aflatoxin molecularly imprinted polymers prepared in example 6 are used as experimental objects, namely, a molecularly imprinted polymer 8 using hematoxylin as a template molecule and a blank molecularly imprinted polymer. Preparing two groups of aflatoxin gradient solutions with concentration gradients of 3 ppm, 5ppm, 10 ppm, 20ppm, 30 ppm, 50ppm, 70ppm and 90ppm, adding 10mg of molecularly imprinted polymer 8 and blank molecularly imprinted polymer into the two groups of aflatoxin gradient solutions, adsorbing for 1 hour, measuring the concentration of aflatoxin B1 in the two groups of aflatoxin gradient solutions after adsorption by using a high performance liquid chromatograph-fluorescence detector, and calculating the adsorption amount of the molecularly imprinted polymer 8 and the blank molecularly imprinted polymer on aflatoxin B1 in the aflatoxin gradient solutions according to the change of the concentrations before and after adsorption by referring to the adsorption amount calculation formula in example 5, which is shown in Table 5 and the specification attached figure 5, wherein the calculation formula is as follows:

TABLE 5 adsorption Performance of molecularly imprinted polymers prepared with different template molecules on aflatoxin B1

According to table 5 and the specification and the attached figure 5, under the same adsorption solution condition, the adsorption amount of the molecularly imprinted polymer 8 taking hematoxylin as a template molecule to aflatoxin B1 in an aflatoxin gradient solution of 5ppm to 90ppm is higher than that of a blank molecularly imprinted polymer, which indicates that the adsorption performance of the molecularly imprinted polymer 8 taking hematoxylin as a template molecule to aflatoxin B1 is higher than that of the blank molecularly imprinted polymer to aflatoxin B1, wherein in an aflatoxin solution of 50ppm to 70ppm, the adsorption amount of the molecularly imprinted polymer 8 to aflatoxin B1 reaches a higher level, and is respectively improved by 10.59%, 8.75% and 27.79% compared with that of the blank molecularly imprinted polymer.

Example 8

The detection method for determining the aflatoxin content in traditional Chinese medicines and foods by using the aflatoxin molecularly imprinted polymer prepared in the embodiment 2 and taking hematoxylin as a template molecule comprises the following steps:

step a, preparing a molecular imprinting-solid phase extraction column:

taking a clean SPE small column, putting a polytetrafluoroethylene gasket at the bottom of the clean SPE small column, adding 0.1g of aflatoxin molecularly imprinted polymer as a filler, uniformly distributing the aflatoxin molecularly imprinted polymer in the column, pressing the polytetrafluoroethylene gasket at the top of the filler again for fixing the filler, and obtaining a molecularly imprinted-solid phase extraction column;

step b, preparing a sample solution to be detected:

taking one of traditional Chinese medicine materials, traditional Chinese medicine preparations or food as a sample to be detected, crushing the sample to be detected, sieving the sample with a second sieve, weighing 15g of sample powder to be detected, placing the sample powder into a homogenizing bottle, adding 75mL of a first extracting agent, carrying out ultrasonic oscillation extraction for 30 minutes, filtering the sample powder by using qualitative filter paper, collecting filtrate, measuring 15mL of the filtrate, placing the filtrate into a 50mL volumetric flask, adding 2.5mL of water, adding a methanol solution with the volume fraction of 60% to a constant volume to reach a scale, and shaking up to obtain a sample extracting solution, wherein the volume ratio of methanol to water in the sample extracting solution is 3:2, and the first extracting agent is a methanol aqueous solution with the volume fraction of 70%;

in the step, a methanol aqueous solution with the volume fraction of 70% is used as a first extracting agent, the effect of extracting aflatoxin from sample powder to be detected is good, a certain amount of water and a methanol aqueous solution with the volume fraction of 60% are added into filtrate, the volume ratio of methanol to water in a sample extracting solution is adjusted to be 3:2, an optimal adsorption condition is provided for a molecularly imprinted polymer to adsorb aflatoxin in the sample extracting solution in subsequent operation, and the molecularly imprinted polymer is convenient to adsorb aflatoxin in the sample extracting solution;

connecting the molecular imprinting-solid phase extraction column to a solid phase extraction device, adding 3.0ml of methanol into the column, soaking and washing the small column, and activating a filler; after filler activation, 3.0ml of sample extracting solution is taken to slowly pass through a molecular imprinting-solid phase extraction column at the speed of 1.0ml/min, air passes through the column, appropriate amount of water is added to elute the column, the air passes through the column, 1.0ml of methanol is added to elute for three times, incubation is needed for 30 seconds during each elution, and eluent is collected to obtain sample solution to be detected;

step c, preparing a reference substance gradient solution to be detected:

measuring a proper amount of aflatoxin B1 standard solution with the concentration of 1.0 mu g/ml, and diluting with 70% methanol solution to obtain control substance gradient solution containing aflatoxin B1 with the concentrations of 0.12ng/ml, 0.25ng/ml, 1.0ng/ml, 2.0ng/ml, 5.0ng/ml and 10.0 ng/ml;

respectively taking 3.0ml of the control gradient solution with each concentration, slowly passing through a molecular imprinting-solid phase extraction column at the speed of 1.0ml/min, allowing air to pass through the column, adding a proper amount of water to elute the column, allowing air to pass through the column, adding 1.0ml of eluent to elute for three times, incubating for 30 seconds during each elution, and collecting the eluent to obtain a control gradient solution to be detected;

and d, carrying out high performance liquid chromatography-tandem mass spectrometry detection:

the liquid chromatography conditions were: a chromatographic column: a chromatographic column with octadecylsilane chemically bonded silica as a filler, and a mobile phase: phase A: 10mmol/L ammonium acetate solution; phase B: methanol, column temperature: 25 ℃, flow rate: 0.3ml/min, gradient elution mode as shown in Table 6:

TABLE 6

Time (minutes)	Mobile phase A (%)	Mobile phase B (%)
			0～4.5	65→15	35→85
4.5～6	15→0	85→100
			6～6.5	0→65	100→35
6.5～10	65	35

The mass spectrum detection conditions are as follows: the electrospray ion source has a positive ion acquisition mode, and the corresponding retention time, monitored ion pair and voltage parameters of the target are shown in table 7:

TABLE 7

And (3) absorbing 5 mul of each solution to be tested of the reference substance gradient, injecting the solution into a high performance liquid chromatography-tandem mass spectrometer, measuring the peak area, drawing a standard curve by taking the peak area as a vertical coordinate and the content concentration of the aflatoxin B1 as a horizontal coordinate, wherein the standard curve is shown in the attached figure 6 of the specification, and the obtained standard curve equation is as follows:

y＝10814.336790x-183.813198，R²0.9983; and (3) additionally sucking 5 mu l of the sample liquid to be detected, injecting the sample liquid into a high performance liquid chromatography-tandem mass spectrometer, measuring the peak area, calculating to obtain the concentration equivalent to the aflatoxin B1 in the sample extracting solution according to a standard curve, and then calculating to obtain the content of the aflatoxin in the sample to be detected.

The detection method of the embodiment has the detection limit of 0.25g/kg for aflatoxin in traditional Chinese medicines and foods, has high detection accuracy for aflatoxin content in traditional Chinese medicines and foods, performs specific adsorption for aflatoxin, effectively extracts aflatoxin in a sample to be detected, ensures the accuracy of a detection result, has the advantages of simple preparation, high temperature resistance, acid and alkali resistance, reusability, greatly reduced cost, and capability of replacing an immunoaffinity column, and solves the problems of complicated operation process, unstable immunoaffinity column, high price, influence of organic solvent on adsorption performance, pH value and temperature and the like in the existing detection method.

The above description is only for the embodiments of the present invention, and it is obvious to those skilled in the art that various changes and modifications can be made without departing from the inventive concept of the present invention, and these changes and modifications are all within the scope of the present invention.