阅读说明：本技术 一种快速检测百菌清及其氧化还原产物的方法 (Method for rapidly detecting chlorothalonil and redox product thereof ) 是由 施婷婷 陆宁 尹程 司雄元 花日茂 高超 殷长玉 檀珮雯 于 2020-04-27 设计创作，主要内容包括：本发明提供了一种快速检测百菌清及其氧化还原产物的方法,包括如下步骤：S1、制备百菌清分子印迹聚合物；S2、将所述百菌清分子印迹聚合物与硅藻土混合装柱制得百菌清分子印迹固相萃取柱；S3、将制得的百菌清分子印迹固相萃取柱用于待测样品处理,得洗脱液；S4、对步骤S3处理所得洗脱液,用色谱法检测洗脱液中的百菌清及其氧化还原产物含量。本发明利用百菌清为模板分子制备百菌清分子印迹聚合物,并将聚合物装填于固相萃取柱中制成萃取柱,对蔬菜与水体样品中的百菌清及其降解产物进行富集,净化,结合色谱法检测,确立一种高效、简单的方法；本发明特异性更高、灵敏度更好、检测速度更快。(The invention provides a method for rapidly detecting chlorothalonil and redox products thereof, which comprises the following steps: s1, preparing a chlorothalonil molecularly imprinted polymer; s2, mixing the chlorothalonil molecularly imprinted polymer with diatomite, and loading the mixture into a column to prepare a chlorothalonil molecularly imprinted solid phase extraction column; s3, using the prepared chlorothalonil molecular imprinting solid-phase extraction column for treating a sample to be detected to obtain an eluent; s4, processing the eluent obtained in the step S3, and detecting the content of chlorothalonil and redox products thereof in the eluent by chromatography. The invention uses chlorothalonil as a template molecule to prepare a chlorothalonil molecularly imprinted polymer, and the polymer is filled in a solid phase extraction column to prepare the extraction column, so that the chlorothalonil and degradation products thereof in vegetable and water samples are enriched, purified and detected by combining with a chromatography, and a high-efficiency and simple method is established; the invention has higher specificity, better sensitivity and higher detection speed.)

1. A method for rapidly detecting chlorothalonil and redox products thereof is characterized by comprising the following steps:

s1, preparing a chlorothalonil molecularly imprinted polymer;

s2, mixing the chlorothalonil molecularly imprinted polymer with diatomite, and loading the mixture into a column to prepare a chlorothalonil molecularly imprinted solid phase extraction column;

s3, using the prepared chlorothalonil molecular imprinting solid-phase extraction column for treating a sample to be detected to obtain an eluent;

s4, processing the eluent obtained in the step S3, and detecting the content of chlorothalonil and redox products thereof in the eluent by chromatography.

2. The method for rapidly detecting chlorothalonil and redox products thereof according to claim 1, wherein the step S3 comprises the following steps:

s31, obtaining a sample extracting solution to be detected;

s32, activating a chlorothalonil molecular imprinting solid-phase extraction column;

s33, passing the extract to be detected through a column and loading the sample;

s34, washing and removing impurities of the molecularly imprinted solid phase extraction column, and then eluting with acetone; obtaining the eluent.

3. The method for rapidly detecting chlorothalonil and redox products thereof as claimed in claim 1, wherein the chlorothalonil and the redox products thereof comprise 4-hydroxychlorothalonil, 5-chloro-1, 3-isophthalonitrile, 2, 5-dichloro-1, 3-isophthalonitrile and 2, 4, 5-trichloro-1, 3-isophthalonitrile.

4. The method for rapidly detecting chlorothalonil and redox products thereof according to claim 1, wherein the preparation method of the chlorothalonil molecularly imprinted polymer is as follows:

the method is characterized in that mother chlorothalonil is used as a template molecule, acetonitrile is used as a pore-foaming agent, acrylamide is used as a functional monomer, ethylene glycol dimethacrylate is used as a cross-linking agent, and azobisisobutyronitrile is respectively used as an initiator, and the method is as follows:

(1) weighing chlorothalonil, dissolving the chlorothalonil in acetonitrile, adding acrylamide, and performing ultrasonic oscillation for 30min at room temperature to completely combine the chlorothalonil and the acrylamide; then, adding ethylene glycol dimethacrylate and azobisisobutyronitrile, and continuing ultrasonic oscillation for 15min to fully and uniformly mix the mixture; filling nitrogen to discharge oxygen, sealing, and heating in a water bath kettle at 60 deg.C for 16h to obtain white solid polymer;

(2) crushing and grinding the white solid polymer obtained in the step (1), and sieving the white solid polymer with a 200-mesh sieve;

(3) soxhlet extracting with organic solvent to elute chlorothalonil in the polymer, eluting with ultrapure water to neutrality, and finally drying the product in a 60 ℃ oven for 24h to obtain the target desired chlorothalonil molecularly imprinted polymer MIPs taking chlorothalonil as a virtual template.

5. The method for rapidly detecting chlorothalonil and redox products thereof according to claim 4, wherein in the step (1), the ratio of chlorothalonil: acrylamide: ethylene glycol dimethacrylate: the molar ratio of azobisisobutyronitrile is 1:7:40: 0.6.

6. the method for rapidly detecting chlorothalonil and redox products thereof according to claim 4, wherein in the step (3), the elution time is 30 hours in the process of soxhlet extraction with the organic solvent to elute the chlorothalonil from the polymer.

7. The method for rapidly detecting chlorothalonil and redox products thereof according to claim 1, wherein the mass ratio of the chlorothalonil in the molecularly imprinted solid phase extraction column is as follows: the mass ratio of the chlorothalonil molecularly imprinted polymer is 1: 1-1: 2.

8. the method as claimed in claim 1, wherein the sample to be tested includes water, vegetables, fruits, and grains.

9. The method according to claim 8, wherein when the test substance is a vegetable, a fruit or a grain, the method further comprises preparing a test solution, specifically, homogenizing the test sample, adding 0.5% HCl acetonitrile solution for extraction, performing vortex oscillation for 2min, performing ultrasonic extraction for 30min, centrifuging at 10000r/min for 10min, and collecting the supernatant; blow-drying with a nitrogen blowing instrument, dissolving with an organic solvent, and diluting with pure water to obtain the solution to be measured.

Technical Field

The invention relates to the field of preparation of molecularly imprinted polymers, in particular to a method for rapidly detecting chlorothalonil and redox products thereof.

Background

Chlorothalonil, with the chemical name of tetrachloroisophthalonitrile, is a non-systemic, broad-spectrum and efficient protective fungicide and has the effect of preventing and treating fungal diseases of various crops. The sterilization mechanism is that the enzyme acts with the phosphoglyceraldehyde dehydrogenase in the fungal cells, and combines with the protein containing cysteine in the dehydrogenase to make the dehydrogenase lose activity and destroy the metabolism of the fungal cells, thereby leading to the death of the fungi.

Since the production of the chlorothalonil is started, the chlorothalonil is used in agricultural production in the world for more than 50 years, the residual chlorothalonil is detected in crops and natural ecological environment due to wide and long-term use, meanwhile, the chlorothalonil is subjected to oxidative degradation and reductive degradation in the nature, and the parent and degradation products of the chlorothalonil have potential threats to the environment and food safety. Chlorothalonil is low in toxicity to human beings and mammals, causes symptoms such as skin inflammation, eye discomfort and gastrointestinal irritation, is high in toxicity to aquatic organisms such as fish and shellfish, and has been listed as one of substances possibly causing carcinogenesis by human beings by the national environmental protection agency of the United states. Chlorothalonil is listed in the monitoring range for the first time in 'sanitary Standard for Drinking Water' revised in 2012 of China. At present, there are many methods for detecting chlorothalonil in crops and natural ecological environment, but the following defects generally exist: low specificity, poor sensitivity and long detection time.

Molecular Imprinting Technology (MIT) is a new technology for preparing polymer materials with recognition function that has recently appeared, and Molecular Imprinted Polymers (MIPs) that perfectly match a certain molecule in spatial structure and binding site can be obtained. The high selectivity of molecular imprinting recognition comes from a large number of binding sites matched with target molecules in the aspects of size, shape, functional groups and the like in an imprinted polymer matrix.

Therefore, there is an urgent need to design a method for rapidly detecting chlorothalonil and redox products thereof, and apply the method to the detection of chlorothalonil in crops and natural ecological environment so as to improve the specificity of detection, increase the sensitivity of detection and shorten the detection time.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a method for rapidly detecting chlorothalonil and redox products thereof, which can rapidly detect chlorothalonil and redox products thereof in an object to be detected, can improve the specificity of detection, increase the sensitivity of detection and shorten the detection time.

The invention adopts the following technical scheme to solve the technical problems:

a method for rapidly detecting chlorothalonil and redox products thereof is characterized by comprising the following steps:

s1, preparing a chlorothalonil molecularly imprinted polymer;

s2, mixing the chlorothalonil molecularly imprinted polymer with diatomite, and loading the mixture into a column to prepare a chlorothalonil molecularly imprinted solid phase extraction column;

s3, using the prepared chlorothalonil molecular imprinting solid-phase extraction column for treating a sample to be detected to obtain an eluent;

s4, processing the eluent obtained in the step S3, and detecting the content of chlorothalonil and redox products thereof in the eluent by chromatography.

Further, the specific step of S3 is as follows:

s31, obtaining a sample extracting solution to be detected;

s32, activating a chlorothalonil molecular imprinting solid-phase extraction column;

s33, passing the extract to be detected through a column and loading the sample;

s34, washing and removing impurities of the molecularly imprinted solid phase extraction column, and then eluting with acetone; obtaining the eluent.

Further, the chlorothalonil and the redox product thereof comprise 4-hydroxychlorothalonil, 5-chloro-1, 3-isophthalonitrile, 2, 5-dichloro-1, 3-isophthalonitrile and 2, 4, 5-trichloro-1, 3-isophthalonitrile.

Further, the preparation method of the chlorothalonil molecularly imprinted polymer is as follows:

the method is characterized in that mother chlorothalonil is used as a template molecule, acetonitrile is used as a pore-foaming agent, acrylamide is used as a functional monomer, ethylene glycol dimethacrylate is used as a cross-linking agent, and azobisisobutyronitrile is respectively used as an initiator, and the method is as follows:

(1) weighing chlorothalonil, dissolving the chlorothalonil in acetonitrile, adding acrylamide, and performing ultrasonic oscillation for 30min at room temperature to completely combine the chlorothalonil and the acrylamide; then, adding ethylene glycol dimethacrylate and azobisisobutyronitrile, and continuing ultrasonic oscillation for 15min to fully and uniformly mix the mixture; filling nitrogen to discharge oxygen, sealing, and heating in a water bath kettle at 60 deg.C for 16h to obtain white solid polymer;

(2) crushing and grinding the white solid polymer obtained in the step (1), and sieving the white solid polymer with a 200-mesh sieve;

(3) soxhlet extracting with organic solvent to elute chlorothalonil in the polymer, eluting with ultrapure water to neutrality, and finally drying the product in a 60 ℃ oven for 24h to obtain the target desired chlorothalonil molecularly imprinted polymer MIPs taking chlorothalonil as a virtual template.

Further, in the step (1), chlorothalonil: acrylamide: ethylene glycol dimethacrylate: the molar ratio of azobisisobutyronitrile is 1:7:40: 0.6.

further, in the step (3), the elution time is 30 hours in the process of soxhlet extraction with an organic solvent to elute the chlorothalonil from the polymer.

Further, in the chlorothalonil molecular imprinting solid phase extraction column, the ratio of diatomite: the mass ratio of the chlorothalonil molecularly imprinted polymer is 1: 1-1: 2.

further, the sample to be detected comprises water, vegetables, fruits and grains.

Further, when the object to be detected is a vegetable, a fruit or grain, the method also comprises the preparation of a liquid to be detected, specifically, a sample to be detected is homogenized, then 0.5% HCl acetonitrile solution is added for extraction, then vortex oscillation is carried out for 2min, ultrasonic extraction is carried out for 30min, centrifugation is carried out for 10min at 10000r/min, and supernate is taken; blow-drying with a nitrogen blower (or rotary evaporating to dryness), dissolving with organic solvent, and diluting with pure water to obtain the solution to be tested.

Compared with the prior art, the invention has the advantages that: aiming at the food safety problem caused by chlorothalonil organochlorine pesticide residue, the invention aims at the rapid analysis and detection of chlorothalonil and degradation product residue in vegetable and water body environments; the method comprises the steps of preparing chlorothalonil molecularly imprinted polymer MIPs by using chlorothalonil as a template molecule, filling polymer powder into a solid-phase extraction column to prepare chlorothalonil molecularly imprinted solid-phase extraction column CHT MISPE, enriching and purifying chlorothalonil and degradation products thereof in vegetable and water samples, and detecting by combining a chromatographic instrument, so that an efficient and simple method is established; compared with the traditional method, the method has the advantages of higher specificity, better sensitivity, higher detection speed and the like.

Drawings

FIG. 1 is a morphological diagram of a white solid polymer in example 1;

FIG. 2 is a morphological diagram of chlorothalonil molecularly imprinted polymers MIPs in example 1;

FIG. 3 is a graph showing the effect of different types of functional monomers on the Q value in example 1;

FIG. 4 is a graph showing the UV absorption of the mixture of the functional monomer and the template molecule at different molar ratios in example 1;

FIG. 5 is a graph showing the effect of different molar ratios of template molecules to initiator on the adsorption Q value of MIPs in example 1;

FIG. 6 is a graph of the results of different elution times on the absorbance values of MIPs in example 1;

FIG. 7 is a graph showing the results of the influence of different adsorption solvent types on the adsorption Q value of MIPs in example 1 (in the graph, a.10% acetonitrile-water; b.30% acetonitrile-water; c.50% acetonitrile-water; d.70% acetonitrile-water; e. acetonitrile);

FIG. 8 is a graph showing the results of the effects of different temperatures on the Q value of the amount of adsorption in example 1;

FIG. 9 is a scanning electron microscope image of MIPs and NIPs in example 2 (in the figure, a is the morphological feature of MIPs and b is the morphological feature of NIPs);

FIG. 10 is a graph of the adsorption of MIPs to substrate at different adsorption times in example 2;

FIG. 11 is adsorption isotherms of MIPs and NIPs on different substrate molecules in example 2;

FIG. 12 is a graph showing the adsorption of 7 substrates by CHT MIPs in example 2 (in the figure, a. chlorothalonil; b.5-chloro-1, 3-isophthalonitrile; c.2, 5-dichloro-1, 3-isophthalonitrile; d.2, 4, 5-trichloro-1, 3-isophthalonitrile; e.4-hydroxychlorothalonil; f. biphenyl; g. glucose);

FIG. 13 is a graph showing the elution profile of the molecularly imprinted solid phase extraction column in example 3;

FIG. 14 is a UV panscan of 5 substances from example 4 (in the figure, a.5-chloro-1, 3-isophthalonitrile; b.2, 5-dichloro-1, 3-isophthalonitrile; c.2, 4, 5-trichloro-1, 3-isophthalonitrile; d. chlorothalonil; e.4-hydroxychlorothalonil);

FIG. 15 is a high performance liquid chromatogram of 5 standards at 236nm in example 4 (in the figure, a.4-hydroxybai fungus; b.5-chloro-1, 3-isophthalonitrile; c.2, 5-dichloro-1, 3-isophthalonitrile; d.2, 4, 5-trichloro-1, 3-isophthalonitrile; e.chlorothalonil);

FIG. 16 is a high performance liquid chromatogram of 5 standards at 220nm in example 4 (in the figure, a.4-hydroxybai fungus; b.5-chloro-1, 3-isophthalonitrile; c.2, 5-dichloro-1, 3-isophthalonitrile; d.2, 4, 5-trichloro-1, 3-isophthalonitrile; e.chlorothalonil);

FIG. 17 is a graph showing the effect of different extractant types in example 5 on the extraction efficiency of chlorothalonil and its degradation products from pakchoi (in the graph, a.0.5% HCl, 80% aqueous methanol solution; b.0.5% HCl, 100% aqueous methanol solution; c.0.5% HCl in acetonitrile);

FIG. 18 is a chromatogram of a blank sample of Brassica campestris in example 5;

FIG. 19 is a chromatogram of a labeled sample of Brassica campestris in example 5 (in the figure, a.4-hydroxyBaijun; b.5-chloro-1, 3-isophthalonitrile; c.2, 5-dichloro-1, 3-isophthalonitrile; d.2, 4, 5-trichloro-1, 3-isophthalonitrile; e.chlorothalonil);

FIG. 20 is a chromatogram of an actual sample of Pinus thunbergii in example 5;

FIG. 21 is a May slow actual sample chromatogram of example 5;

FIG. 22 is a GC-MS chromatogram of an actual sample of Pinus densiflora of example 5 (in the figure, a. sample MRM diagram; b.2, 4, 5-trichloro-1, 3-isophthalonitrile retention site; c.2, 4, 5-trichloro-1, 3-isophthalonitrile corresponding to ion pair diagram; d. chlorothalonil ion pair diagram);

FIG. 23 is a chromatogram of the photolysis of chlorothalonil in aqueous solution according to example 7;

FIG. 24 is a chromatogram of chlorothalonil and its redox products from photolysis of an aqueous solution with anthocyanins from example 7;

FIG. 25 is a chromatogram of chlorothalonil and its redox products from photolysis of procyanidins in aqueous solution as in example 7;

FIG. 26 is a chromatogram of a 30% acetonitrile aqueous solution in example 7.

Detailed Description

The following examples are given for the detailed implementation and specific operation of the present invention, but the scope of the present invention is not limited to the following examples.