Preparation method and application of fumonisin molecularly imprinted polymer

Abstract

The present invention discloses the preparation of a fumonisin molecular imprinting polymer and its application in the field of food safety detection. The present invention adopts a double substitution template method to improve the similarity between the substitution template and the target molecule, increase the affinity and selectivity of the molecular imprinting material to fumonisin, and reduce the material preparation cost. Based on the principle of solid phase extraction, a fumonisin molecular imprinting solid phase extraction technology is established to achieve specific recognition, efficient separation and enrichment of fumonisin, and finally realize the detection of fumonisin contamination in agricultural products and feed.

Description

Technical Field

[0001] The invention relates to the technical field of food safety detection, and more particularly to a preparation method and application of a fumonisin molecular imprinting polymer. Background Art

[0002] Fumonisins (FBs), also known as fumonisins, are small molecule secondary metabolites produced by Fusarium moniliforme and Fusarium verticillium under specific temperature and humidity. They are detected at a high rate in cereal crops such as corn, rice, and sorghum, among which corn is most susceptible to infection.

[0003] Fumonisins are a class of structurally similar diester compounds composed of polyhydric alcohols and tricarbamic acid, including a fatty backbone consisting of 20 carbon atoms and a hydrophilic side chain connected by two ester bonds. There are more than a dozen fumonisins discovered so far, among which fumonisin B1 (FB1) is its main component.

[0004] FB1 is a water-soluble fungal toxin that is heat-stable and not easily destroyed by cooking. FB1 is a structural analog of sphingosine and dihydrosphingosine, which can compete with ceramide synthase, leading to the accumulation of sphingosine and dihydrosphingosine, thereby reducing the synthesis of complex sphingosine esters. The toxicity of FB1 mainly includes neurotoxicity, immunotoxicity, reproductive toxicity, organ toxicity and carcinogenicity. It can cause equine leukomalacia and porcine pulmonary edema, and is also associated with human esophageal cancer and neural tubular defect.

[0005] The International Agency for Research on Cancer has classified FB1 as a Class 2B carcinogen. Currently, the detection method for fumonisin mainly uses solvent extraction followed by solid phase extraction purification followed by liquid chromatography-mass spectrometry detection. However, the detection samples of fumonisin are mainly cereal crops such as corn, which have complex matrices and severe interference. Therefore, developing a sample pretreatment technology with high affinity and selectivity for FB1 is of great significance and role in improving the sensitivity and accuracy of FB1 contamination detection in agricultural products and feed.

[0006] Molecular Imprinting Polymers (MIPs) technology, which simulates the interaction between enzymes and substrates, receptors and ligands in nature, has developed rapidly in recent years and has been applied to the field of food safety detection technology. This technology uses the target analyte or its structural analog as a template. First, it uses the covalent or non-covalent binding between the functional monomer and the template molecule to form a monomer-template molecule complex; then the complex is "frozen" in a three-dimensional polymer structure through a polymerization reaction; finally, the template molecule is removed from the polymer to form a cavity that is complementary to the template molecule. Compared with traditional solid phase extraction methods, molecular imprinting technology can achieve selective recognition and capture of target molecules, and thus has better specificity.

[0007] Compared with biological molecules such as antibodies and nucleic acid aptamers, molecular imprinting materials have better chemical stability, simpler preparation process, lower cost, can be used for the pretreatment of complex samples, and can be reused many times.

[0008] However, since molecular imprinting polymers require a large amount of target compounds as template molecules during the preparation process and fumonisin standards are expensive, there are few studies on molecular imprinting polymer materials targeting fumonisin.

[0009] In addition, traditional molecular imprinting polymer templates are difficult to completely elute, and template leakage may occur during the pretreatment process, resulting in inaccurate test results. The use of alternative templates can effectively solve these problems. The molecular imprinting technology using FB1 structural analogs as alternative templates combined with chromatography-mass spectrometry is expected to play an important role in sample pretreatment in FB1 qualitative and quantitative analysis.

[0010] Therefore, the present invention adopts a double replacement template to improve the similarity between the template and FB1 and increase the binding sites of the polymer, prepares FB1 molecular imprinting polymer by free radical polymerization, and establishes a FB1 molecular imprinting solid phase extraction-liquid chromatography-mass spectrometry method based on solid phase extraction technology to realize the detection of FB1 contamination in agricultural products. Further verification shows that the material is also suitable for sample pretreatment for the detection of fumonisins B2 and B3 in agricultural products. Summary of the invention

[0011] The object of the present invention is to provide a molecular imprinting polymer for detecting fumonisin, wherein the molecular imprinting polymer can realize the specific recognition, capture and enrichment of fumonisin in agricultural products through molecular imprinting recognition and solid phase extraction technology.

[0012] In order to achieve the above object, the present invention adopts the following technical solution:

[0013] A method for preparing a fumonisin molecularly imprinted polymer comprises the following steps:

[0014] (1) dissolving succinic acid and D-sphingosine in acetonitrile, adding diethylaminoethyl methacrylate after dissolution and shaking to mix, then adding trimethylolpropane trimethacrylate and azobisisobutyronitrile, ultrasonically mixing and removing bubbles, sealing and irradiating under ultraviolet light to obtain a blocky polymer solid;

[0015] (2) Grinding and sieving the obtained block polymer solid to make fine powder, and then eluting the template with formic acid-methanol solvent to obtain the fumonisin molecularly imprinted polymer.

[0016] Preferably, the molar ratio of diethylaminoethyl methacrylate to succinic acid and D-sphingosine in step (1) is 23:8:3.

[0017] Preferably, the molar ratio of trimethylolpropane trimethacrylate and azobisisobutyronitrile to succinic acid and D-sphingosine in step (1) is 86:6:8:3.

[0018] Preferably, in step (1), the oscillation time is 4-6 min; the wavelength of the ultraviolet lamp is 360-370 nm; and the irradiation time is 23-25 ​​h.

[0019] Preferably, the grinding in step (2) is performed using an agate mortar; the sieving is performed using a 200-mesh sieve; and the elution is performed using a Soxhlet extractor.

[0020] Preferably, the volume ratio of methanol to formic acid in the formic acid-methanol solvent in step (2) is 99:1.

[0021] Another object of the present invention is to provide the use of the fumonisin molecularly imprinted polymer prepared by the preparation method of the fumonisin molecularly imprinted polymer in the detection of fumonisins in agricultural products.

[0022] The above application specifically includes: aspirating 900 μL of sample into a centrifuge tube, adding 100 μL of 10 μg / mL fumonisin standard solution, vortexing thoroughly, and standing for 10 minutes. As the test solution, accurately weigh 20 mg of fumonisin molecular imprinting polymer, fill it into a blank solid phase extraction column, activate it alternately with methanol-water, directly load the test solution after activation to ensure its natural dripping, rinse with pure water, elute with acidified methanol, blow dry with nitrogen at 30-50°C, and then dissolve it in methanol-water solution, pass through a 0.22 μm filter membrane and detect it on an LC-MS / MS machine.

[0023] The use of a fumonisin molecular imprinted polymer in the detection of fumonisins in agricultural products according to claim 8, characterized in that the acidified methanol is acidified with 1% volume fraction of formic acid; and the methanol aqueous solution is 50% volume fraction of methanol.

[0024] It can be seen from the above technical solution that compared with the prior art, the present invention has the following beneficial effects:

[0025] The present invention adopts a double replacement template method to improve the similarity between the replacement template and the target molecule, increase the affinity and selectivity of the molecular imprinting material to fumonisin, and reduce the material preparation cost. Based on solid phase extraction technology, a fumonisin molecular imprinting solid phase extraction technology is established to realize the detection of fumonisin contamination in agricultural products, and realize the specific identification, efficient separation and enrichment of fumonisin in agricultural products. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0027] Figure 1 is a scanning electron microscopy image of fumonisin molecularly imprinted polymer.

[0028] Figure 2 The adsorption rate bar graph of fumonisin B1

[0029] Figure 3 The adsorption rate bar graph of fumonisin B2

[0030] Figure 4 It is the bar graph of the adsorption rate of fumonisin B3.

[0031] Figure 5 This is the infrared spectrum of the synthesized fumonisin molecularly imprinted polymer. DETAILED DESCRIPTION

[0032] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0033] Example 1

[0034] Preparation of Fumonisin Molecularly Imprinted Polymer

[0035] Take a certain proportion of succinic acid and D-sphingosine standards and dissolve them in acetonitrile. Add diethylaminoethyl methacrylate according to the molar ratio of diethylaminoethyl methacrylate to succinic acid and D-sphingosine of 23:8:3, vortex and shake for 5 minutes, add trimethylolpropane trimethacrylate (with azobisisobutyronitrile, succinic acid and D-sphingosine in a molar ratio of 86:6:8:3) and azobisisobutyronitrile (10 mg), and ultrasonicate for 15 minutes. After sealing, place it under 365nm ultraviolet light for 24 hours. After the reaction is completed, pour the block solid polymer into an agate mortar, grind it into fine powder, and elute it with formic acid-methanol (1:99, v / v) solvent on a Soxhlet extractor for 48 hours.

[0036] Example 2

[0037] Molecularly imprinted solid phase extraction method for fumonisins

[0038] Accurately pipette 900 μL of corn flour extract sample into a 10 mL centrifuge tube, add 100 μL of 10 μg / mL fumonisin standard solution, vortex thoroughly, and let stand for 10 min. As the test solution, accurately weigh 20 mg of fumonisin molecularly imprinted polymer, fill it into a blank solid phase extraction column, activate it alternately with methanol-water, directly load the sample after activation to ensure its natural dripping, rinse with pure water, and after it is dried, elute with methanol-formic acid solution (1% formic acid), blow nitrogen at 40°C, and dissolve it in methanol-water solution (50% methanol), pass through a 0.22 μm filter membrane, and detect it by LC-MS / MS.

[0039] Data Experiment

[0040] (1) Linear range, adsorption rate and detection limit of the method

[0041] Corn flour was selected as the actual sample. After the sample was purified by solid phase extraction (SPE), the blank sample extract was used to prepare the FB1 standard solution with a concentration of 100-1000 ng / mL. The FB1 peak area and concentration showed a good linear relationship, and the obtained correlation linear coefficient (r 2 ) were all greater than 0.99. As shown in Table 1, the minimum detection limit of FB1 was 5.79 ng / mL.

[0042] (2) Add recovery experiment

[0043] Corn flour was selected as the actual sample, and four concentrations of 100 ng / mL, 200 ng / mL, 500 ng / mL, and 1000 ng / mL were set. The sample was purified and enriched by solid phase extraction, and LC-MS / MS was used for determination, and the matrix external standard method was used for quantification. The recovery rate results showed (see Table 1 for details) that the recovery rate of FB1 was between 92% and 117%.

[0044] Table 1 Adsorption rate, addition recovery and detection limit of fumonisin in corn flour samples

[0045]

[0046] In this specification, each embodiment is described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the embodiments can be referred to each other. For the device disclosed in the embodiment, since it corresponds to the method disclosed in the embodiment, the description is relatively simple, and the relevant parts can be referred to the method part.

[0047] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing a fumonisin molecularly imprinted polymer, characterized in that: The following steps are involved: (1) Succinic acid and D -sphingosine is dissolved in acetonitrile, and after being dissolved, diethylaminoethyl methacrylate is added and shaken to mix, and then trimethylolpropane trimethacrylate and azobisisobutyronitrile are added, ultrasonically mixed and bubbles are removed, and the mixture is sealed and irradiated under ultraviolet light to obtain a block polymer solid; the molar ratio of diethylaminoethyl methacrylate to succinic acid and D-sphingosine is 23:8:3; The molar ratio of trimethylolpropane trimethacrylate and azobisisobutyronitrile to succinic acid and D-sphingosine is 86:6:8:3; (2) Grinding and sieving the obtained block polymer solid to make fine powder, and then eluting the template with formic acid-methanol solvent to obtain the fumonisin molecularly imprinted polymer.

2. The method for preparing a fumonisin molecularly imprinted polymer according to claim 1, characterized in that: The shaking time in step (1) is 4-6 minutes; the wavelength of the ultraviolet lamp is 360-370nm; and the irradiation time is 23-25 ​​hours.

3. The method for preparing a fumonisin molecularly imprinted polymer according to claim 1, characterized in that: In step (2), the grinding is performed by using an agate mortar; the sieving is performed by using a 200-mesh sieve; and the elution is performed by using a Soxhlet extractor.

4. The method for preparing a fumonisin molecularly imprinted polymer according to claim 1, characterized in that: The volume ratio of methanol to formic acid in the formic acid-methanol solvent in step (2) is 99:

1.

5. Use of the fumonisin molecularly imprinted polymer prepared by the preparation method of the fumonisin molecularly imprinted polymer according to any one of claims 1 to 4 in the detection of fumonisins in agricultural products.

6. The use of a fumonisin molecularly imprinted polymer in the detection of fumonisins in agricultural products according to claim 5, characterized in that: The method comprises: drawing a sample into a centrifuge tube, adding a fumonisin standard solution with a concentration of 1 μg / mL, vortexing thoroughly, and letting it stand as a test solution, weighing a fumonisin molecular imprinting polymer, filling it into a blank solid phase extraction column, activating it alternately with methanol-water, directly loading the test solution after activation to ensure its natural dripping, rinsing with pure water, eluting with acidified methanol, drying with nitrogen at 30-50°C, re-dissolving with methanol-water solution, filtering the membrane, and detecting on an LC-MS / MS machine.

7. The use of a fumonisin molecularly imprinted polymer in the detection of fumonisins in agricultural products according to claim 6, characterized in that: The acidified methanol is acidified with 1% volume fraction of formic acid; The methanol aqueous solution contains 50% methanol by volume.

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