CN-121988299-A - Aptamer functionalized COF material and application thereof in aflatoxin detection

Abstract

The invention belongs to the technical field of analytical chemistry, and particularly relates to an aptamer functionalized COF material and application thereof in aflatoxin detection. Specifically, the invention takes vinyl COF (Tpb-Dva-COF) as a substrate, introduces amino through post-modification, and covalently fixes the aflatoxin aptamer on the surface by using a chemical crosslinking method. The invention also establishes a method for detecting aflatoxin B 1 、B 2 、G 1 、G 2 in foods and traditional Chinese medicinal materials based on the material serving as a Solid Phase Extraction (SPE) adsorbent and combining ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), and provides a high-efficiency and reliable solution for accurately detecting trace aflatoxins in complex matrixes, so that the method has good practical application value.

Inventors

• XIN JUNHONG
• NI ZIYUE
• TANG ZHIXIN
• JIANG HAIQIANG
• LI YUNLUN

Assignees

• 山东中医药大学

Dates

Publication Date

20260508

Application Date

20260331

Claims (10)

1. 1. The aptamer functionalized COF material is characterized in that the aptamer functionalized COF material takes a vinyl covalent organic framework material as a substrate, an aflatoxin nucleic acid aptamer is modified on the surface of the aptamer functionalized COF material, and the vinyl covalent organic framework material is synthesized by reacting monomers of 1,3, 5-tris (4-aminophenyl) benzene and 1, 4-dialdehyde-2, 5-divinylbenzene.
2. 2. The aptamer functionalized COF material of claim 1, wherein the vinyl covalent organic framework material is linked to the aptamer via a sulfo-SMCC cross-linker.
3. 3. The aptamer functionalized COF material of claim 1, wherein the aptamer is capable of specifically recognizing aflatoxins B 1 、B 2 、G 1 and G 2 , and preferably the nucleotide sequence of the aptamer is 5'-GTT GGG CAC GTG TTG TCT CTC TGT GTC TCG TGC CCT TCG CTA GGC CCA CA-SH-3' (SEQ ID NO: 1).
4. 4. A method of preparing an aptamer functionalized COF material according to any one of claims 1 to 3, comprising: Reacting a vinyl covalent organic framework material Tpb-Dva-COF with 4-amino thiophenol to obtain an aminated covalent organic framework material (NH 2 -COF), reacting the obtained aminated covalent organic framework material with a cross-linking agent sulfo-SMCC to obtain an activated material, activating a sulfhydryl modified aflatoxin nucleic acid aptamer, and reacting the activated material with the obtained activated material to obtain the aptamer functionalized covalent organic framework material.
5. 5. The preparation method of claim 4, wherein the specific method for reacting Tpb-Dva-COF with 4-aminophenylsulfiol comprises the steps of adding Tpb-Dva-COF, 4-aminophenylsulfol and azobisisobutyronitrile into acetonitrile solution, heating and reacting under inert gas atmosphere, and purifying to obtain the aminated covalent organic framework material; the mass ratio of the Tpb-Dva-COF to the 4-amino thiophenol to the azodiisobutyronitrile is 1-10:10-20:1-5; The inert gas is nitrogen, and the specific conditions of the heating reaction are that stirring and heating are carried out at 70-80 ℃ for 10-40 h; the purification includes the steps of acetone washing and drying.
6. 6. The preparation method of claim 4, wherein the specific conditions for the reaction of the aminated covalent organic framework material and the cross-linking agent sulfo-SMCC are that NH 2 -COF is dispersed in a buffer solution, sulfo-SMCC solution is added, after the oscillation reaction at room temperature, the mixture is centrifuged and washed by the buffer solution, and then the mixture is resuspended.
7. 7. The method according to claim 4, wherein the thiol-modified aflatoxin aptamer is activated and then reacted with the activated material, the method comprising mixing the thiol-modified aflatoxin aptamer solution with the activated material, shaking overnight at room temperature, centrifuging after the reaction to remove the supernatant, and washing with pure water.
8. 8. A solid phase extraction adsorbent comprising at least an aptamer functionalized COF material according to any one of claims 1 to 3.
9. 9. Use of the aptamer functionalized COF material according to any one of claims 1 to 3 or the solid-phase extraction adsorbent according to claim 8 for the enrichment and/or detection of aflatoxins, further comprising one or more of aflatoxins B 1 、B 2 、G 1 and G 2 .
10. 10. A method for detecting aflatoxin is characterized by comprising the steps of pretreating a sample to be detected by using the aptamer functionalized COF material according to any one of claims 1-3 or the solid phase extraction adsorbent according to claim 8, separating and enriching aflatoxin; the sample to be tested comprises food and Chinese medicinal materials, and further comprises semen Platycladi, semen Cassiae, fructus Jujubae, semen Nelumbinis, semen Scaphii Lychnophori, semen Persicae, coicis semen, semen Arachidis Hypogaeae, semen Maydis and jowar.

Description

Aptamer functionalized COF material and application thereof in aflatoxin detection Technical Field The invention belongs to the technical field of analytical chemistry, and particularly relates to an aptamer functionalized COF material and application thereof in aflatoxin detection. Background The disclosure of this background section is only intended to increase the 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 already known to those of ordinary skill in the art. Aflatoxins are a class of highly carcinogenic secondary metabolites produced by fungi such as aspergillus flavus and aspergillus parasiticus, and are listed as class I carcinogens by the international cancer research institute. The pollution of the plant is widely existing in grain and oil crops and seed traditional Chinese medicinal materials, and the plant constitutes a serious threat to public health. In order to control risks, strict limit standards are formulated in various countries, and extremely high requirements are put on the sensitivity and reliability of detection technology. However, the food and Chinese medicinal materials have complex matrixes, are rich in oil, pigment and other interferents, and can generate a strong matrix effect to influence the accurate quantification of the target. Therefore, the realization of efficient purification and accurate enrichment of aflatoxin is a prerequisite for accurate detection. At present, the detection method of aflatoxin mainly comprises an immunological method and a chromatography-mass spectrometry combination method. Among them, liquid chromatography-tandem mass spectrometry (LC-MS/MS) technology has become the first choice for trace analysis due to its high sensitivity and high specificity. However, prior to instrument analysis, efficient sample pretreatment techniques must be employed to remove matrix interference. Solid Phase Extraction (SPE) is the dominant pretreatment means. The current SPE strategy is mainly divided into two types, namely an immunoaffinity column based on antigen-antibody specificity recognition, but the core monoclonal antibody has the limitations of high cost, sensitivity to organic solvents and pH, poor batch stability and the like, and a reversed phase or mixed mode polymer adsorbent relying on non-specific interaction has insufficient selectivity, so that the target and the interference component are difficult to effectively distinguish from a complex matrix. Disclosure of Invention Aiming at the defects of the prior art, the invention provides an aptamer functionalized COF material and application thereof in aflatoxin detection. Specifically, the invention takes vinyl COF (Tpb-Dva-COF) as a substrate, introduces amino through post-modification, and utilizes a chemical crosslinking method to covalently fix the aflatoxin aptamer on the surface of the substrate. The invention also establishes a method for detecting aflatoxin B 1、B2、G1、G2 in foods and traditional Chinese medicinal materials based on the material serving as a Solid Phase Extraction (SPE) adsorbent and combining ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), and provides a high-efficiency and reliable scheme for accurately detecting trace aflatoxins in complex matrixes. Based on the above results, the present invention has been completed. In order to achieve the technical purpose, the invention adopts the following technical scheme: In a first aspect of the invention, an aptamer functionalized COF material (Apt@NH 2 -COF) is provided, wherein the aptamer functionalized COF material takes a vinyl covalent organic framework material as a substrate, and an aflatoxin nucleic acid aptamer is modified on the surface of the aptamer functionalized COF material, and the vinyl covalent organic framework material is synthesized by reacting monomers of 1,3, 5-tris (4-aminophenyl) benzene (Tpb) and 1, 4-dialdehyde-2, 5-divinylbenzene (Dva), and is named as Tpb-Dva-COF. The Tpb-Dva-COF is used as a substrate, so that the advantages of a porous crystal structure, large specific surface area, adjustable pore diameter and good chemical stability of the COF material are effectively realized, and sufficient sites and good mass transfer environment are provided for the adsorption of aflatoxin. In a second aspect of the present invention, there is provided a method for preparing the above-described aptamer functionalized COF material (apt@nh 2 -COF), the method comprising: Reacting a vinyl covalent organic framework material (Tpb-Dva-COF) with 4-amino thiophenol to obtain an aminated covalent organic framework material (NH 2 -COF), reacting the obtained aminated covalent organic framework material with a cross-linking agent sulfo-SMCC to obtain an activated material, activating a sulfhydryl modified aflatoxin nucleic acid aptamer, and reacting the activ