Method for preparing a visual solid phase extraction column based on acidochromic molecularly imprinted covalent organic framework composite material

Abstract

This invention discloses a method for preparing a visualization solid-phase extraction column based on an acid-chromic molecularly imprinted covalent organic framework composite material. The method involves sequentially mixing 40-100 mg of the acid-chromic molecularly imprinted covalent organic framework composite material and 160-400 mg of conventional solid-phase extraction packing material with 2-10 mL of dispersion, wherein the volume ratio of chloroform to isopropanol in the dispersion is 1:9. The mixture is then sequentially transferred into an empty solid-phase extraction column, and both ends are sealed with a sieve plate. The column is then rinsed with methanol and purged with an inert gas to remove residual organic solvents. After drying, the desired product is obtained. This invention features a simple and low-cost preparation process. The resulting acid-chromic molecularly imprinted covalent organic framework composite material solid-phase extraction column exhibits uniform distribution and stable properties. It demonstrates high selectivity and large adsorption capacity for trace amounts of residual steroid hormones in samples, enabling rapid and visualized analysis and detection of trace steroid hormones in animal-derived food samples.

Description

Technical Field

[0001] This invention relates to the field of solid-phase extraction column preparation technology, and more particularly to a method for preparing a visualization solid-phase extraction column based on an acid-induced colorimetric molecularly imprinted covalent organic framework composite material. Background Technology

[0002] Steroid hormones are a class of hormone drugs containing steroidal structures. These hormones are stable, but once they enter the human body through the food chain, they can cause serious harm, including cancer, damage to reproductive function, and disorders of the nervous and immune systems. Trace amounts of steroid hormones remaining in animal-derived foods pose a potential risk to human health. Current methods for detecting trace amounts of steroid hormones in animal-derived foods include liquid chromatography and liquid chromatography-tandem mass spectrometry. However, these methods require large analytical instruments, time-consuming sample pretreatment, and have long analysis cycles, and cannot meet the need for rapid detection of trace amounts of steroid hormones in large quantities of animal-derived foods. Therefore, developing a rapid detection method for trace amounts of steroid hormones in animal-derived foods is of great practical significance for routine monitoring of these residues.

[0003] Visualized rapid detection technology is based on color changes caused by various reactions, allowing for the analysis of analytes by observing the color changes of the reaction system with the naked eye. Currently, visualized colorimetric analysis based on nanozymes is one of the research hotspots in visualized rapid detection technology. However, nanozymes themselves do not have light absorption properties; they require the introduction of an oxidant and a chromogenic substrate as a catalyst to catalyze the substrate reaction and cause color changes. Furthermore, their poor color development time greatly limits the further application of this technology. Stimulus-responsive materials can exhibit reversible changes in physical or chemical properties under external stimuli such as pH, light, humidity, and temperature. Among them, covalent organic framework materials, as a class of stimulus-responsive materials with two-dimensional or three-dimensional structures, are widely used in sensor applications, catalysis, and energy storage. Due to their efficient energy transfer performance between external stimuli and active sites, as well as their clear structure-property relationship, they hold promise as ideal materials for developing acid-induced colorimetric systems and applying them to visualized rapid detection technology.

[0004] Theoretically, acid-induced color-changing properties of covalent organic framework materials (COFrames) can be achieved by designing appropriate framework structures. Alternatively, reversible color-changing properties can be achieved by adding specific guest molecules to the pores of COFrames to form a doping system. However, acid-induced color-changing COFrames currently lack high selectivity for target analytes, severely limiting their further application in visual rapid detection technologies. Molecularly imprinted polymers can simulate the molecular recognition interaction between antigens and antibodies. Their internal molecularly imprinted pores not only exhibit excellent binding performance to template molecules but also possess specific recognition selectivity. Combining the unique high selectivity of molecularly imprinted polymers with acid-induced color-changing COFrame composites holds promise for developing visual colorimetric analysis sensors with specific recognition responses to steroid hormones. Therefore, developing an acid-induced color-changing molecularly imprinted covalent organic framework composite material that combines the specific recognition properties of molecularly imprinted polymers with the reversible color-changing properties of acid-induced color-changing covalent organic framework composites, and using it as a packing material for solid-phase extraction columns for rapid, visual, and colorimetric analysis of trace steroid hormone residues in animal-derived foods, has broad application prospects. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies by providing a method for preparing a visual solid-phase extraction column based on an acid-chromic molecularly imprinted covalent organic framework composite material. This invention features a simple preparation process, and the solid-phase extraction column based on the acid-chromic molecularly imprinted covalent organic framework composite material exhibits high selectivity for trace steroid hormones, large adsorption capacity, and enables rapid visual analysis and detection of residual trace steroid hormones in animal-derived foods.

[0006] The objective of this invention is achieved through the following technical solution: a method for preparing a visualization solid-phase extraction column based on an acid-induced color-changing molecularly imprinted covalent organic framework composite material, comprising the following steps:

[0007] (1) Mix 40-100 mg of acid-induced color-changing molecularly imprinted covalent organic framework composite material with 2-10 mL of dispersion, wherein the volume ratio of chloroform and isopropanol in the dispersion is 1:9. Stir with a glass rod until the dispersion is uniform, transfer to a solid-phase extraction column made of polypropylene, and seal both ends with a sieve plate.

[0008] (2) Mix 160-400 mg of traditional solid phase extraction packing with 2-10 mL of dispersion, wherein the volume ratio of chloroform and isopropanol in the dispersion is 1:9. Stir with a glass rod until the dispersion is uniform, transfer to the solid phase extraction column of step (1), seal the top with a sieve plate, and then wash the column with 20-100 mL of methanol.

[0009] (3) The solid phase extraction column from step (2) was purged with an inert gas to remove the residual organic solvent, and then dried under vacuum at 40°C for 24 hours to obtain a visualized solid phase extraction column based on an acid-induced colorimetric molecularly imprinted covalent organic framework composite material.

[0010] Furthermore, the acid-induced color-changing molecularly imprinted covalent organic framework composite material is prepared through the following steps:

[0011] (1.1) 60 mg of 3,5-dicarboxyphenylboronic acid, 30 mg of 1,3,5-tris(4-aminophenyl)benzene, 5 mL of 0.6 mol / L trifluoroformic acid aqueous solution, 3 mL of o-xylene, 0.5 mL of acetone and steroidal hormone template molecules were added to a high-pressure reactor and ultrasonically dispersed for 10 min until uniform dispersion. The temperature was raised to 120 °C and reacted for 72 h. The mixture was then cooled to 30 °C and washed several times with ultrapure water until the pH reached 7.0.

[0012] (1.2) The product obtained in step (1) was ultrasonically washed several times with methanol until the template molecules were no longer detectable, and then vacuum dried at 60°C for 24 hours to obtain an acid-induced color-changing molecularly imprinted covalent organic framework composite material with reversible color-changing properties.

[0013] Furthermore, the steroidal sex hormone template molecule is at least one selected from testosterone, dehydroepiandrosterone, estradiol, estriol, 20α-hydroxyprogesterone, and 17α-hydroxyprogesterone.

[0014] Furthermore, the conventional solid-phase extraction packing material is at least one of octaalkylsilane-bonded silica gel (C8), neutral alumina, and N-propylethylenediamine solid-phase adsorbent (PSA).

[0015] Preferably, the conventional solid-phase extraction packing material is C8.

[0016] Furthermore, in step (2), the mass ratio of the acid-induced color-changing molecularly imprinted covalent organic framework composite material to the traditional solid-phase extraction filler is 1:4 to 1:10.

[0017] The beneficial effects of this invention are that it has the advantages of simple preparation process, high selectivity for steroid hormones, large adsorption capacity, and the ability to achieve rapid visual analysis and detection of trace steroid hormone residues in animal-derived foods. Attached Figure Description

[0018] Figure 1 This is a scanning electron microscope image of the molecularly imprinted covalent organic framework composite material based on acid-induced color change according to Embodiment 1 of the present invention. Detailed Implementation

[0019] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0020] The present invention provides a method for preparing a visualization solid-phase extraction column based on an acid-chromic molecularly imprinted covalent organic framework composite material, which specifically includes the following steps:

[0021] (1) Mix 40–100 mg of the acid-chromic molecularly imprinted covalent organic framework composite material with 2–10 mL of dispersion, wherein the volume ratio of chloroform to isopropanol in the dispersion is 1:9. Stir with a glass rod until uniformly dispersed, then transfer to a solid-phase extraction column made of polypropylene and seal both ends with sieve plates. The acid-chromic molecularly imprinted covalent organic framework composite material can be prepared by the following steps:

[0022] (1.1) 60 mg of 3,5-dicarboxyphenylboronic acid, 30 mg of 1,3,5-tris(4-aminophenyl)benzene, 5 mL of 0.6 mol / L trifluoroformic acid aqueous solution, 3 mL of o-xylene, 0.5 mL of acetone and steroidal hormone template molecules were added to a high-pressure reactor and ultrasonically dispersed for 10 min until uniform dispersion. The temperature was raised to 120 °C and reacted for 72 h. The mixture was then cooled to 30 °C and washed several times with ultrapure water until the pH reached 7.0.

[0023] Furthermore, the steroidal sex hormone template molecule is at least one of testosterone, dehydroepiandrosterone, estradiol, estriol, 20α-hydroxyprogesterone, and 17α-hydroxyprogesterone.

[0024] (1.2) The product obtained in step (1) was ultrasonically washed several times with methanol until the template molecules were no longer detectable, and then vacuum dried at 60°C for 24 hours to obtain an acid-induced color-changing molecularly imprinted covalent organic framework composite material with reversible color-changing properties.

[0025] (2) Mix 160-400 mg of traditional solid phase extraction packing with 2-10 mL of dispersion, wherein the volume ratio of chloroform and isopropanol in the dispersion is 1:9. Stir with a glass rod until the dispersion is uniform, transfer to the solid phase extraction column of step (1), seal the top with a sieve plate, and then wash the column with 20-100 mL of methanol.

[0026] Furthermore, the conventional solid-phase extraction packing material can be at least one of C8, neutral alumina, and PSA.

[0027] Preferably, the conventional solid-phase extraction packing material is C8.

[0028] Furthermore, the mass ratio of the acid-induced color-changing molecularly imprinted covalent organic framework composite material to the traditional solid-phase extraction filler is 1:4 to 1:10.

[0029] (3) The solid phase extraction column from step (2) was purged with an inert gas to remove the residual organic solvent, i.e., to remove the residual dispersion and methanol, and then vacuum dried at 40°C for 24 hours to obtain a solid phase extraction column based on an acid-induced colorimetric molecularly imprinted covalent organic framework composite material.

[0030] In this invention, the performance of a solid-phase extraction column based on an acid-induced colorimetric molecularly imprinted covalent organic framework composite material is evaluated by testing actual chicken samples.

[0031] Specifically, 1.0 g of homogenized blank chicken sample and spiked chicken sample (steroid hormone spiked at 10 μg / kg) were weighed into two 50 mL centrifuge tubes, respectively. 5.0 mL of methanol / water solution (7:3 v / v) was added to each tube, and the mixture was vortexed for 5.0 min. After centrifugation at 15000 r / min for 2 min, the supernatant was transferred to another 50 mL centrifuge tube, and the extraction was repeated once with 5.0 mL of methanol / water solution (7:3 v / v). The supernatants were then combined, and 100 μL of formic acid was added to each tube. The mixture was vortexed for 1.0 min and allowed to stand. Two solid-phase extraction (SPE) columns, No. 1 and No. 2, based on acid-chromic molecularly imprinted covalent organic framework (MFC) composites, were activated with 2.0 mL of methanol / water solution (7:3 v / v). The MFC composite layer based on acid-chromic molecularly imprinted covalent organic frameworks showed a yellow color. The extracts from the blank chicken sample and the spiked sample were transferred to the MFC composite SPE columns No. 1 and No. 2, respectively. In the No. 1 SPE column, the MFC composite layer changed from yellow to orange-red, while in the No. 2 SPE column, the MFC composite layer remained yellow. The steroid hormone content in the chicken sample was identified based on the color of the system (yellow-orange-red). The results showed that the sensitivity of this acid-chromic MFC composite SPE column for detecting steroid hormones in animal-derived foods can reach 10 μg / kg.

[0032] The principle is as follows: When the sample extract does not contain steroid hormones, under the action of acid, the conjugated groups in the acid-induced color-changing molecularly imprinted covalent organic framework composite material undergo a red shift, causing its color to change from yellow to orange-red. When the sample extract contains trace amounts of steroid hormones, the steroid hormones rapidly enter the imprinted pores of the molecularly imprinted covalent organic framework composite material. The conjugated groups in the molecularly imprinted covalent organic framework composite material cannot move in the long-wavelength direction, thus not causing a color change in the material. The steroid hormone content in chicken samples can be identified based on the color (yellow-orange-red) of the acid-induced color-changing molecularly imprinted covalent organic framework composite material, enabling rapid and visual analysis of steroid hormones. The results show that the solid-phase extraction column based on acid-induced color change of molecularly imprinted covalent organic framework composite material prepared in this invention has both strong reversible color change and high selectivity. After 10 uses, the solid-phase extraction column based on acid-induced color change of molecularly imprinted covalent organic framework composite material has good selective retention ability for trace amounts of residual steroid hormones in the sample, and the detection sensitivity of steroid hormones can reach 10 μg / kg.

[0033] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so as to highlight the advantages and beneficial effects of the present invention. However, the present invention is not limited to the following embodiments.

[0034] Example 1: Preparation of a solid-phase extraction column based on an acid-induced colorimetric molecularly imprinted covalent organic framework composite material

[0035] (1) Mix 40 mg of acid-induced color-changing molecularly imprinted covalent organic framework composite material with 5 mL of dispersion, wherein the volume ratio of chloroform and isopropanol in the dispersion is 1:9. Stir with a glass rod until the dispersion is uniform, transfer to a solid phase extraction column made of polypropylene, and seal both ends with a sieve plate.

[0036] (2) Mix 360mg of powdered C8 filler with 10mL of dispersion, wherein the volume ratio of chloroform and isopropanol in the dispersion is 1:9. Stir with a glass rod until the dispersion is uniform, transfer to the solid phase extraction column of step (1), seal the top with a sieve plate, and then wash the column with 50mL of methanol.

[0037] (3) The solid phase extraction column obtained in step (2) is purged with an inert gas to remove the residual organic solvent, that is, to remove the residual dispersion and methanol, and then vacuum dried at 40°C for 24 hours to obtain a solid phase extraction column based on an acid-induced color change molecularly imprinted covalent organic framework composite material.

[0038] Performance evaluation of solid-phase extraction columns based on acid-chromic molecularly imprinted covalent organic framework composite materials:

[0039] (1) Weigh 1.0g of homogenized blank chicken sample into a 50mL centrifuge tube, add 5.0mL of methanol / water solution (7:3v / v), vortex for 5.0min; centrifuge at 15000r / min for 2min, transfer the supernatant to another 50mL centrifuge tube, and repeat the extraction once with 5.0mL of methanol / water solution (7:3v / v), combine the supernatants, add 100μL of formic acid, vortex for 1.0min, and wait for loading.

[0040] (2) The solid-phase extraction column of acid-induced colorimetric molecularly imprinted covalent organic framework composite material was activated with 2.0 mL of methanol / water solution (7:3 v / v). The layer of acid-induced colorimetric molecularly imprinted covalent organic framework composite material showed a yellow color. The sample extract was loaded onto the column at a flow rate of 1.0 mL / min. After the loading was completed, the color change of the packing material in the acid-induced colorimetric molecularly imprinted covalent organic framework composite material solid-phase extraction column was observed with the naked eye to identify the content of steroid hormones.

[0041] The operation steps of Examples 2 to 9 are the same as those of Example 1. The raw materials, raw material formulas and preparation conditions of Examples 1 to 9 are shown in Table 1.

[0042] Table 1: Raw material components and preparation parameters of Examples 1-9 of the present invention

[0043]

[0044] The solid-phase extraction column based on acid-induced colorimetric molecularly imprinted covalent organic framework composite material described in this invention has been experimentally demonstrated to have the following characteristics: The preparation process is simple and low-cost; the resulting solid-phase extraction column is uniformly distributed and stable; it exhibits high selectivity and large adsorption capacity for trace amounts of residual steroid hormones in animal-derived food samples, enabling rapid and visual analysis and detection of these hormones, with a detection sensitivity reaching 10 μg / kg.

[0045] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a visualization solid-phase extraction column based on an acid-chromic molecularly imprinted covalent organic framework composite material, characterized in that, Includes the following steps: (1) Mix 40~100mg of acid-induced color-changing molecularly imprinted covalent organic framework composite material with 2~10mL of dispersion, wherein the volume ratio of chloroform and isopropanol in the dispersion is 1:

9. Stir with a glass rod until the dispersion is uniform, transfer to a solid phase extraction column made of polypropylene, and seal both ends with a sieve plate. The acid-induced color-modulated molecularly imprinted covalent organic framework composite material was prepared through the following steps: (1.1) 60 mg of 3,5-dicarboxyphenylboronic acid, 30 mg of 1,3,5-tris(4-aminophenyl)benzene, 5 mL of 0.6 mol / L trifluoroformic acid aqueous solution, 3 mL of o-xylene, 0.5 mL of acetone and steroidal sex hormone template molecules were added to a high-pressure reactor and ultrasonically dispersed for 10 min until uniform dispersion. The temperature was raised to 120 °C and reacted for 72 h. The mixture was then cooled to 30 °C and washed several times with ultrapure water until the pH was 7.

0. (1.2) The product obtained in step (1) was ultrasonically washed several times with methanol until the template molecules were no longer detectable, and then vacuum dried at 60°C for 24 hours to obtain an acid-induced color-changing molecularly imprinted covalent organic framework composite material with reversible color-changing properties. (2) Mix 160~400mg of traditional solid phase extraction packing with 2~10mL of dispersion, wherein the volume ratio of chloroform and isopropanol in the dispersion is 1:

9. Stir with a glass rod until the dispersion is uniform, transfer to the solid phase extraction column of step (1), seal the top with a sieve plate, and then wash the column with 20~100mL of methanol. (3) The solid phase extraction column from step (2) was purged with an inert gas to remove the residual organic solvent, and then dried under vacuum at 40°C for 24 hours to obtain a visualized solid phase extraction column based on an acid-induced colorimetric molecularly imprinted covalent organic framework composite material.

2. The method for preparing a visualized solid-phase extraction column based on an acid-induced colorimetric molecularly imprinted covalent organic framework composite material according to claim 1, characterized in that, The steroidal sex hormone template molecule is at least one of testosterone, dehydroepiandrosterone, estradiol, estriol, 20α-hydroxyprogesterone, and 17α-hydroxyprogesterone.

3. The method for preparing a visualized solid-phase extraction column based on an acid-induced colorimetric molecularly imprinted covalent organic framework composite material according to claim 1, characterized in that, The conventional solid-phase extraction packing material is at least one of octaalkylsilane-bonded silica gel, neutral alumina, and N-propylethylenediamine solid-phase adsorbent.

4. The method for preparing a visualized solid-phase extraction column based on an acid-induced colorimetric molecularly imprinted covalent organic framework composite material according to claim 3, characterized in that, The conventional solid-phase extraction packing material is octaalkylsilane-bonded silica gel.

5. The method for preparing a visualized solid-phase extraction column based on an acid-induced colorimetric molecularly imprinted covalent organic framework composite material according to claim 1, characterized in that, In step (2), the mass ratio of the acid-induced color-changing molecularly imprinted covalent organic framework composite material to the traditional solid-phase extraction filler is 1:4 to 1:10.

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