Apatamer-modified magnetic covalent organic framework composite material and preparation method and application thereof

Abstract

This invention discloses a magnetic covalent organic framework composite material modified with nucleic acid aptamers, its preparation method, and its applications. The composite material uses magnetic iron oxide microspheres as the core, with the surface of the core coated with a shell of covalent organic framework material. Nucleic acid aptamers capable of specifically recognizing tetracycline antibiotics are stably assembled onto the shell surface through non-covalent interactions. This invention fully utilizes the large specific surface area and electron-rich framework of magnetic covalent organic framework materials as efficient carriers for nucleic acid aptamers, effectively overcoming the shortcomings of traditional adsorption materials in terms of poor selectivity and limited adsorption capacity in complex matrices, while avoiding cumbersome covalent modification processes. This composite material exhibits excellent adsorption capacity and superior selectivity for tetracycline antibiotics, and is particularly suitable for rapid and efficient magnetic solid-phase extraction of trace tetracycline antibiotics in complex animal-derived food matrices, significantly improving the detection sensitivity and accuracy of subsequent instrumental analysis.

Description

Technical Field

[0001] This invention relates to the fields of nanomaterials science and food safety testing technology, specifically to a method for preparing a nucleic acid aptamer-functionalized magnetic covalent organic framework composite material based on a dual recognition mechanism, and the application of this material as a magnetic solid-phase extraction adsorbent in the detection of trace tetracycline antibiotics in complex food matrices such as milk and livestock meat. Background Technology

[0002] Tetracycline antibiotics (TCs) are a class of broad-spectrum antibiotics, mainly including tetracycline (TC), chlortetracycline (CTC), oxytetracycline (OTC), and doxycycline (DC). Due to their broad antibacterial spectrum, low cost, and ease of use, TCs are widely used in animal husbandry and aquaculture for the prevention and treatment of bacterial infections and to promote animal growth. However, the overuse and improper use of antibiotics have led to serious food safety problems. TC residues in animal-derived foods (such as milk, pork, chicken, and beef) can not only cause allergic reactions and liver and kidney toxicity in humans, but more seriously, long-term low-dose intake can induce the development of drug-resistant strains and even lead to the emergence of "superbugs," posing a significant threat to global public health. Therefore, countries and organizations such as China, the European Union, and the United States have established strict maximum residue limits (MRLs) for TCs in food (usually at the µg / kg level).

[0003] Magnetic solid-phase extraction (MSPE) is an emerging sample pretreatment technique that utilizes magnetic adsorbents dispersed in a sample solution to capture target analytes, followed by rapid separation using an external magnetic field. It offers advantages such as simplicity and speed. Magnetic covalent organic frameworks (MCOFs) combine the large specific surface area and well-organized pore structure of COF materials with the superparamagnetism of magnetic nanoparticles, making them highly promising adsorbents for MSPE. However, the capture of target molecules by MCOFs relies primarily on non-specific interactions such as π-π stacking and hydrophobic interactions. This results in insufficient selectivity when processing complex food matrices, and they are prone to co-extracting large amounts of interfering substances, severely impacting the accuracy of subsequent trace analysis.

[0004] To address this challenge, the introduction of specific recognition elements has become crucial. Aptamers, as single-stranded oligonucleotides for in vitro screening, can bind to target molecules with extremely high affinity and specificity through their specific three-dimensional structure, and are hailed as "chemical antibodies."

[0005] In view of this, the present invention aims to organically combine the high loading advantage of MCOF with the high specificity recognition capability of nucleic acid aptamers to construct a novel functionalized magnetic adsorption material. This material aims to effectively solve the technical bottlenecks of large matrix interference and low purification efficiency faced by existing technologies in enriching trace amounts of tetracycline antibiotics in complex food matrices through a "dual recognition" mechanism. Summary of the Invention

[0006] The main objective of this invention is to provide a magnetic covalent organic framework composite material modified with nucleic acid aptamers, its preparation method, and its application, so as to overcome the defects of existing materials and effectively solve the technical problems such as large matrix interference and low purification efficiency faced by existing technologies when enriching trace amounts of tetracycline antibiotics in complex food matrices.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] In a first aspect, the present invention provides a nucleic acid aptamer-modified magnetic covalent organic framework composite material, comprising:

[0009] The core body is an amino-functionalized magnetic iron oxide microsphere;

[0010] The shell, which covers the surface of the core, is a covalent organic framework material composed of repeating units as shown in Formula I; and

[0011] A recognition element, wherein the recognition element is a nucleic acid aptamer that specifically recognizes tetracycline antibiotics, which is assembled onto the surface of the shell through non-covalent interactions; the sequence of the nucleic acid aptamer is:

[0012] 5'-ACGTTGACGYTGGTGCCCGGTTGTGGTGCGAGTKKTGTG-3', where the italicized part is the core sequence, K is G or T, Y is C or T, and it can specifically bind to tetracycline antibiotics.

[0013] The nucleic acid aptamer-modified magnetic covalent organic framework composite material of the present invention possesses mesoporous properties and can serve as an adsorbent for specifically binding tetracycline antibiotics. This composite material uses magnetic iron oxide microspheres (i.e., magnetic nanoparticles) as the core, the surface of which is coated with a shell of covalent organic framework material with abundant conjugated structures. Nucleic acid aptamers that specifically recognize tetracycline antibiotics are stably assembled onto the surface of this magnetic covalent organic framework shell through non-covalent interactions. This invention fully utilizes the large specific surface area and electron-rich framework of magnetic covalent organic framework materials as efficient carriers for nucleic acid aptamers, constructing a dual recognition mechanism of "high-capacity adsorption-specific capture," effectively overcoming the shortcomings of traditional adsorbent materials with poor selectivity and limited adsorption capacity in complex matrices, while avoiding cumbersome covalent modification processes. This composite material exhibits excellent adsorption capacity and superior selectivity for tetracycline antibiotics, and is particularly suitable for rapid and efficient magnetic solid-phase extraction of trace tetracycline antibiotics in complex animal-derived food matrices (such as milk, pork, chicken, and beef), significantly improving the detection sensitivity and accuracy of subsequent instrumental analysis.

[0014] In this invention, the CGYTGGTG and GTKKTGT portions of the nucleic acid aptamer sequence are the core sequences and are key to achieving specific binding of tetracycline antibiotics. CGYTGGTG and GTKKTGT can be nucleotide sequences that have undergone one or more substitutions and / or deletions and / or additions, as long as the sequences have the same or similar ability to specifically bind tetracyclines.

[0015] In this invention, GTKKTGT is selected from GTGGTGT, GTTTTGT, GTGTTGT, or GTTGTGT. Preferably, GTKKTGT is GTGTTGT.

[0016] In this invention, CGYTGGTG is CGCTGGTG or CGTTGGTG. Preferably, CGYTGGTG is CGCTGGTG.

[0017] Preferably, the average diameter of the nucleus is 350-600 nm.

[0018] Preferably, the shell thickness of the unassembled nucleic acid aptamer is 35-40 nm.

[0019] Preferably, the shell thickness after assembling the nucleic acid aptamer is 55-60 nm.

[0020] Preferably, the covalent organic framework material is prepared by reacting an aldehyde monomer and an amino monomer, wherein the aldehyde monomer is 1,3,5-benzenetriformaldehyde and the amino monomer is 5',5''-bis(4-aminophenyl)-[1,1':3',1'':3'',1'''-tetraphenyl]-4,4'''-diamine.

[0021] Preferably, the molar ratio of the aldehyde monomer to the amino monomer is (2-4):1.

[0022] Preferably, the sequence of the nucleic acid aptamer is 5'-ACGTTGACGCTGGTGCCCGGTTGTGGTGCGAGTGTTGTG-3'.

[0023] Furthermore, the non-covalent interactions include π-π stacking interactions, electrostatic interactions, and hydrogen bonding interactions.

[0024] Preferably, the average pore size of the composite material is 22-26 nm.

[0025] Preferably, the specific surface area of ​​the composite material is 31-35 m². 2 / g.

[0026] Preferably, the saturation magnetization of the composite material is 25-28 emu / g.

[0027] Preferably, the adsorption capacity of the nucleic acid aptamer-modified magnetic covalent organic framework composite material for the tetracycline antibiotic is 160-190 mg / g. -1 .

[0028] Preferably, the adsorption equilibrium time of the tetracycline antibiotic by the nucleic acid aptamer-modified magnetic covalent organic framework composite material is 10-30 min.

[0029] Furthermore, in the X-ray powder diffraction data, the composite material exhibits crystal diffraction peaks corresponding to Fe3O4 at 2θ angles of 30.2°, 35.6°, 43.2°, 53.5°, 57.1°, and 62.7°.

[0030] Secondly, the present invention provides a method for preparing the above-described nucleic acid aptamer-modified magnetic covalent organic framework composite material, comprising the following steps:

[0031] (1) Preparation of core-shell type magnetic covalent organic framework material: Amino-functionalized magnetic iron oxide microspheres were prepared by solvothermal method. Covalent organic framework shells composed of condensation of aldehyde monomers and amino monomers were grown in situ on the surface of the amino-functionalized magnetic iron oxide microspheres by seed growth method.

[0032] The seed growth method includes: first, dissolving amino-functionalized magnetic iron oxide microspheres and aldehyde monomers in an organic solvent, then sonicating and stirring the reaction to anchor the aldehyde monomer molecules on the surface of the amino-functionalized magnetic iron oxide microspheres to form a seed layer; then adding an organic solvent solution containing aldehyde monomers and amino monomers and an acetic acid catalyst to carry out a polymerization reaction to obtain the magnetic covalent organic framework material.

[0033] (2) Nucleic acid aptamer pretreatment: The lyophilized nucleic acid aptamer powder was dissolved in binding buffer and activated by heating or incubation to promote proper refolding, resulting in a refolded nucleic acid aptamer solution.

[0034] (3) Non-covalent assembly: The magnetic covalent organic framework material obtained in step (1) is mixed with the refolded nucleic acid aptamer solution obtained in step (2) and incubated at room temperature so that the nucleic acid aptamer is fixed on the surface of the magnetic covalent organic framework material through non-covalent interaction. After magnetic separation, washing and drying, the composite material is obtained.

[0035] In step (1) of the method described above, the seed growth method is an innovative preparation method. The core of the core-shell MCOF is the precise coating of the magnetic core (i.e., magnetic iron oxide microspheres or magnetic Fe3O4 microspheres) with the covalent organic framework material (COF) shell. Currently, the commonly used preparation methods in this field are mainly direct mixing and in-situ polymerization (one-step / two-step). The direct mixing method physically mixes the magnetic core with the pre-made COF powder. It is the simplest to operate, but it has no core-shell structure and is only a simple complex. The interfacial bonding is weak and the dispersibility is poor, so it has been gradually phased out. The in-situ polymerization method disperses the magnetic core in the monomer reaction system of COF and allows COF to directly polymerize and grow on the surface of the magnetic core. It is currently the most mainstream method for preparing core-shell MCOF (the two-step in-situ polymerization method is more commonly used. First, the surface of the magnetic core is modified to introduce active sites, and then COF polymerization is initiated).

[0036] In the preparation method of this invention, a functionalized magnetic seed layer is first prepared, and then a COF shell is directionally grown using the seed as the core. The boundaries between the two processes are clear. Compared with the existing classical processes, this method achieves directional, uniform, and controllable thick-walled coating of the COF shell on the Fe3O4 surface. The prepared MCOF retains more than 90% of its performance after six cycles of use. The specific purpose and function of each step in the seed growth method include:

[0037] The first stage is the directional construction of the seed layer (i.e., the nucleation stage): This involves a covalent anchoring step between separately designed amino-functionalized magnetic iron oxide microspheres (Fe3O4-NH2) and the aldehyde monomer 1,3,5-benzenetriformaldehyde (Bz), rather than directly mixing all monomers with the magnetic core. Utilizing the Schiff base condensation between the -NH2 group on the Fe3O4-NH2 surface and the -CHO group on Bz, a layer of Bz molecules is grafted onto the magnetic core surface as active seed sites, forming a uniform COF growth "substrate." This solves the problems of uneven nucleation and the tendency for bare nuclei to form on the magnetic core surface in conventional methods, and is the core step of the seed growth method.

[0038] Then comes the seed-centered shell growth stage: the subsequently added 1,3,5-benzenetriformaldehyde (Bz) and 5',5''-bis(4-aminophenyl)-[1,1':3',1'':3'',1'''-tetraphenyl]-4,4'''-diamine (BAPQ) monomers are not randomly polymerized in the system, but rather take the Bz seed sites already anchored on the magnetic core surface as the sole growth starting point, and achieve directional, layer-by-layer growth of COF crystal layers through interfacial Schiff base condensation, ultimately forming a core-shell structure (Fe3O4-NH2)-shell (COF), rather than a random complex.

[0039] Finally, there is the interface confinement reaction stage: the growth of the entire COF shell is completed in the interface region on the surface of the magnetic seed. The introduction of the catalyst acetic acid only acts on the condensation reaction at the seed interface, avoiding the homogeneous polymerization of COF in the solution to form free COF powder, thus ensuring the purity and integrity of the core-shell structure.

[0040] Preferably, in step (1), the preparation of amino-functionalized magnetic iron oxide microspheres by solvothermal method includes: dissolving 10 g of ferric chloride hexahydrate, 2.0 g of sodium acetate and 5 mL of hexamethylenediamine in 30 mL of ethylene glycol and stirring vigorously for 30 minutes to obtain a uniform precursor solution; then sealing the mixture in a 50 mL polytetrafluoroethylene-lined autoclave and heating it at 190°C for 6 hours; after natural cooling, collecting the obtained black precipitate by magnetic force, washing and purifying it repeatedly with 3 × 50 mL of ultrapure water and ethanol, then drying it in an oven at 60°C for 12 hours, and then storing it in the dark.

[0041] Preferably, in step (1), the seed growth method includes: firstly, dissolving amino-functionalized magnetic iron oxide microspheres and aldehyde monomers in an organic solvent, wherein the mass ratio of the amino-functionalized magnetic iron oxide microspheres to the aldehyde monomers is (3-3.5):1; after ultrasonic treatment, stirring reaction is carried out at a stirring speed of 250-350 rpm / min, so that the aldehyde monomer molecules are anchored on the surface of the amino-functionalized magnetic iron oxide microspheres to form a seed layer; wherein, in the ultrasonic treatment, the ultrasonic power is 50-60 kHz, the ultrasonic frequency is 100-300 W, and the ultrasonic time is 30-60 min; subsequently, adding an organic solvent solution containing dissolved aldehyde monomers and amino monomers and an acetic acid catalyst, wherein the molar ratio of the aldehyde monomers to the amino monomers is (2-3):1, and performing a Schiff base condensation reaction to obtain the magnetic covalent organic framework material.

[0042] Based on the preferred conditions of the above seed growth method, in step (1), the mass ratio of the amino-functionalized magnetic iron oxide microspheres to the aldehyde monomer is (3-3.5):1. During the nucleation stage, the mass ratio of Fe3O4-NH2 to Bz is (3-3.5):1, essentially to match the amount of -CHO in Bz with the total amount of -NH2 sites on the surface of Fe3O4-NH2, ensuring that the magnetic core surface is uniformly covered by aldehyde seed sites. More preferably, the mass ratio is 3.2:1.

[0043] More preferably, in step (1), the reaction is stirred at a stirring speed of 300 rpm / min.

[0044] Based on the preferred conditions of the above seed growth method, in step (1), the ultrasonic power in the ultrasonic treatment is 50~60 kHz. The ultrasonic frequency determines the uniformity of the seed layer Bz anchorage. If the frequency is too high, it cannot effectively disperse the magnetic nucleus agglomerates, the agglomerate ratio increases, and the subsequent COF shell is prone to "thick spots" or bare nuclei, resulting in a decrease in magnetic separation recovery rate. If the ultrasonic frequency is too low, large-sized cavitation bubbles are easily generated, which impact the magnetic microspheres and cause Fe3O4-NH2 to break.

[0045] Based on the preferred conditions of the above seed growth method, in step (1), the ultrasonic frequency in the ultrasonic treatment is 100~300 W. Ultrasonic power: 100~300 W (intermittent ultrasound is better to avoid local high temperature); of which 200~300 W is used for seed layer dispersion and 100~200 W is used for shell monomer dissolution. The power determines the dispersion efficiency and system stability. If the power is too low, the magnetic attraction between Fe3O4-NH2 cannot be overcome and the agglomerates cannot be dispersed; if the power is too high, local high temperature will be generated, resulting in slight volatilization of THF, and at the same time, it may damage the -NH2 functional groups on the surface of Fe3O4-NH2 (loss of active sites), and reduce the covalent anchoring efficiency of the seed layer.

[0046] Based on the preferred conditions of the above seed growth method, in step (1), the ultrasonic treatment time is 30~60 min. The core of setting the ultrasonic time to 30~60 min is to ensure "complete dispersion + initial contact". If the time is too short, the aggregates will not be completely dispersed and the seed layer will be unevenly anchored; if the time is too long, there will be no additional dispersion effect, but the system temperature will rise (>40℃) due to ultrasonic heat, which will prematurely trigger a small amount of Schiff base condensation, forming irregular seed layer nuclei, and the subsequent COF shell growth will be disordered.

[0047] Based on the preferred conditions of the above seed growth method, in step (1), the molar ratio of the aldehyde monomer to the amino monomer is (2-3):1, and a Schiff base condensation reaction is performed. During the shell growth stage, the molar ratio of the aldehyde monomer Bz to the amino monomer BAPQ is (2-3):1. However, in practice, the aldehyde monomer should be in moderate excess to offset the functional group loss in the heterogeneous reaction, ensuring that all -NH2 groups of BAPQ can fully react with -CHO, thus achieving uniform and continuous growth of the COF shell and avoiding material performance defects caused by amino residues. More preferably, the above molar ratio is 2.86:1.

[0048] Preferably, in step (1), during the entire synthesis of the magnetic covalent organic framework material, the total molar ratio of aldehyde monomer to amino monomer is (2-4):1, more preferably 3.7:1.

[0049] Preferably, in step (1), during the entire process of synthesizing the magnetic covalent organic framework material, the total mass ratio of the amino-functionalized magnetic iron oxide microspheres to the aldehyde monomer is 1:1-2, more preferably 1:1.4.

[0050] Preferably, in step (1), the temperature of the Schiff base condensation reaction is 50-70°C, more preferably 60°C.

[0051] Preferably, in step (1), the Schiff base condensation time is 1-4 hours, more preferably 3 hours.

[0052] Preferably, in step (1), the organic solvent is a tetrahydrofuran solution.

[0053] Preferably, step (2) includes: dissolving the lyophilized nucleic acid aptamer powder in a binding buffer, wherein the binding buffer is a mixed aqueous solution of 10 mM 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) and 10 mM magnesium chloride (MgCl2), preparing a nucleic acid aptamer solution, and activating it at room temperature to allow for appropriate refolding.

[0054] Preferably, the volume ratio of 10 mM HEPES to 10 mM MgCl2 in the binding buffer is (0.5-2):1, more preferably 1:1.

[0055] Preferably, in step (2), the pH value of the binding buffer is 6.0-8.0, more preferably 7.0.

[0056] Preferably, in step (2), the activation reaction temperature is 20-30°C, more preferably 25°C.

[0057] Preferably, in step (2), the activation reaction time is 0.5-2 hours, more preferably 1 hour.

[0058] Preferably, in step (2), the lyophilized nucleic acid aptamer powder is dissolved in a binding buffer to form a 50-200 nmol / mL solution, wherein the binding buffer is a mixed solution of 10 mM HEPES and 10 mM MgCl2. The nucleic acid aptamer solution is prepared and activated at room temperature to achieve appropriate refolding. In the above suitable binding buffer, appropriate refolded secondary and tertiary structures will naturally form. More preferably, step (2) further includes suspending 100 mg of magnetic covalent organic framework (MCOF) powder in 5-10 mL of ethanol, sonicating for 10-20 minutes, and then washing with ultrapure water at least 3 times by magnetic separation to equilibrate the material.

[0059] Preferably, in step (3), the incubation conditions include: incubation with shaking at room temperature and in the dark, the shaking time being 1-3 hours and the shaking frequency being 100-300 rpm; the washing step is performed using the binding buffer. More preferably, in step (3), the shaking time is 2 hours and the shaking frequency is 200 rpm.

[0060] Thirdly, the present invention provides the application of the nucleic acid aptamer-modified magnetic covalent organic framework composite material or the nucleic acid aptamer-modified magnetic covalent organic framework composite material prepared by the above-described method in the enrichment of tetracycline antibiotics or the preparation of products for enriching tetracycline antibiotics.

[0061] Preferably, the tetracycline antibiotics include at least one of tetracycline, chlortetracycline, oxytetracycline, and doxycycline.

[0062] Fourthly, the present invention provides a product enriched with tetracycline antibiotics, comprising the above-described nucleic acid aptamer-modified magnetic covalent organic framework composite material or the nucleic acid aptamer-modified magnetic covalent organic framework composite material prepared by the above-described methods.

[0063] Fifthly, the present invention provides a method for detecting tetracycline antibiotics, comprising the following steps: pretreatment of the sample to be tested using the above-described nucleic acid aptamer-modified magnetic covalent organic framework composite material, the nucleic acid aptamer-modified magnetic covalent organic framework composite material prepared by the above-described preparation method, or the above-described product enriched with tetracycline antibiotics.

[0064] (a) The composite material or the product enriched with tetracycline antibiotics is mixed with the extract of the sample to be tested to form a magnetic solid phase extraction system, and the extraction of tetracycline antibiotics is completed within 1 to 5 minutes.

[0065] (b) Separating the composite material or product enriched with tetracycline antibiotics from the solution by applying an external magnetic field.

[0066] (c) Tetracycline antibiotics were eluted from the composite material using an organic solvent, filtered through a 0.22 μm filter membrane, and then detected by HPLC-MS / MS.

[0067] Preferably, the tetracycline antibiotics include at least one of tetracycline, chlortetracycline, oxytetracycline, and doxycycline.

[0068] Preferably, in step (a), the amount of the nucleic acid aptamer-modified magnetic covalent organic framework composite material used is 1-3 mg, more preferably 2 mg, based on a 5 mL liquid sample.

[0069] Preferably, in step (a), the extraction time is 1 to 4 minutes, more preferably 2 minutes;

[0070] Preferably, in step (a), the pH of the extraction system is 6-9, more preferably 7.0-7.4;

[0071] Preferably, in step (c), elution is performed using the organic solvent acetonitrile;

[0072] Preferably, in step (c), the elution time is 0.5 to 2 minutes, more preferably 1 minute.

[0073] Preferably, in step (c), the volume of the eluent is 1 to 3 mL, more preferably 2 mL.

[0074] Preferably, in step (c), the HPLC-MS / MS method includes: separation of tetracycline antibiotics using a C18 column at a column temperature of 40°C; a mobile phase consisting of phase A (acetonitrile) and phase B (water containing 0.1% formic acid), with a flow rate of 0.4 mL / min and an injection volume of 3.0 µL; a gradient elution program including: 0-2 min, 5-25% A; 2-4 min, 25-50% A; 4-6 min, 50-95% A; 6-8 min, 95% A, hold; 8.0-8.1 min, 95-5% A; 8.1-10 min, 5% A, hold; and multiple reaction monitoring (MRM) mass spectrometry conditions including: mass spectrometry detection using an electrospray ionization (ESI) source in positive ion mode (ESI+), with a spray voltage of 4500 V; an ion source temperature of 500°C; a curtain gas pressure of 30 psi; and a collision gas pressure of 9 psi.

[0075] Preferably, the sample to be tested is a food sample, and the food includes at least one of milk, beef, pork, chicken, mutton, pork liver, goat milk, and eggs. More preferably, the food is at least one of beef, pork, and chicken.

[0076] It is worth noting that the adsorption equilibrium time used to investigate the adsorption mechanism in this invention is fundamentally different from the extraction time in actual sample pretreatment. In adsorption kinetic characterization, this invention places the adsorbent in a high-concentration pure standard solution for adsorption. By recording the adsorption amount at different time points, a kinetic model is fitted to obtain the adsorption rate constant and theoretical equilibrium adsorption capacity to infer the adsorption mechanism. The adsorption equilibrium time here is a theoretically determined value derived from the plateau phase of the fitted curve. In contrast, the extraction time in actual pretreatment refers to an empirical value optimized to meet the methodological requirements of the detection under conditions of a complex matrix and low-concentration spiking. Too short a time will result in incomplete adsorption of the target analyte, leading to a low recovery rate; too long a time will result in saturation and ineffective adsorption, wasting time.

[0077] The beneficial effects of the present invention include at least the following:

[0078] 1. High specificity and high selectivity: By covalently modifying the surface of MCOF with nucleic acid aptamers that specifically recognize tetracycline antibiotics, the adsorbent can accurately capture target substances from complex mixtures, with a selectivity that is 20-30% higher than that of unmodified MCOF.

[0079] 2. Optimized material structure and physicochemical properties: The prepared adsorbent has a high molecular weight (31-35 μm) 2 With a specific surface area of ​​ / g and an average pore size of 22-26 nm, it provides an ideal platform for high-density immobilization of nucleic acid aptamers and rapid mass transfer of target analytes.

[0080] 3. Rapid kinetics and simple operation: The adsorption process can reach equilibrium within 2 minutes, and the extraction time is short; the material has a saturation magnetization of 25-28 emu / g, and solid-liquid separation can be achieved within seconds under an external magnetic field, which greatly simplifies the operation process.

[0081] 4. Significant application results: When applied to the detection of four tetracycline antibiotics in milk, beef, pork and chicken samples, recoveries of 88.63%-108.68% and detection limits as low as 0.19 µg / kg were obtained, demonstrating its reliability and high sensitivity in practical applications.

[0082] This invention provides a magnetic covalent organic framework composite material modified with nucleic acid aptamers, which can serve as an adsorbent. The covalently linked nucleic acid aptamers possess a specific three-dimensional spatial structure, enabling them to interact with tetracycline antibiotic molecules through various non-covalent bonds such as hydrogen bonds, spatial conformation matching, and hydrophobic interactions, forming stable complexes. This forms the technical basis for the highly selective recognition and adsorption of the material. Simultaneously, the covalent organic framework, as the substrate, provides an ideal platform for high-density immobilization of nucleic acid aptamers due to its large specific surface area and abundant π-electron conjugation system, further enhancing the affinity for tetracycline antibiotic molecules with conjugated structures through π-π stacking interactions. Fe3O4-NH2 nanoparticles loaded on the surface of the covalent organic framework endow the entire composite material with excellent superparamagnetism, enabling rapid solid-liquid separation under an applied magnetic field. This solves the problems of cumbersome operation and easy clogging of traditional adsorbents (such as SPE packing materials), as well as the difficulty in recovering non-magnetic nanomaterials.

[0083] The synergistic effect of the composite material of this invention as an adsorbent—namely, the specific recognition sites provided by the nucleic acid aptamer, the large specific surface area adsorption platform provided by the covalent organic framework, and the rapid magnetic separation capability provided by the Fe3O4-NH2 nanoparticles—jointly ensures that the material of this invention exhibits high adsorption capacity, excellent selectivity, and rapid adsorption kinetics for tetracycline antibiotics. The nucleic acid aptamer-modified magnetic covalent organic framework composite material of this invention can achieve rapid and efficient enrichment and purification of trace tetracycline antibiotics in complex matrices such as milk, effectively eliminating matrix interference and thus significantly improving the sensitivity and accuracy of subsequent instrumental analysis. Attached Figure Description

[0084] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0085] Figure 1 This is a schematic diagram of the synthesis reaction and extraction process of the nucleic acid aptamer-modified magnetic covalent organic framework composite material as an adsorbent, as provided in Example 1 of the present invention.

[0086] Figure 2 This is a graph showing the batch-to-batch repeatability test results of the adsorbent provided in Example 2 of the present invention.

[0087] Figure 3 The figure shows the results of the selective adsorption experiment of the adsorbent provided in Example 2 of the present invention.

[0088] Figure 4 The images provided in Embodiment 3 of this invention are scanning electron microscope (SEM) images, transmission electron microscope (TEM) images, and elemental distribution (EDS) diagrams of the material. Figure 4 Figures A and D show Fe3O4-NH2 microspheres. Figure 4 Figures B and E in the figure are MCOFs. Figure 4 Figures C and F in the figure are APT / MCOF. Figure 4 Figures G and L in the diagram represent APT / MCOF.

[0089] Figure 5 The image shows an X-ray photoelectron spectroscopy (XPS) spectrum of the material provided in Embodiment 3 of the present invention. Figure 5 Figures A, C, E, and G are MCOF, while Figures B, D, F, H, and I are APT / MCOF.

[0090] Figure 6 This is a comprehensive material characterization diagram provided in Embodiment 3 of the present invention, wherein... Figure 6 Figure A in the diagram is a hysteresis loop diagram. Figure 6 Figure B in the figure is a thermogravimetric analysis (TGA) graph. Figure 6 Figure C in the image shows a fluorescence verification diagram of nucleic acid aptamer ligation. Figure 6 Figure D in the image shows the Fourier transform infrared (FT-IR) spectrum. Figure 6 Figure E in the figure shows the nitrogen adsorption-desorption isotherm. Figure 6 Figure F in the figure is an X-ray diffraction (XRD) pattern.

[0091] Figure 7 This is a graph showing the optimization results of magnetic solid-phase extraction parameters provided in Example 4 of the present invention, wherein... Figure 7 Figure A in the figure shows the optimization of extraction time. Figure 7 Figure B shows the optimization of elution time. Figure 7 Figure C shows the optimization of nucleic acid aptamer dosage. Figure 7 Figure D shows the optimization of adsorbent dosage. Figure 7 Figure E in the figure shows the optimization of elution solvent types. Figure 7Figure F in the figure shows the optimization of elution solvent volume. Figure 7 Figure G in the figure shows the pH optimization of the extraction solution.

[0092] Figure 8 This is a schematic diagram illustrating the adsorption performance of the adsorbent for tetracycline antibiotics provided in Example 5 of the present invention, wherein... Figure 8 Figure A in the diagram is a static adsorption diagram. Figure 8 Figure B in the diagram is a dynamic adsorption diagram.

[0093] Figure 9 This demonstrates that the MCOF structure described in Example 1 of the present invention exhibits significantly better adsorption performance for the target analyte.

[0094] Figure 10 This demonstrates that the nucleic acid aptamer used in Example 1 of the present invention exhibits significantly better adsorption specificity for the target analyte.

[0095] Figure 11 The MCOF synthesized by the synthesis process described in Example 1 of this invention has a significantly larger specific surface area than the MCOF synthesized by the synthesis process described in Example 3. Detailed Implementation

[0096] Unless otherwise defined herein, the scientific and technical terms used in conjunction with this invention shall have the meanings commonly understood by one of ordinary skill in the art. The meaning and scope of terms shall be clear; however, in any case of potential ambiguity, the definitions provided herein shall prevail over any dictionary or foreign definitions. In this application, unless otherwise stated, the use of "or" means "and / or". Furthermore, the use of the term "comprising" and other forms is non-limiting.

[0097] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, 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. Unless otherwise specified, all reagents used are commercially available analytical grade or superior grade products. All instruments used are conventional instruments in the art.

[0098] Example 1

[0099] The preparation of nucleic acid aptamer-modified magnetic covalent organic framework composites (APT / MCOF) specifically includes the following steps:

[0100] (1) Preparation of core-shell magnetic covalent organic framework (MCOF) materials:

[0101] Synthesis of amino-functionalized magnetic iron(III) oxide (Fe3O4-NH2) microspheres:

[0102] Amino-functionalized Fe3O4 microspheres were prepared by combining core synthesis with in-situ surface functionalization using a one-pot solvothermal method. Ferric chloride hexahydrate (10 g), sodium acetate (2.0 g), and hexamethylenediamine (5 mL) were sequentially dissolved in ethylene glycol (30 mL) and stirred vigorously for 30 minutes to obtain a homogeneous precursor solution. The mixture was then sealed in a 50 mL PTFE-lined autoclave and heated at 190°C for 6 hours. After natural cooling, the resulting black precipitate was collected magnetically and purified by repeated washing with ultrapure water and ethanol (3 × 50 mL each). The precipitate was then dried in a 60°C oven for 12 hours and stored in the dark.

[0103] Synthesis of magnetic covalent organic frameworks (MCOFs):

[0104] Core-shell MCOF magnetic composites were prepared using a seed growth method. To form a seed layer, 125 mg of Fe3O4-NH2 microspheres and 39 mg of 1,3,5-benzenetriformaldehyde (Bz) were dispersed in 20 mL of tetrahydrofuran (THF) in a 50 mL two-necked round-bottom flask and sonicated for 60 min (ultrasonic power 50 kHz, ultrasonic frequency 200 W). Subsequently, the suspension was stirred in a water bath at 60°C (300 rpm) for 30 min to promote the covalent anchoring of Bz molecules to the amino-functionalized magnetic core. For subsequent covalent organic framework (COF) shell growth, a solution obtained by sonicating 140 mg Bz and 156 mg 5',5''-bis(4-aminophenyl)-[1,1':3',1'':3'',1'''-tetraphenyl]-4,4'''-diamine (BAPQ) in 10 mL THF was added dropwise to the seed suspension. Subsequently, 2 mL of acetic acid (AcOH) was introduced as a catalyst. The reaction mixture was continuously stirred at 60°C (300 rpm) for 3 hours to promote interfacial Schiff base condensation. After cooling to room temperature, the product was collected by magnetic separation, purified by washing with tetrahydrofuran, methanol, and acetonitrile (three times each), and dried in an oven at 60°C for 12 hours to obtain the MCOF material.

[0105] (2) Synthesis of nucleic acid aptamer-modified magnetic covalent organic framework composite material (APT / MCOF):

[0106] 100 mg of magnetic covalent organic framework (MCOF) powder was suspended in 5 mL of ethanol, sonicated for 10 min, and then washed three times with ultrapure water by magnetic separation to equilibrate the material. Meanwhile, 100 nanomolar lyophilized aptamer powder was dissolved in 1.0 mL of binding buffer (10 mM HEPES and 10 mM MgCl2, volume ratio 1:1), vortexed (500 rpm) for 1 min, and activated at room temperature for 1 hour to achieve proper refolding.

[0107] Subsequently, 500 μL of the refolded aptamer solution was added to the equilibrated magnetic COF. The mixture was gently agitated (200 rpm) at room temperature for 2 hours (protected from light) to promote immobilization. Finally, the nucleic acid aptamer-modified magnetic covalent organic framework composite (APT / MCOF) was collected by magnetic separation. The synthetic route is as follows: Figure 1 As shown.

[0108] Example 2

[0109] This embodiment aims to evaluate the batch-to-batch reproducibility and selectivity of the nucleic acid aptamer-modified magnetic covalent organic framework composite material prepared according to the present invention as an adsorbent, so as to assess the stability and reliability of the preparation method of the present invention and the performance of the obtained composite material as an adsorbent in practical applications in complex samples. The specific details are as follows:

[0110] (1) To evaluate the batch-to-batch reproducibility of the materials, six batches of nucleic acid aptamer-modified magnetic covalent organic framework composite material (APT / MCOF) were independently prepared as adsorbents using the method described in Example 1. Each batch of adsorbent was then used to perform magnetic solid-phase extraction experiments on spiked samples containing four tetracycline antibiotics. Figure 2 As shown, the extraction recoveries of the six different batches of adsorbent for the four tetracycline antibiotics did not show significant differences, with relative standard deviations (RSDs) ranging from 2.21% to 4.93%, all within acceptable limits. These results indicate that the preparation method provided by this invention is stable, reproducible, and ensures that the prepared adsorbent exhibits uniform and reliable performance.

[0111] (2) To evaluate the selectivity of the adsorbent, the APT / MCOF adsorbent prepared in this invention and a magnetic covalent organic framework (MCOF) without nucleic acid aptamer modification were used as a control group to adsorb a mixed standard solution containing four tetracycline antibiotics and four common interfering antibiotics (sulfadiazine (SMZ), sulfadiazine (SD), enrofloxacin (ENR), and norfloxacin (NOR)). Figure 3As shown, compared with the control group MCOF, the APT / MCOF adsorbent of the present invention exhibits 20%-30% higher adsorption efficiency for four tetracycline antibiotics, while showing extremely low adsorption for four interfering mycotoxins. Experiments demonstrate that by covalently linking nucleic acid aptamers, the adsorbent of the present invention achieves excellent targeted recognition capabilities, preferentially capturing tetracycline antibiotics even in the presence of multiple mycotoxins, thus achieving highly selective enrichment.

[0112] Example 3

[0113] The nucleic acid aptamer-modified magnetic covalent organic framework composite material (APT / MCOF) prepared in Example 1 was systematically characterized to verify its chemical structure and physical properties. The specific details are as follows:

[0114] (1) Morphology analysis: The microstructure of the material was analyzed using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). For example... Figure 4 As shown in Figures A-F, Fe3O4-NH2 appears as spherical nanoparticles with a slightly rough surface. After coating, although the particle size of the MCOF and APT / MCOF composite materials increases and the surface becomes smoother, they still maintain good sphericity. TEM images clearly reveal the core-shell structure of the material: the dark Fe3O4 magnetic core is tightly wrapped by a uniformly thick and light-colored organic shell, and the shell thickness increases slightly with the modification of the aptamer, visually confirming the success of layer-by-layer assembly.

[0115] (2) Elemental composition analysis: The elemental distribution of the final composite material APT / COF was analyzed using energy dispersive spectroscopy (EDS). For example... Figure 4 As shown in Figures G-L, the surface scan results indicate that Fe is mainly distributed in the core region, while C, N, and O are uniformly distributed throughout the microspheres, confirming the existence of the organic shell. More importantly, P, a characteristic element of nucleic acid aptamers, exhibits a uniform and dense distribution on the material surface, proving that the aptamer was successfully and homogeneously grafted onto the magnetic COF surface.

[0116] (3) Surface chemical composition analysis: X-ray photoelectron spectroscopy (XPS) was used to characterize the chemical state of the material surface. For example... Figure 5 As shown, a new P 1s peak appeared in the full spectrum of APT / COF. In the high-resolution spectrum, the N 1s spectrum showed enhanced -NH2 and NH⁺ signals characteristic of the aptamer bases; the O 1s and P 2p spectra showed characteristic peaks of P=O / POC and PO / P=O bonds, respectively. These results strongly confirm from the perspective of chemical bonding that the nucleic acid aptamer has been successfully covalently immobilized on the magnetic COF surface.

[0117] (4) Magnetic property analysis: The magnetic properties of the material were evaluated using a vibrating sample magnetometer (VSM). For example... Figure 6 As shown in Figure A, both Fe3O4-NH2 and MCOF as the core, and the final product APT / MCOF, exhibit typical "S"-shaped hysteresis loops with negligible coercivity and remanence, confirming their excellent superparamagnetism. With the coating of non-magnetic shells (COF and aptamers), the saturation magnetization of the materials gradually decreases from 65.08 emu / g for Fe3O4-NH2. Even so, the final composite material APT / MCOF still retains a saturation magnetization of 26.15 emu / g, sufficient to achieve rapid solid-liquid separation within seconds under an external magnetic field, greatly simplifying the operation process.

[0118] (5) Thermal stability analysis: To investigate the material's stability at high temperatures and the amount of surface modification, thermogravimetric analysis (TGA) was used to study the mass change of the material during the heating process. For example... Figure 6 As shown in Figure B, the original COF exhibits good thermal stability below 600℃, with a weight loss of only 7.17%. In contrast, the APT / COF composite material shows a more significant weight loss (15.58%) within the same temperature range. This additional mass loss is mainly attributed to the thermal decomposition of the nucleic acid aptamers grafted onto the material surface, thus quantitatively confirming the successful loading of the aptamers.

[0119] (6) Fluorescence spectroscopy analysis: To further verify the successful connection of the aptamers, the composite material modified with the FAM fluorescent group was detected using a fluorescence spectrometer. Figure 6 As shown in Figure C, the APT / COF suspension exhibits a strong characteristic fluorescence emission peak at 518 nm, which is typical of FAM fluorescent dyes. This result provides direct evidence that the fluorescently labeled nucleic acid aptamers have been successfully covalently bound to the surface of the magnetic composite material.

[0120] (7) Functional group structure analysis: Fourier transform infrared spectroscopy (FT-IR) was used to analyze the functional groups of the products at each stage of the synthesis process. For example... Figure 6 As shown in Figure D, monomers BAPQ and Bz are located at 3400-3200 cm⁻¹. -1 and 1691 cm -1 Characteristic amino and aldehyde absorption peaks are observed at this point. In the COF spectrum, these monomer peaks disappear, replaced by a peak at 1624 cm⁻¹. -1 The newly emerging strong absorption peak is attributed to the stretching vibration of the C=N imine bond, confirming the occurrence of the Schiff base condensation reaction. After functionalization, the spectrum of APT / COF is at 1148 cm⁻¹. -1A new and significant absorption peak appeared, corresponding to the PO bond vibration in the aptamer's phosphate backbone, further confirming the successful modification of the aptamer.

[0121] (8) Pore structure analysis: such as Figure 6 Figure E shows the evaluation of the porosity characteristics of the composite materials. Nitrogen adsorption-desorption isotherms show that both COF and APT / COF exhibit typical Type IV isotherms and Type H3 hysteresis loops, indicating that the materials possess a mesoporous structure. The measured BET specific surface area of ​​APT / COF is 33.03 m². 2 / g, with an average pore size of 24.03 nm. The slight decrease in specific surface area and pore size compared to unmodified COF is attributed to the aptamer molecules occupying part of the pores, which is consistent with the conclusion of successful aptamer loading.

[0122] (9) Crystal structure analysis: The crystal structure of the material is analyzed using X-ray diffraction (XRD). For example... Figure 6 As shown in Figure F, clear diffraction peaks were observed at 2θ angles of 30.2°, 35.6°, 43.2°, 53.5°, 57.1°, and 62.7° in the diffraction pattern of APT / COF. These peak positions perfectly match the standard card for cubic spinel Fe3O4, and the sharp peak shapes indicate that the crystal structure of the magnetic core remained intact and maintained good crystallinity during the multi-step modification process. Simultaneously, the broad peaks appearing at low angles suggest the presence of an amorphous organic layer on the surface.

[0123] Example 4

[0124] In this embodiment, the magnetic covalent organic framework composite material modified with nucleic acid aptamers prepared in Example 1 was used as an adsorbent for magnetic solid-phase extraction of four tetracycline antibiotics. The difference lies in the variation of several parameters, including the amount of MCOF, the amount of APT, the extraction time, the pH of the extraction solution, the type of elution solvent, the elution time, and the elution volume. The effects of different magnetic solid-phase extraction conditions on the extraction recovery rate were investigated. Specifically, the effects of extraction time (1-6 min), elution time (0.5-5 min), APT amount (5-50 μL), MCOF amount (1-5 mg), elution solvent (methanol, acetonitrile, ethanol, ethyl acetate, isopropanol, n-hexane), elution volume (0.5-6 mL), and extraction solution pH (4-10) on the extraction recovery rate of the four tetracycline antibiotics were investigated. It should be noted that when the above parameters were varied, the specific operating steps of Example 1 or below were followed. If the conditions mentioned are different from those in Example 1 or below, the corresponding conditions are substituted. The method for enriching tetracycline antibiotics includes the following specific operational steps:

[0125] Preparation of the sample extract: Accurately weigh 1.0 g ± 0.01 g of homogenized solid sample or transfer 1.0 mL of liquid sample into a 50 mL polypropylene centrifuge tube, and add 10 mL of acetonitrile for extraction; only milk samples require the addition of 500 mg of sodium chloride to promote protein precipitation and produce a salting-out effect; after vigorous vortexing (2500 r / min) for 5 minutes, sonicate (100 W) for 5 minutes; then centrifuge at 4℃ and 8000 rpm for 10 minutes, take 10.0 mL of clear supernatant and transfer it to a clean test tube, and gently dry it with nitrogen at 40℃; the dried residue is reconstituted with 5.0 mL of deionized water, and vortexed at 2500 r / min for 1 minute to ensure complete dissolution;

[0126] Before magnetic solid phase extraction (MSPE), the resulting aqueous solution was filtered through a 0.22 μm needle filter to remove residual insoluble particles, and the final filtrate was collected for subsequent MSPE purification steps.

[0127] Activation of the adsorbent: Take 2.0 mg of APT / Fe3O4-NH2@Bz-BAPQ adsorbent, add 2.0 mL of binding buffer (10 mM HEPES buffer containing 10 mM MgCl2 (volume ratio 1:1), and activate at 25℃ and pH 7.4 for 1 hour to ensure proper folding of the nucleic acid aptamer;

[0128] MSPE purification: After magnetic separation and discarding the supernatant, the activated adsorbent is mixed with 5.0 mL of the sample extract to form a magnetic solid phase extraction system, and vortexed for 2 minutes to adsorb the target analyte;

[0129] Magnetic separation was performed with the help of an external magnet, and then the supernatant was discarded. 2 mL of acetonitrile eluent was added to the precipitate, and tetracycline antibiotics were eluted by sonication for 1 min.

[0130] After the final magnetic separation, the eluent was filtered through a 0.22 μm needle filter and then analyzed by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS).

[0131] Finally, HPLC-MS / MS was used for detection. A Shimadzu Prominence LC-30 CE HPLC chromatographic analysis system (Shimadzu Corporation, Kyoto, Japan) was used, which was connected to an AB SCIEX 6500+ triple quadrupole mass spectrometer (AB SCIEX, Redwood, USA).

[0132] Tetracycline antibiotics (TCs) were isolated using an Aglient ZORBAX Eclipse Plus C18 column (2.1 mm × 100 mm, 3.5 µm) at 40 °C. The mobile phase consisted of acetonitrile (A) and water (B) containing 0.1% formic acid, with a flow rate of 0.4 mL / min. The injection volume was 3.0 µL.

[0133] The gradient elution program is as follows: 0-2 minutes, 5-25% A; 2-4 minutes, 25-50% A; 4-6 minutes, 50-95% A; 6-8 minutes, 95% A (hold); 8.0-8.1 minutes, 95-5% A; 8.1-10 minutes, 5% A (hold).

[0134] Multiple response monitoring (MRM) mass spectrometry conditions: Mass spectrometry detection was performed using an electrospray ionization (ESI) source in positive ion mode (ESI+). Spray voltage: 4500 V; ion source temperature: 500°C; desolvation temperature: 550°C; gas curtain: 30 psi; collision gas: 9 psi. Data acquisition was performed using MRM mode. Optimized MRM parameters for four tetracycline antibiotics (TC, CTC, OTC, and DC), including parent ion mass-to-charge ratio, daughter ion mass-to-charge ratio, cone voltage, and collision energy, are shown in Table 1.

[0135]

[0136] *Quantitative Ions

[0137] All experiments were set up in triplicate, and the results were averaged. The error bars in the graph represent the standard deviation between the parallel data.

[0138] Experimental results are as follows Figure 7 As shown, specifically, as Figure 7 As shown in Figure A, the dosage of MCOF in APT / MCOF is 2 mg, as follows: Figure 7 As shown in Figure B, when the APT dosage was 10 μL, the extraction recoveries of the four tetracycline antibiotics tended to stabilize at 88-96%, indicating that only a small amount of adsorbent is needed to achieve efficient enrichment; Figure 7 As shown in Figure C, the extraction recoveries of the four tetracycline antibiotics tended to stabilize when the extraction time was 2 min; Figure 7 As shown in Figure D, different organic solvents exhibit significant differences in elution efficiency for the four tetracycline antibiotics. When acetonitrile is used as the elution solvent, the extraction recovery rates of the four tetracycline antibiotics are relatively ideal. Figure 7 As shown in Figure E, the extraction recovery rates of the four tetracycline antibiotics were optimal when the elution volume was 2 mL; Figure 7 As shown in Figure F, the extraction recoveries of the four tetracycline antibiotics tended to stabilize when the elution time was 1 min; Figure 7 As shown in Figure G, the extraction recovery rates of the four tetracycline antibiotics reached their highest at pH 7.

[0139] Under optimized experimental conditions, several key parameters of the method were evaluated. The quantitative performance of the method was verified, including the coefficient of determination (R²). 2 The matrix effect (ME), limit of detection (LOD), limit of quantitation (LOQ), accuracy, and precision were investigated. The linearity of the method was examined, and the matrix effect was evaluated by comparing the slopes of calibration curves obtained from analytes in the matrix and in the solvent. LOD and LOQ were determined using the baseline noise method, based on signal-to-noise ratios of 3 and 10, respectively. Relevant parameters of the detection method are shown in Table 2.

[0140] In summary, the composite material of the present invention, as a magnetic solid-phase extraction adsorbent, has the advantages of low material consumption, short extraction time, low organic reagent consumption, simple operation, and high selectivity, and has a very broad application prospect.

[0141] Example 5

[0142] The adsorption behavior of the nucleic acid aptamer-modified magnetic covalent organic framework composite material (APT / MCOF) prepared in Example 1 on four tetracycline antibiotics was studied, as follows:

[0143] The adsorption performance of APT / MCOF prepared in Example 1 for tetracycline antibiotic compounds of different concentrations at different times was studied by adsorption isotherm and adsorption kinetic experiments.

[0144] For the adsorption isotherm experiment, 1 mg of APT / MCOF was added to 2 mL of aqueous solutions of four tetracycline antibiotics at different concentrations (1 mg·L⁻¹). -1 5 mg·L -1 10 mg·L -1 20 mg·L -1 30 mg·L -1 40 mg·L -1 50 mg·L -1 60 mg·L -1 80 mg·L -1 100 mg·L -1 120 mg·L -1 140 mg·L -1The adsorbent was vortexed for 2 hours to ensure full contact between the adsorbent and the target analyte, and the adsorption capacity Qe (mg / g) of tetracycline antibiotics on the APT / MCOF adsorbent was studied.

[0145] For adsorption kinetics experiments, 1 mg of APT / MCOF was added to 2 mL of a solution of four tetracycline antibiotics (120 μg / mL). -1 The APT / MCOF was vortexed to allow adsorption at different times (1 min, 5 min, 10 min, 20 min, 30 min, 40 min, 60 min, and 90 min). The maximum adsorption capacity of APT / MCOF for four tetracycline antibiotic solutions was calculated.

[0146] Assume the mass of the adsorbent is m (mg), the volume of the solution is V (mL), and the initial concentration of the target analyte is C0 (μg·mL⁻¹). -1 At a certain moment (t) in the solution, the concentration of the target analyte is C. e (μg·mL) -1 Ignoring changes in solution volume, the adsorption capacity Qe (mg / g) of APT / MCOF can be calculated using the following formula:

[0147]

[0148] The static equilibrium adsorption curves of APT / MCOF for four tetracycline antibiotics at different initial concentrations are shown below. Figure 8 As shown, Figure 8 Figure A shows that when the amount of adsorbent is 1.0 mg, the adsorption capacity of the magnetic adsorbent for tetracycline antibiotics is within 0 μg·mL⁻¹. -1 Up to 140 μg·mL -1 The concentration range gradually increased, reaching 120 μg·mL -1 Adsorption equilibrium was reached at this time. The calculated maximum adsorption capacities for TC, CTC, OTC, and DC were 166.83 mg·g⁻¹. -1 176.62 mg·g -1 181.25 mg·g -1 and 182.91 mg·g -1 The dynamic equilibrium adsorption curves of APT / MCOF for four tetracycline antibiotics at different adsorption times are shown below. Figure 8As shown in Figure B, the adsorption of four tetracycline antibiotics by APT / MCOF is a rapid process, reaching adsorption equilibrium within 10–20 minutes. APT / MCOF exhibits excellent adsorption capacity and extremely fast adsorption equilibrium for these four tetracycline antibiotics, which stems from the synergistic mechanism between the magnetic covalent organic framework matrix and the surface-covalently linked nucleic acid aptamers. First, the magnetic covalent organic framework, as the matrix, with its large specific surface area and regular mesoporous structure, constructs a highly efficient physical adsorption platform, providing a wealth of active binding sites and unimpeded mass transfer pathways for the target molecules. More importantly, the abundant π-π conjugated system in its framework can undergo strong π-π stacking interactions with the planar aromatic rings of tetracycline molecules, thus achieving rapid primary enrichment of the target analytes. Second, the surface-anchored nucleic acid aptamers play a decisive recognition role. The aptamers fold into a specific three-dimensional conformation, forming a high-affinity "molecular pocket," which accurately recognizes and locks onto tetracycline molecules through multiple forces, including hydrogen bonds, electrostatic attraction, and hydrophobic effects. This specific binding is the fundamental reason why this material differs from traditional adsorbents and achieves ultra-high selectivity. In summary, the efficient physical enrichment of MCOF and the precise molecular recognition of the aptamer complement each other, jointly endowing the APT / MCOF adsorbent with excellent performance of high capacity, high selectivity and fast kinetics.

[0149] Comparative Example 1

[0150] The adsorption effects of the MCOF material (BAPQ-Bz) synthesized with Bz as the aldehyde monomer described in Example 1 (BAPQ-Tp, BAPQ-Nap) on four target tetracyclines (TC, CTC, OTC, DC) were compared with those synthesized with other structures as aldehyde monomers. The adsorbent described in Example 1 showed a significantly higher recovery rate of the target analytes than the other adsorbents, such as... Figure 9 As shown in the figure, the MCOF structure described in Example 1 has significantly better adsorption performance for the target analyte. The different MCOF materials and monomer compositions are shown in Table 3.

[0151]

[0152] Comparative Example 2

[0153] The adsorption effects of the nucleic acid aptamer (TCs1) modified magnetic covalent organic framework adsorbent described in Example 1 and other nucleic acid aptamers (TCs2-5) modified magnetic covalent organic framework adsorbents on four target tetracyclines (TC, CTC, OTC, DC) were compared. The adsorbent described in Example 1 showed a significantly higher recovery rate of the target analytes than the other adsorbents, demonstrating that the nucleic acid aptamer used in Example 1 has significantly better adsorption specificity for the target analytes. Figure 10 As shown in Table 4, the sequences of different nucleic acid aptamers are shown in Table 4.

[0154]

[0155] Comparative Example 3

[0156] The specific surface area of ​​MCOF synthesized using the MCOF synthesis process described in Example 1 was compared with that synthesized using the following process. The synthesis process of this comparative example is as follows:

[0157] (1) Disperse or dissolve Fe3O4-NH2 (0.4 mmol, 92.6 mg) and trimethylolpropionate (Bz) (0.3 mmol, 64.9 mg) in 20 mL of tetrahydrofuran solvent in a double-necked round-bottom flask (100 mL) and sonicate for 20 min.

[0158] (2) The mixture was then transferred to a water bath at 60 °C and mechanically stirred at 300 rpm for 30 min. 5',5''-bis(4-aminophenyl)-[1,1':3',1'':3'',1'''-tetraphenyl]-4,4'''-diamine (BAPQ) (0.4 mmol, 155.4 mg) was dissolved in tetrahydrofuran (10 mL) and slowly added to the above system.

[0159] (3) Acetic acid (2 mL) was added dropwise to the flask as a catalyst to accelerate the reaction. The mixture was mechanically stirred at 300 rpm for 4 h at room temperature.

[0160] (4) With the help of an external magnet, the obtained magnetic brown precipitate was washed alternately with MeOH and ACN until the supernatant was clear. Finally, the material was dried in an oven at 60°C for 8 h (yield ≥83%) to obtain magnetic brown powder particles Fe3O4-NH2@Bz-BAPQ.

[0161] Compared to the comparative example, the BET specific surface area of ​​MCOF in Example 1 was significantly increased, indicating that the synthesis conditions of Example 1 facilitated the directional, uniform, and stable formation of the COF porous structure on the Fe3O4 surface. Figure 11 As shown.

[0162] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; 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 or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A magnetic covalent organic framework composite material modified with nucleic acid aptamers, characterized in that, include: The core body is an amino-functionalized magnetic iron oxide microsphere; The shell, which covers the surface of the core, is a covalent organic framework material composed of repeating units as shown in Formula I; and ， A recognition element, wherein the recognition element is a nucleic acid aptamer that specifically recognizes tetracycline antibiotics, which is assembled onto the surface of the shell through non-covalent interactions; the sequence of the nucleic acid aptamer is: 5'-ACGTTGACGYTGGTGCCCGGTTGTGGTGCGAGTKKTGTG-3', where the italicized part is the core sequence, K is G or T, Y is C or T, and it can specifically bind to tetracycline antibiotics.

2. The composite material according to claim 1, characterized in that, The average diameter of the nucleus is 350-600 nm; and / or The shell thickness of the unassembled nucleic acid aptamer is 35-40 nm; and / or The shell thickness after assembling the nucleic acid aptamer is 55-60 nm; and / or The covalent organic framework material is prepared by reacting an aldehyde monomer and an amino monomer, wherein the aldehyde monomer is 1,3,5-benzyltricarboxaldehyde, and the amino monomer is 5',5''-bis(4-aminophenyl)-[1,1':3',1'':3'',1'''-tetraphenyl]-4,4'''-diamine; and / or The molar ratio of the aldehyde monomer to the amino monomer is (2-4):1; and / or The sequence of the nucleic acid aptamer is 5'-ACGTTGACGCTGGTGCCCGGTTGTGGTGCGAGTGTTGTG-3'; The non-covalent interactions include π-π stacking interactions, electrostatic interactions, and hydrogen bonding; and / or The composite material has an average pore size of 22-26 nm; and / or The specific surface area of the composite is 31-35 m 2 / g; and / or The saturation magnetization of the composite material is 25-28 emu / g; and / or The adsorption capacity of the nucleic acid aptamer modified magnetic covalent organic framework composite material to the tetracycline antibiotic is 160~190mg·g -1 ; and / or The adsorption equilibrium time for the tetracycline antibiotic in the nucleic acid aptamer-modified magnetic covalent organic framework composite material is 10-30 min; and / or In X-ray powder diffraction data, the composite material exhibits crystal diffraction peaks corresponding to Fe3O4 at 2θ angles of 30.2°, 35.6°, 43.2°, 53.5°, 57.1°, and 62.7°.

3. A method for preparing a magnetic covalent organic framework composite material modified with nucleic acid aptamers as described in claim 1, characterized in that, Includes the following steps: (1) Preparation of core-shell type magnetic covalent organic framework material: Amino-functionalized magnetic iron oxide microspheres were prepared by solvothermal method. Covalent organic framework shells composed of condensation of aldehyde monomers and amino monomers were grown in situ on the surface of the amino-functionalized magnetic iron oxide microspheres by seed growth method. The seed growth method includes: first, dissolving amino-functionalized magnetic iron oxide microspheres and aldehyde monomers in an organic solvent, then sonicating and stirring the reaction to anchor the aldehyde monomer molecules on the surface of the amino-functionalized magnetic iron oxide microspheres to form a seed layer; then adding an organic solvent solution containing aldehyde monomers and amino monomers and an acetic acid catalyst to carry out a polymerization reaction to obtain the magnetic covalent organic framework material. (2) Nucleic acid aptamer pretreatment: The lyophilized nucleic acid aptamer powder was dissolved in binding buffer and heated or incubated to promote proper refolding, resulting in a refolded nucleic acid aptamer solution. (3) Non-covalent assembly: The magnetic covalent organic framework material obtained in step (1) is mixed with the refolded nucleic acid aptamer solution obtained in step (2) and incubated at room temperature so that the nucleic acid aptamer is fixed on the surface of the magnetic covalent organic framework material through non-covalent interaction. After magnetic separation, washing and drying, the composite material is obtained.

4. The method according to claim 3, characterized in that, In step (1), the seed growth method includes: firstly, dissolving amino-functionalized magnetic iron oxide microspheres and aldehyde monomers in an organic solvent, wherein the mass ratio of the amino-functionalized magnetic iron oxide microspheres to the aldehyde monomers is (3-3.5):1; after ultrasonic treatment, stirring is performed at a stirring speed of 250-350 rpm / min to anchor the aldehyde monomer molecules on the surface of the amino-functionalized magnetic iron oxide microspheres to form a seed layer; wherein, in the ultrasonic treatment, the ultrasonic power is 50-60 kHz, the ultrasonic frequency is 100-300 W, and the ultrasonic time is 30-60 min; subsequently, adding an organic solvent solution containing dissolved aldehyde monomers and amino monomers and an acetic acid catalyst, wherein the molar ratio of the aldehyde monomers to the amino monomers is (2-3):1, and performing a Schiff base condensation reaction to obtain the magnetic covalent organic framework material; and / or In step (1), during the entire synthesis of the magnetic covalent organic framework material, the total molar ratio of aldehyde monomers to amino monomers is (2-4):1, preferably 3.7:1; and / or In step (1), throughout the synthesis of the magnetic covalent organic framework material, the total mass ratio of the amino-functionalized magnetic iron oxide microspheres to the aldehyde monomer is 1:1-2, preferably 1:1.4; and / or In step (1), the Schiff base condensation reaction is carried out at a temperature of 50-70°C, preferably 60°C; and / or In step (1), the Schiff base condensation time is 1-4 hours, preferably 3 hours; and / or In step (1), the organic solvent is a tetrahydrofuran solution.

5. The method according to claim 3, characterized in that, Step (2) includes: dissolving the lyophilized nucleic acid aptamer powder in a binding buffer, wherein the binding buffer is a mixed aqueous solution of 10 mM 4-hydroxyethylpiperazine ethanesulfonic acid and 10 mM magnesium chloride, preparing a nucleic acid aptamer solution, and activating it at room temperature to allow for appropriate refolding; and / or In step (2), the volume ratio of 10 mM HEPES to 10 mM MgCl2 in the binding buffer is (0.5-2):1; and / or In step (2), the pH of the binding buffer is 6.0-8.0; and / or In step (2), the activation reaction temperature is 20-30°C; and / or In step (2), the activation reaction time is 0.5-2 hours; and / or In step (3), the incubation conditions include: incubation at room temperature and in the dark with shaking for 1-3 hours at 100-300 rpm; the washing step is performed using the binding buffer.

6. The use of the nucleic acid aptamer-modified magnetic covalent organic framework composite material according to claim 1 or claim 2, or the nucleic acid aptamer-modified magnetic covalent organic framework composite material prepared by the method according to any one of claims 3-5, in the enrichment of tetracycline antibiotics or the preparation of products for enriching tetracycline antibiotics.

7. A product enriched with tetracycline antibiotics, characterized in that, This includes magnetic covalent organic framework composite materials modified with nucleic acid aptamers according to claim 1 or claim 2, or magnetic covalent organic framework composite materials modified with nucleic acid aptamers prepared by the method according to any one of claims 3-5.

8. A method for detecting tetracycline antibiotics, characterized in that, The sample to be tested is pretreated using the nucleic acid aptamer-modified magnetic covalent organic framework composite material according to claim 1 or claim 2, the nucleic acid aptamer-modified magnetic covalent organic framework composite material prepared by the preparation method according to any one of claims 3-5, or the product enriched with tetracycline antibiotics according to claim 8, and the pretreatment includes the following steps: (a) The composite material or the product enriched with tetracycline antibiotics is mixed with the extract of the sample to be tested to form a magnetic solid phase extraction system, and the extraction of tetracycline antibiotics is completed within 1 to 5 minutes. (b) By applying an external magnetic field, the composite material or product enriched with tetracycline antibiotics adsorbed with tetracycline antibiotics is separated from the solution. (c) Tetracycline antibiotics were eluted from the composite material using an organic solvent, filtered through a 0.22 μm filter membrane, and then detected by HPLC-MS / MS.

9. The method for enriching tetracycline antibiotics according to claim 8, characterized in that, The tetracycline antibiotics include at least one of tetracycline, chlortetracycline, oxytetracycline, and doxycycline; and / or In step (a), based on a 5 mL liquid sample, the amount of the nucleic acid aptamer-modified magnetic covalent organic framework composite material used is 1–3 mg; and / or In step (a), the extraction time is 1-4 min; and / or In step (a), the pH of the extraction system is 6-9; and / or In step (c), elution is performed using the organic solvent acetonitrile; and / or In step (c), the elution process takes 0.5 to 2 minutes; and / or In step (c), the volume of the eluent is 1-3 mL; and / or In step (c), the HPLC-MS / MS method includes: the separation of tetracycline antibiotics using a C18 column at a column temperature of 40°C; the mobile phase consisting of phase A acetonitrile and phase B water containing 0.1% formic acid, with a flow rate of 0.4 mL / min and an injection volume of 3.0 µL; The gradient elution program includes: 0-2 minutes, 5-25% A; 2-4 minutes, 25-50% A; 4-6 minutes, 50-95% A; 6-8 minutes, 95% A, hold; 8.0-8.1 minutes, 95% A; 8.1-10 minutes, 5% A, hold; Multiple reaction monitoring (MRM) mass spectrometry conditions included: mass spectrometry detection was performed using an electrospray ionization (ESI) source in positive ion mode (ESI+), with a spray voltage of 4500 V; ion source temperature of 500°C; curtain gas of 30 psi; and collision gas of 9 psi.

10. The method according to claim 4, characterized in that, The sample to be tested is a food sample, and the food includes at least one of milk, beef, pork, chicken, mutton, pork liver, goat milk, and eggs.

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