Interface modified magnetic MOF-polymer brush composite nanomaterials, methods of making and using the same

Abstract

The application provides an interface modified magnetic MOF-polymer brush composite nanomaterial and a preparation method and application thereof, and belongs to the technical field of molecular biology detection. The application constructs a multilayer composite structure by taking FS as a magnetic core, growing a U6N metal organic framework shell in situ, and then grafting a cationic PVBTAC polymer brush through interface RAFT polymerization. The material effectively weakens the matrix interference of casein, fat, metal ions and the like in milk, greatly improves the nucleic acid extraction efficiency, and relies on the rich DNA binding sites of MOF, electrostatic enrichment and anti-fouling performance of the polymer brush. The application can complete the nucleic acid extraction of pathogenic bacteria in a milk sample in 5 minutes in cooperation with a lysis buffer, and the extracted DNA can be directly used for LAMP amplification detection. The material is combined with fluorescent LAMP technology, and the detection limit of Escherichia coli in milk can reach 19.2 CFU / mL, the whole set of method is simple to operate, does not need large and precise instruments, is suitable for on-site rapid screening of complex food matrix, and has a good application prospect in the field of dairy product safety detection.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology detection technology, specifically relating to interface-modified magnetic MOF-polymer brush composite nanomaterials, their preparation methods, and applications. Background Technology

[0002] Milk is a high-quality, nutritious dairy product consumed daily by the general public, rich in protein, calcium, and various vitamins. However, its nutritional matrix is ​​also highly susceptible to bacterial growth. Related surveys show that approximately 85% of raw milk has bacterial contamination issues. Raw milk is easily contaminated by mastitis pathogens and microorganisms from the dairy farming environment. Inadequate cleaning of equipment during processing and damaged packaging can also lead to secondary contamination. Pathogenic bacteria can induce foodborne illnesses, seriously threatening public health and safety.

[0003] Traditional microbial detection methods, including plate culture, enzyme-linked immunosorbent assay (ELISA), and flow cytometry, generally suffer from drawbacks such as long detection cycles, low sensitivity, reliance on large instruments, and complex operation, failing to meet the needs of rapid on-site screening of dairy products. Molecular detection technology has become mainstream due to its high sensitivity and specificity, and nucleic acid extraction is the primary rate-limiting step in molecular detection; the extraction efficiency and purity directly determine the accuracy of subsequent detection results.

[0004] Milk is a highly complex matrix containing a large number of impurities such as casein micelles, fat globules, calcium ions, and phosphate ions. These impurities readily bind to or adsorb onto the surface of the extraction material, causing severe non-specific interference and inhibiting nucleic acid separation and subsequent amplification. Traditional organic extraction and centrifugation column methods are cumbersome, and while magnetic bead extraction is simple, commercially available ordinary magnetic nanoparticles have low adsorption capacity and poor anti-fouling ability, resulting in a significant decrease in extraction efficiency in milk matrices.

[0005] Metal-organic frameworks (MOFs) are a class of porous materials with high specific surface area and tunable pores. Among them, zirconium-based UiO-66-NH2 exhibits excellent hydrolytic stability and biocompatibility, and can efficiently capture nucleic acids through coordination and hydrogen bonding. However, pure UiO-66-NH2 is easily contaminated by proteins and fats in complex systems such as milk, and the active sites are occupied, limiting its extraction performance. Cationic polymer brush poly(vinylbenzyltrimethylammonium chloride) (PVBTAC) can enrich negatively charged nucleic acids through electrostatic interactions, while utilizing steric hindrance to reduce non-specific protein adsorption, exhibiting excellent interfacial antifouling capabilities.

[0006] Based on the shortcomings of existing technologies, this invention designs a magnetic MOF-cationic polymer brush multilayer composite nanomaterial that integrates the three major advantages of magnetic separation, high-efficiency nucleic acid adsorption of MOF, and resistance to matrix interference by polymer brush. Combined with a special lysis buffer, it enables ultra-rapid extraction of nucleic acids from pathogenic bacteria in milk. Furthermore, by combining LAMP isothermal amplification technology, a pathogenic bacteria screening system suitable for on-site detection without the need for large instruments is constructed. Summary of the Invention

[0007] To address the problems of weak anti-interference ability, slow extraction speed, and difficulty in field application of existing nucleic acid extraction methods for pathogenic bacteria in milk, this invention provides a magnetic MOF-polymer brush composite nucleic acid extraction material FS@U6N@PVBTAC, along with its preparation process, a matching rapid nucleic acid extraction method, and a pathogen detection scheme based on LAMP technology, achieving rapid extraction of pathogenic bacteria nucleic acid from complex milk matrices within 5 minutes and highly sensitive field detection.

[0008] To achieve the above effects, the present invention adopts the following technical solution: An interface-modified magnetic MOF-polymer brush composite nanomaterial includes the following steps: (1) Preparation of FS magnetic core: Fe3O4 nanoparticles were prepared by co-precipitation method, and then coated with silica to obtain FS magnetic core; the average particle size of the FS magnetic core was 12-15 nm; (2) Preparation of FS@U6NMOF composite material: UiO-66-NH2 (U6N) layer is grown in situ on the surface of FS magnetic core by solvothermal method, and the particle size of the FS@U6NMOF composite material is 180-235nm; (3) RAFT chain transfer agent grafting: RAFT chain transfer agent CPCP was grafted onto the surface of FS@U6N to obtain FS@U6N-CPCP intermediate; (4) PVBTAC polymer brush polymerization: Using VBTAC as monomer, polymer brushes were grafted through surface-initiated RAFT polymerization reaction, and FS@U6N@PVBTAC was obtained after purification.

[0009] Preferably, the interface-modified magnetic MOF-polymer brush composite nanomaterial includes the following steps: (1) Preparation of FS magnetic core: Fe3O4 nanoparticles were prepared by coprecipitation method. Fe3O4 was dispersed in ethanol, and deionized water and ammonia were added in sequence to obtain a mixed system. Tetraethyl orthosilicate (TEOS) was dissolved in ethanol to obtain a tetraethyl orthosilicate solution, which was then added dropwise to the mixed system. After stirring at room temperature, the mixture was washed and dried to obtain the FS magnetic core. (2) Preparation of FS@U6NMOF composite material: The FS magnetic core, zirconium tetrachloride and 2-aminoterephthalic acid were uniformly dispersed in DMF; after adding acetic acid, a high-temperature solvothermal reaction was carried out. After the reaction was completed, the product was collected by magnetic separation, washed and vacuum dried to obtain FS@U6NMOF composite material. (3) RAFT chain transfer agent grafting: 4-cyano-4-(phenylcarbothio)valerate (CPCP) and N-hydroxysuccinimide (NHS) were dissolved in dichloromethane (DCM), and then dicyclohexylcarbodiimide was added and stirred to complete the CPCP activation, resulting in an activated CPCP solution; the FS@U6NMOF composite material was evenly dispersed in a solvent, and the activated CPCP solution was added, and the reaction was carried out at room temperature in the dark. back The product collected by magnetic separation was washed and vacuum dried to obtain FS@U6N-CPCP intermediate; (4) Polymer brush polymerization of PVBTAC: FS@U6N-CPCP, vinylbenzyltrimethylammonium chloride (VBTAC) monomer, azobis(4-cyanovaleric acid) (ACVA) initiator, and N-hydroxysuccinimide (DCC) were added to deionized water and mixed. After nitrogen gas was introduced to remove oxygen, the mixture was sealed and reacted. After the reaction was completed, the mixture was cooled to room temperature, washed, magnetically separated and collected, vacuum dried and dispersed in deionized water for storage, thus obtaining the interface-modified magnetic MOF-polymer brush composite nanomaterial FS@U6N@PVBTAC.

[0010] Preferably, the method for preparing Fe3O4 nanoparticles by coprecipitation in step (1) includes the following steps: Under nitrogen protection, ferrous chloride tetrahydrate and ferric chloride hexahydrate were dissolved in deionized water, heated to react, and ammonia water was added dropwise to continue the reaction. After washing and drying, Fe3O4 was obtained. The mass-to-volume ratio of ferrous chloride tetrahydrate, ferric chloride hexahydrate, deionized water, and ammonia is 0.3-0.4 g : 0.9-1.0 g : 20-25 mL : 2-3 mL; the concentration of the ammonia is 20-25%. The heating reaction is carried out at a temperature of 50-60℃ for 30-35 minutes. The continued reaction is carried out at a temperature of 50-60℃ for 30-35 minutes.

[0011] Preferably, in the mixed system described in step (1), the mass-to-volume ratio of Fe3O4, ethanol, deionized water, and ammonia is 0.1-0.2g:100-120mL:25-30mL:1-2mL, and the concentration of the ammonia is 20-25%; the mass-to-volume ratio of tetraethyl orthosilicate solution to ethanol in the tetraethyl orthosilicate solution is 100-110μL:5-7mL; The ratio of tetraethyl orthosilicate to Fe3O4 is 100-110 μL: 0.1-0.2 g.

[0012] Preferably, the stirring reaction time at room temperature in step (1) is 8-12 hours; the washing is performed by washing with deionized water and ethanol 2-3 times in sequence; and the drying is performed by drying in a vacuum oven at 50-60°C for 2-3 hours.

[0013] Preferably, the ratio of FS magnetic core, zirconium tetrachloride, 2-aminoterephthalic acid, DMF and acetic acid in step (2) is 30-35 mg: 0.45-0.5 mmol: 0.45-0.5 mmol: 30-35 mL: 70-75 mmol.

[0014] Preferably, the temperature of the high-temperature solvothermal reaction in step (2) is 100-120℃, and the reaction time is 20-24h; The intensity of the magnetic separation is 0.3-0.5T; the washing is performed by washing with DMF and deionized water 2-3 times respectively; the vacuum drying temperature is 50-60℃ and the drying time is 2-3h.

[0015] Preferably, the ratio of CPCP, NHS, DCM, DCC and FS@U6NMOF composite material in step (3) is: 2.5-3 mmol: 2.5-3 mmol: 5-7 mL: 2.5-3 mmol: 10-12 mg; The FS@U6NMOF composite material was dispersed in a solvent, DCM, and the ratio of the solvent to the FS@U6NMOF composite material was 5 mL: 10 mg. The reaction time at room temperature in the dark is 20-24 hours, the intensity of magnetic separation is 0.3-0.5T, the washing is 1-3 times with ethanol, and the vacuum drying temperature is 50-60℃, and the drying time is 2-3 hours.

[0016] Preferably, in step (4), the ratio of FS@U6N-CPCP, VBTAC monomer, ACVA initiator, CPCP, and deionized water is 10-12 mg: 17-18 mmol: 1.4 × 10⁻⁶ mg. -2 -1.5×10 -2 mmol: 1.3×10 -1 -1.4×10 -1 mmol: 10-12 mL.

[0017] Preferably, in step (4), the nitrogen gas is introduced for 30-40 minutes; the temperature of the sealed reaction is 70°C and the time is 20-24 hours; the washing is performed by washing with ethanol and deionized water 1-3 times in sequence to remove residual monomers and impurities; the intensity of the magnetic separation is 0.3-0.5T; the temperature of the vacuum drying is 50-60°C and the drying time is 2-3 hours.

[0018] This invention also provides an interface-modified magnetic MOF-polymer brush composite nanomaterial, wherein the composite nanomaterial has a multi-layer core-shell structure, consisting of an FS magnetic core, a UiO-66-NH2 metal-organic framework intermediate layer, and a cationic PVBTAC polymer brush outer layer from the inside out.

[0019] The present invention also provides an application of the composite nanomaterial, which is used to detect Escherichia coli in milk; the detection method is fluorescence LAMP or visual colorimetric LAMP.

[0020] Preferably, the application of the composite nanomaterial includes the following steps: Nucleic acid was extracted from milk using a suspension of FS@U6N@PVBTAC magnetic particles, and the results were determined by fluorescence detection or visual colorimetry in a LAMP reaction system.

[0021] More preferably, the application of the composite nanomaterial includes the following steps: Step 1: Nucleic acid extraction (1) Take a milk sample and add lysis buffer and mix well; the lysis buffer is an aqueous solution containing 0.5-0.6M NaOH and 0.1-0.2M EDTA; (2) Add FS@U6N@PVBTAC magnetic particle suspension to the system and incubate at room temperature to complete nucleic acid adsorption; (3) Collect magnetic particles using a 0.3-0.5T magnetic field, discard the supernatant, and wash the particles with 70% ethanol; (4) Add deionized water to elute DNA, use a 0.3-0.5T magnetic field for magnetic separation, collect the eluent, and complete nucleic acid extraction; The entire extraction process takes ≤5 minutes. The experimental parameters are as follows: 1-2 mL of milk sample, 15-16 μL of FS@U6N@PVBTAC suspension (15-20 mg / mL), 400-420 μL of lysis buffer, incubation (lysis) at room temperature for 2-5 min, and DNA elution time for 2-4 min.

[0022] Step 2: LAMP detection: (1) Prepare the LAMP reaction system and amplify it at 65℃ using the extracted DNA as a template; (2) The results are determined by fluorescence detection or visual colorimetry. The fluorescence method determines positive / negative based on the amplification fluorescence curve. The visual method uses Chrome Black T as an indicator. If the solution changes from purple to blue, it is considered positive. If the solution does not change from purple, it is considered negative.

[0023] 1. Composite material integral structure The FS@U6N@PVBTAC provided by this invention has a three-layer core-shell structure: the inner layer is an FS magnetic core, providing rapid magnetic separation capability and structural stability; the middle layer is a U6NMOF shell, which provides a large number of nucleic acid binding sites through coordination bonds and hydrogen bonds, thereby increasing the nucleic acid adsorption capacity; the outer layer is a cationic PVBTAC polymer brush, which enhances nucleic acid enrichment through electrostatic interaction, while blocking the adsorption of impurities such as proteins, fats, and ions in milk, thus improving the material's matrix tolerance and anti-fouling properties. The synergistic effect of the three layers solves the problems of severe interference and low extraction efficiency of traditional magnetic beads and pure MOF materials in milk matrices.

[0024] 2. Rapid Nucleic Acid Extraction System This invention uses lysis buffer (0.5M NaOH + 0.1M EDTA) and optimizes the parameters of magnetic bead dosage, lysis time, washing conditions, and elution time to compress the entire nucleic acid extraction process to within 5 minutes. The extracted DNA recovery rate can reach 84%, and the purity meets the requirements for LAMP amplification.

[0025] 3. Visual Detection System for Pathogenic Bacteria The extracted nucleic acid is used directly as a template to detect E. coli in milk using either fluorescent LAMP or visual colorimetric LAMP techniques. Fluorescent LAMP has high sensitivity and a detection limit as low as 19.2 CFU / mL. Visual LAMP uses Chrome Black T as an indicator and relies on color changes to determine the results. It does not require fluorescent equipment and is suitable for screening scenarios at the grassroots level, on-site, and at home.

[0026] Compared with the prior art, the present invention has the following beneficial effects: 1. Excellent material properties This invention, FS@U6N@PVBTAC, combines the advantages of both MOF and polymer brush, achieving a DNA recovery rate of 84%, far exceeding that of MOF composites consisting of single magnetic particles and ungrafted polymer brushes. The material surface is positively charged, resulting in strong electrostatic adsorption of nucleic acids, with a maximum equilibrium adsorption capacity of 124.85 μg / mg. The adsorption process is monolayer specific adsorption with high selectivity. Furthermore, the material exhibits superparamagnetism, enabling rapid magnetic separation and reusability.

[0027] 2. Extremely high extraction efficiency With a dedicated lysis buffer and optimized process, the extraction of pathogenic bacteria nucleic acid from milk samples can be completed within 5 minutes. Compared with the traditional extraction method that takes several hours, the efficiency is significantly improved, which is fully adapted to the needs of rapid detection. The extracted nucleic acid has low impurity content and no amplification inhibitors, and can be directly used for LAMP isothermal amplification.

[0028] 3. Strong resistance to matrix interference The outer PVBTAC polymer brush effectively inhibits the non-specific adsorption of casein, fat, and inorganic salt ions in milk through steric hindrance and electrostatic repulsion, solving the problem of interference from complex dairy product matrices and significantly improving the material's matrix tolerance.

[0029] 4. Flexible testing solutions and wide applicability Dual detection modes: Fluorescent LAMP offers high sensitivity, suitable for precise quantification in laboratories; Visual colorimetric LAMP requires only a constant temperature heating device and relies on visual interpretation of results, exhibiting low equipment dependence and wide applicability in scenarios such as farmers' markets, dairy processing plants, grassroots testing institutions, and on-site food safety sampling. The entire method is simple to operate, low in cost, and highly sensitive, with a detection limit as low as 19.2 CFU / mL.

[0030] 5. The process is simple and easy to promote. The raw materials for material synthesis are all commercially available conventional reagents, the preparation process is mature, and the reaction conditions are easy to control; the nucleic acid extraction and detection steps are standardized, eliminating the need for professional molecular biology operators to learn complex skills, which facilitates industrialization and large-scale application. Attached Figure Description

[0031] Figure 1 A schematic diagram of the synthesis route for FS@U6N@PVBTAC multilayer composite materials; Figure 2 Transmission electron microscopy (TEM) characterization images of different materials; Figure 3 The Fourier Transform Infrared (FTIR) spectra of materials at different synthesis stages; Figure 4 The hysteresis loop (VSM) spectra of Fe3O4 and FS@U6N@PVBTAC; Figure 5 A graph showing the change in Zeta potential of the material at each stage; Figure 6 The LAMP fluorescence amplification curves under different lysis buffers; Figure 7 This is a comparison of DNA recovery rates of FS, FS@U6N, and FS@U6N@PVBTAC in milk matrix with LAMP threshold time in Example 1. Figure 8 DNA adsorption isotherm and fitting curves of Langmuir and Freundlich models; Figure 9 Amplification curves (A) and corresponding Tt values ​​(B) of fluorescent LAMP on milk samples with different concentrations of Escherichia coli; Figure 10 These are real-life photos showing the color changes of samples with different bacterial concentrations in a visually chromogenic LAMP assay. Detailed Implementation

[0032] This invention provides an interface-modified magnetic MOF-polymer brush composite nanomaterial, comprising the following steps: (1) Preparation of FS magnetic core: Fe3O4 nanoparticles were prepared by co-precipitation method, and then coated with silica to obtain FS magnetic core; the average particle size of the FS magnetic core was 12-15 nm; (2) Preparation of FS@U6NMOF composite material: U6N layer is grown in situ on the surface of FS magnetic core by solvothermal method, and the particle size of the FS@U6NMOF composite material is 180-235nm; (3) RAFT chain transfer agent grafting: RAFT chain transfer agent CPCP was grafted onto the surface of FS@U6N to obtain FS@U6N-CPCP intermediate; (4) PVBTAC polymer brush polymerization: Using VBTAC as monomer, polymer brushes were grafted through surface-initiated RAFT polymerization reaction, and FS@U6N@PVBTAC was obtained after purification.

[0033] Preferably, the interface-modified magnetic MOF-polymer brush composite nanomaterial includes the following steps: (1) Preparation of FS magnetic core: Fe3O4 nanoparticles were prepared by coprecipitation method. Fe3O4 was dispersed in ethanol, and deionized water and ammonia were added in sequence to obtain a mixed system. Tetraethyl orthosilicate (TEOS) was dissolved in ethanol to obtain a tetraethyl orthosilicate solution, which was then added dropwise to the mixed system. After stirring at room temperature, the mixture was washed and dried to obtain the FS magnetic core. (2) Preparation of FS@U6NMOF composite material: The FS magnetic core, zirconium tetrachloride and 2-aminoterephthalic acid were uniformly dispersed in DMF; after adding acetic acid, a high-temperature solvothermal reaction was carried out. After the reaction was completed, the product was collected by magnetic separation, washed and vacuum dried to obtain FS@U6NMOF composite material. (3) RAFT chain transfer agent grafting: CPCP and NHS were dissolved in dichloromethane (DCM), and DCC was added and stirred to complete the CPCP activation, resulting in an activated CPCP solution; the FS@U6NMOF composite material was dispersed evenly in a solvent, and the activated CPCP solution was added, and the reaction was carried out at room temperature in the dark. back The product collected by magnetic separation was washed and vacuum dried to obtain FS@U6N-CPCP intermediate; (4) PVBTAC polymer brush polymerization: FS@U6N-CPCP, vinylbenzyltrimethylammonium chloride (VBTAC) monomer, azobis(4-cyanopentanoic acid) ACVA initiator, and CPCP were added to deionized water and mixed. After nitrogen gas was introduced to remove oxygen, the mixture was sealed and reacted. After the reaction was completed, the mixture was cooled to room temperature, washed, magnetically separated and collected, vacuum dried and dispersed in deionized water for storage, thus obtaining the interface-modified magnetic MOF-polymer brush composite nanomaterial FS@U6N@PVBTAC.

[0034] Preferably, the method for preparing Fe3O4 nanoparticles by coprecipitation in step (1) includes the following steps: Under nitrogen protection, ferrous chloride tetrahydrate and ferric chloride hexahydrate were dissolved in deionized water, heated to react, and ammonia water was added dropwise to continue the reaction. After washing and drying, Fe3O4 was obtained. The mass-to-volume ratio of ferrous chloride tetrahydrate, ferric chloride hexahydrate, deionized water, and ammonia is 0.3-0.4 g : 0.9-1.0 g : 20-25 mL : 2-3 mL; the concentration of the ammonia is 25%. The heating reaction is carried out at a temperature of 50-60℃ for 30-35 minutes. The continued reaction is carried out at a temperature of 50-60℃ for 30-35 minutes.

[0035] Preferably, in the mixed system described in step (1), the mass-to-volume ratio of Fe3O4, ethanol, deionized water and ammonia is 0.3g:120mL:30mL:2mL, and the concentration of the ammonia is 25%; the mass-to-volume ratio of tetraethyl orthosilicate solution and ethanol in the tetraethyl orthosilicate solution is 100-110μL:5-7mL. The ratio of tetraethyl orthosilicate to Fe3O4 is 100 μL: 0.2 g.

[0036] Preferably, the stirring reaction time at room temperature in step (1) is 8-12 hours; the washing is performed by washing with deionized water and ethanol 2-3 times in sequence; and the drying is performed by drying in a vacuum oven at 50-60°C for 2-3 hours.

[0037] Preferably, the ratio of FS magnetic core, zirconium tetrachloride, 2-aminoterephthalic acid, DMF and acetic acid in step (2) is 30-35 mg: 0.45-0.5 mmol: 0.45-0.5 mmol: 30-35 mL: 70-75 mmol.

[0038] Preferably, the temperature of the high-temperature solvothermal reaction in step (2) is 100-120℃, and the reaction time is 20-24h; The intensity of the magnetic separation is 0.3T; the washing is performed by washing with DMF and deionized water 2-3 times respectively; the vacuum drying temperature is 50-60℃ and the drying time is 2-3h.

[0039] Preferably, the ratio of CPCP, NHS, DCM, DCC and FS@U6NMOF composite material in step (3) is: 2.5-3 mmol: 2.5-3 mmol: 5-7 mL: 2.5-3 mmol: 10-12 mg; The FS@U6NMOF composite material was dispersed in a solvent, DCM, and the ratio of the solvent to the FS@U6NMOF composite material was 5 mL: 10 mg. The reaction time at room temperature in the dark is 20-24 hours, the intensity of magnetic separation is 0.3T, the washing is 1-3 times with ethanol, and the vacuum drying temperature is 50-60℃, and the drying time is 2-3 hours.

[0040] Preferably, in step (4), the ratio of FS@U6N-CPCP, VBTAC monomer, ACVA initiator, CPCP, and deionized water is 10-12 mg: 17-18 mmol: 1.4 × 10⁻⁶ mg. -2 -1.5×10 -2 mmol: 1.3×10 -1 -1.4×10 -1 mmol: 10-12 mL.

[0041] Preferably, in step (4), the nitrogen gas is introduced for 30-40 minutes; the temperature of the sealed reaction is 70°C and the time is 20-24 hours; the washing is performed by washing with ethanol and deionized water 1-3 times in sequence to remove residual monomers and impurities; the intensity of the magnetic separation is 0.3T; the temperature of the vacuum drying is 50-60°C and the drying time is 2-3 hours.

[0042] This invention also provides an interface-modified magnetic MOF-polymer brush composite nanomaterial, wherein the composite nanomaterial has a multi-layer core-shell structure, consisting of an FS magnetic core, a UiO-66-NH2 metal-organic framework intermediate layer, and a cationic PVBTAC polymer brush outer layer from the inside out.

[0043] The present invention also provides an application of the composite nanomaterial, which is used to detect Escherichia coli in milk; the detection method is fluorescence LAMP or visual colorimetric LAMP.

[0044] Preferably, the application of the composite nanomaterial includes the following steps: Step 1: Nucleic acid extraction (1) Take a milk sample and add lysis buffer and mix well; the lysis buffer is an aqueous solution containing 0.5-0.6M NaOH and 0.1-0.2M EDTA; (2) Add FS@U6N@PVBTAC magnetic particle suspension to the system and incubate at room temperature to complete nucleic acid adsorption; (3) Collect magnetic particles using a 0.3T magnetic field, discard the supernatant, and wash the particles with 70% ethanol; (4) Add deionized water to elute DNA, use a 0.3T magnetic field for magnetic separation, collect the eluent, and complete nucleic acid extraction; The entire extraction process takes ≤5 minutes. The experimental parameters are as follows: 1-2 mL of milk sample, 15-16 μL of FS@U6N@PVBTAC suspension (15-20 mg / mL), 400-420 μL of lysis buffer, 2-5 min of room temperature incubation (lysis), and 2-4 min of DNA elution.

[0045] Step 2: LAMP detection: (1) Prepare the LAMP reaction system and amplify it at 65℃ using the extracted DNA as a template; (2) The results are determined by fluorescence detection or visual colorimetry. The fluorescence method determines positive / negative based on the amplification fluorescence curve. The visual method uses Chrome Black T as an indicator. If the solution changes from purple to blue, it is considered positive. If the solution does not change from purple, it is considered negative.

[0046] The technical solution of the present invention will be described in detail below through specific examples.

[0047] Example 1 Preparation of FS@U6N@PVBTAC composite magnetic materials Step 1: Preparation of FS magnetic cores (1) Synthesis of Fe3O4: Take 20 mL of deionized water and purge with nitrogen for 5 min to remove oxygen; weigh 0.34 g of ferrous chloride tetrahydrate and 0.95 g of ferric chloride hexahydrate and add them to the water, and sonicate for 5 min; heat the system to 50 °C and keep it at that temperature for 30 min, then add 2 mL of 25% ammonia dropwise and continue the reaction at 50 °C for 30 min. After the reaction is complete, use a 0.3 T magnetic field to magnetically separate and collect Fe3O4 particles, wash them repeatedly with deionized water and ethanol, and dry them for later use.

[0048] (2) Silica coating: 0.2 g Fe3O4 was ultrasonically dispersed in 120 mL ethanol, and 30 mL deionized water and 2 mL ammonia were added sequentially to obtain a mixed system; 100 μL of tetraethyl orthosilicate (TEOS) was dissolved in 5 mL ethanol to obtain a tetraethyl orthosilicate solution, which was then added dropwise to the mixed system and stirred at room temperature for 8 h. The product was washed alternately with deionized water and ethanol, and dried in a vacuum oven at 60 °C for 2 h to obtain the FS magnetic core; Step 2: Preparation of FS@U6NMOF composite material Weigh 30 mg of FS magnetic core, 0.45 mmol of zirconium tetrachloride, and 0.45 mmol of 2-aminoterephthalic acid, disperse them in 30 mL of DMF, and sonicate for 5 min. Add 72 mmol of acetic acid, transfer the mixture to a polytetrafluoroethylene-lined reactor, and solvothermically react at 120 °C for 24 h. After the reaction, collect the product by magnetic separation using a 0.3 T magnetic field, wash three times with DMF and deionized water, and vacuum dry at 60 °C for 5 h to obtain FS@U6N.

[0049] Step 3: RAFT chain transfer agent grafting (1) Activation of chain transfer agent: Dissolve 2.5 mmol CPC and 2.5 mmol NHS in 5 mL of dichloromethane (DCM), add 2.5 mmol DCC, stir at 25 °C for 24 h to complete CPC activation and obtain activated CPC solution; (2) Grafting reaction: 10 mg of FS@U6N was ultrasonically dispersed in 5 mL of LCM, and the above activated CPC solution was added. The reaction was carried out at room temperature in the dark for 24 h. The product was collected by magnetic separation, washed with ethanol, and dried under vacuum at 60 °C for 5 h to obtain the FS@U6N-CPCP intermediate.

[0050] Step 4: PVBTAC polymer brush polymerization Take 10 mg FS@U6N-CPCP, 17.5 mmol BVTAC monomer, and 1.429 × 10 -2 mmolACVA initiator, 1.339 × 10 -1 mmolCPCP was added to 10 mL of deionized water and mixed; nitrogen gas was purged for 30 min to remove oxygen, and the mixture was sealed and reacted at 70 °C for 24 h. After the reaction was completed, the mixture was cooled to room temperature and washed twice with ethanol and deionized water to remove residual monomers and impurities. The product was collected by magnetic separation using a 0.3-0.5 T magnetic field, dried under vacuum at 60 °C for 5 h, and dispersed in 1 mL of deionized water for storage, thus obtaining the target material FS@U6N@PVBTAC.

[0051] A schematic diagram of the synthesis route for FS@U6N@PVBTAC multilayer composite materials is shown below. Figure 1 .from Figure 1It can be seen that the material was prepared by a stepwise core-shell construction strategy with layer-by-layer modification: firstly, magnetic nanoparticles of iron(III) oxide (Fe3O4) were used as the initial core, and the silica surface was modified by TEOS to obtain FS microspheres. The particle surface was enriched with a large number of silanol groups (Si-OH); the abundant silanol groups can anchor Zr. 4+ Precursor formation FS-Zr 4+ The intermediate is then synthesized via solvothermal processes, inducing heterogeneous nucleation and in-situ growth of UiO-66-NH2 (U6N) on the particle surface to obtain the core-shell structure FS@U6N. The abundant amino groups on the U6N surface provide active sites for subsequent grafting modification. Subsequently, through an amidation reaction, a RAFT chain transfer reagent is covalently bonded to the FS@U6N surface to obtain FS@U6N-CPCP. Finally, a surface-initiated RAFT polymerization reaction is used to polymerize VBTAC monomers on the MOF shell surface, covalently grafting a dense cationic PVBTAC polymer brush, ultimately yielding the three-layer hierarchical magnetic composite material FS@U6N@PVBTAC. In terms of nucleic acid adsorption, the material captures DNA through a triple synergistic effect: the outer positively charged PVBTAC brush rapidly binds negatively charged nucleic acids via electrostatic interactions; the Zr6 oxygen clusters in the intermediate U6N backbone form coordination bonds with nucleic acid phosphate groups, and the amino ligands simultaneously generate hydrogen bonds to enhance adsorption; simultaneously, the polymer brush can shield protein and lipid impurities within the dairy product matrix, significantly reducing non-specific adsorption.

[0052] Transmission electron microscopy (TEM) characterization images of different materials in Example 1 are shown below. Figure 2 :in Figure 2 (A) is Fe3O4 nanoparticles, Figure 2 (B) represents FS@U6N@PVBTAC. Figure 2 (C) is the high-resolution lattice diagram of FS@U6N@PVBTAC. From Figure 2 It can be seen that the pure iron(III) oxide nanoparticles have a regular morphology and clear boundaries, with an average particle size of approximately 13.5 ± 1.77 nm. After multiple steps of continuous surface modification, the morphology of the FS@U6N@PVBTAC composite microspheres changed significantly, transforming from loose aggregation of primary nanoparticles into a cluster structure, with the overall particle size increasing to 208.3 ± 25.56 nm. Simultaneously, the in-situ growth of U6N significantly improved the surface roughness and porosity of the composite material, effectively increasing the specific surface area and accessible active sites, which is beneficial for the adsorption and enrichment of target nucleic acid molecules. Furthermore, a low-contrast, cloud-like thin layer can be observed around the particles, corresponding to the PVBTAC polymer layer grafted onto the surface. These morphological changes corroborate the successful construction of the stepwise core-shell structure in this experiment. High-resolution transmission electron microscopy (HRTEM) image (… Figure 2C) Regular lattice fringes were clearly observed in FS@U6N@PVBTAC, with a crystal plane spacing of 0.658 nm, which is highly consistent with the theoretical crystal plane spacing of the (310) cubic U6N crystal. The clearly discernible lattice features prove that the U6N crystal is completely and stably loaded on the surface of the magnetic carrier, further confirming the successful preparation of the ternary magnetic composite material.

[0053] The infrared (FTIR) spectra of different materials in Example 1 are as follows: Figure 3 .from Figure 3 It can be seen that the pure Fe3O4 sample only contains characteristic peaks of Fe–O and hydroxyl groups; after modification with a TEOS silicon layer to obtain FS, the peak value is 1055 cm⁻¹. -1 The appearance of the Si–O–Si characteristic peak directly proves that the SiO2 shell coating is complete; after loading UiO-66-NH2, the curve adds 631 cm⁻¹. -1 1568cm -1 The MOF-specific characteristic peaks indicate successful in-situ growth of the UiO-66-NH2 framework; after grafting the RAFT reagent CPCP, the spectrum shows C–S characteristic peaks and significantly broadened hydroxyl / amino peaks, verifying that the amidation reaction achieves covalent fixation of the RAFT reagent; in the final product FS@U6N@PVBTAC, the peak at 1612 cm⁻¹ is [missing value]. -1 The absorption peaks were enhanced and quaternary ammonium salt characteristic vibration signals appeared. The progressive changes of the infrared characteristic peaks layer by layer completely confirmed the stepwise synthesis process of Fe3O4→FS→FS@U6N→FS@U6N-CPCP→FS@U6N@PVBTAC. Each layer structure was successfully modified on the surface of the magnetic carrier.

[0054] The hysteresis loop (VSM) spectra of Fe3O4 and FS@U6N@PVBTAC in Example 1 are as follows: Figure 4 ;from Figure 4 It can be seen that Fe3O4 nanoparticles possess higher saturation magnetization. After SiO2 coating, in-situ growth of UiO-66-NH2, and multilayer modification via PVBTAC polymer brush grafting, the saturation magnetization (Ms) of the material decreased from 61 emu / g in pure iron(III) oxide to 45 emu / g in FS@U6N@PVBTAC. This is a normal phenomenon caused by a large amount of non-magnetic shell material encapsulating a magnetic core. Both materials exhibit no significant hysteresis loops, extremely low coercivity and remanence, demonstrating excellent superparamagnetism, ensuring no spontaneous aggregation of particles after magnetic separation. The final product's saturation magnetization of 45 emu / g is sufficient to support efficient magnetic capture and separation of samples in a short time, fully meeting the operational requirements for nucleic acid pretreatment in dairy product matrices, proving that this magnetic composite material is suitable for magnetic separation and extraction systems.

[0055] The Zeta potential variation diagrams for different materials in Example 1 are shown below. Figure 5;from Figure 5 It can be seen that the original Fe3O4 and the initial modified FS and FS@U6N materials all exhibit negative potentials, mainly due to the enrichment of negatively charged functional groups such as hydroxyl and carboxyl groups on the particle surface. With the completion of RAFT reagent grafting and VBTAC monomer polymerization modification, a large number of quaternary ammonium salt cationic groups were successfully introduced into the material surface, resulting in a significant shift in the surface potential of FS@U6N@PVBTAC from negative to positive. The final product has a positive potential as high as +29.6 mV, endowing the material with excellent electrostatic adsorption capabilities, enabling efficient capture of nucleic acid molecules with negative phosphate backbones in the system. Simultaneously, the stepwise change in potential fully confirms the success of the layer-by-layer functionalization modification of the material, and also explains the core reason for the excellent nucleic acid adsorption and enrichment performance of this composite material from the perspective of charge interaction mechanisms.

[0056] Example 2 To determine the optimal lysis buffer system suitable for this magnetic composite material, this invention conducted optimization experiments on the types of lysis buffers. Keeping other extraction parameters constant, only the types of lysis buffers were changed (10 mM Tris-Cl, 20% Triton-X, with pH adjusted to 5.5 and 8.8 using a hydrochloric acid / ammonia gradient; 0.5 M NaOH, 0.1 M EDTA). This study used LAMP fluorescence amplification curves as the core evaluation index.

[0057] like Figure 6 It can be seen that, considering the peak speed, Tt value, and fluorescence peak results of the LAMP amplification curves of each group, the optimal lysis buffer for nucleic acid extraction from milk matrix suitable for this material was finally determined to be an aqueous solution containing 0.5M NaOH and 0.1M EDTA.

[0058] Rapid extraction of E. coli nucleic acid from milk samples (1) Sample preparation: Take 1 mL of milk sample artificially contaminated with Escherichia coli; (2) Lysis: Add 400 μL of lysis buffer (0.5 M NaOH + 0.1 M EDTA) and mix well; add 20 μL of 15 mg / mL FS@U6N@PVBTAC magnetic suspension and incubate at room temperature for 2 min to complete nucleic acid adsorption; (3) Washing: Collect magnetic beads by magnetic field adsorption, discard the supernatant, and wash the magnetic beads 3 times with 70% ethanol; (4) Elution: Add 50 μL of deionized water, elute at room temperature for 2 min, and after magnetic separation, take out the supernatant, which is the purified E. coli DNA solution (DNA template).

[0059] Total time: 4.5 min. The DNA recovery rate was 84%. The A260 / A280 and A260 / A230 ratios were within the normal range, and there was no obvious protein or polysaccharide contamination.

[0060] The comparison of DNA recovery rates of FS, FS@U6N, and FS@U6N@PVBTAC in milk matrix with LAMP threshold time in Example 1 is shown in the figure below. Figure 7 . Figure 7 A represents the original fluorescence amplification curve of the recovered DNA using LAMP. Figure 7 B is a graph showing the purity analysis of the recovered DNA. Figure 7 C is a bar chart showing the DNA recovery rate and LAMP amplification Tt threshold for different materials.

[0061] Figure 7 A shows the fluorescence amplification curves of the three groups of samples. The FS curve showed the latest peak; the FS@U6N curve showed significant improvement; and the FS@U6N@PVBTAC curve showed the earliest peak and combined with... Figure 7 B. Purity analysis showed that the A260 / A280 ratio in the DNA extracted by FS@U6N@PVBTAC was within an acceptable range, which directly confirms that the purity and concentration of the extracted nucleic acid were excellent.

[0062] from Figure 7 C allows for intuitive reading of DNA recovery values ​​for the three groups of materials: the DNA recovery rate of single FS magnetic microspheres is only 46%, the recovery rate of FS@U6N coated with U6N increases to 70%, and the recovery rate of FS@U6N grafted with PVBTAC polymer brush reaches 84%. The data intuitively demonstrate that the nucleic acid capture ability continues to improve after multilayer modification. Combined with the analysis of amplification Tt data, the FS group has the largest Tt value, indicating that the amount of extracted nucleic acid is small and the amplification initiation is delayed; the Tt of FS@U6N is shortened; FS@U6N@PVBTAC has the lowest Tt value, representing the highest concentration of extracted target DNA and the weakest matrix inhibition.

[0063] The results from the three subplots show a clear trend of progressively improving nucleic acid extraction performance among the three materials. The single FS magnetic microstructure lacks specific nucleic acid adsorption capabilities and is severely affected by the matrix of proteins and lipids in milk, resulting in the lowest DNA recovery rate, the longest amplification threshold time, and the worst amplification efficiency. After introducing the U6N functional layer, the nucleic acid capture ability of FS@U6N is improved to some extent, relying on the porous structure, coordination, and hydrogen bond adsorption effect of the MOF material, thus improving both the recovery rate and amplification efficiency. Furthermore, grafting a PVBTAC cationic polymer brush allows FS@U6N@PVBTAC to rapidly enrich negatively charged nucleic acids through strong electrostatic interactions. Simultaneously, the interface shielding effect of the polymer brush effectively reduces non-specific adsorption interference in the milk matrix, ultimately achieving the highest DNA recovery rate of 84% and the shortest LAMP amplification threshold time. These results fully demonstrate that the multilayer synergistic structure greatly optimizes the nucleic acid extraction performance of the materials, enabling FS@U6N@PVBTAC to extract nucleic acids efficiently and with high quality from complex dairy matrices, meeting the requirements for subsequent rapid isothermal amplification.

[0064] Example 3 Detection of Escherichia coli in milk using fluorescent LAMP method (1) Preparation of LAMP reaction system (total system 25 μL): 2× WarmStart 12.5 μL of LAMP premix (New England Biolabs, M1800L), 5.5 μL of a mixture of outer primers, inner primers, and circular primers (Sangon Biotech), 0.5 μL of 50× fluorescent dye (New England Biolabs, M1712S), 2 μL of DNA template extracted in Example 2, and deionized water added to a final volume of 25 μL; final primer concentrations: 0.2 μM for outer primers, 1.6 μM for inner primers, and 0.4 μM for circular primers.

[0065] (2) Amplification reaction: The reaction system was placed in a real-time fluorescence quantitative PCR instrument and amplified at 65℃ for 60 min, and the fluorescence signal was collected in real time.

[0066] (3) Result determination: The presence of a typical amplification fluorescence curve indicates that Escherichia coli is positive, and the absence of a fluorescence amplification curve indicates that it is negative.

[0067] Gradient spiking experiments validated that the limit of detection for Escherichia coli in milk using this method was 19.2 CFU / mL, and the repeatability was good.

[0068] Example 4 Visual colorimetric LAMP method for detecting Escherichia coli in milk The reaction system, amplification temperature, and time were the same as in Example 3, except that the fluorescent dye was replaced with Chrome Black T chromogenic reagent (Ron, R094341). After amplification, the color was observed visually: if the system changed from purple to blue, it was considered positive for E. coli; if the solution remained purple, it was considered negative. This method does not require fluorescence detection equipment and is suitable for rapid on-site screening.

[0069] Example 5 Adsorption isotherm performance test Using E. coli plasmid DNA as the target, DNA solutions with gradient concentrations of 10–200 μg / mL were prepared. FS@U6N@PVBTAC was added for batch adsorption experiments, and adsorption was allowed to reach equilibrium at room temperature for 2 min. The residual DNA concentration in the supernatant was measured, and the equilibrium adsorption capacity Qe was calculated.

[0070] Data fitting results: Figure 8 DNA adsorption isotherms and fitting curves for the Langmuir and Freundlich models; it can be seen that the adsorption process conforms to the Langmuir monolayer adsorption model, R 2=0.9989, theoretical maximum adsorption capacity 196.07μg / mg, experimentally measured maximum adsorption capacity 124.85μg / mg; Langmuir separation factor RL is always between 0 and 1, proving that the adsorption process of DNA by this material is thermodynamically spontaneous and has excellent adsorption performance.

[0071] Figure 9 Amplification curves (A) and corresponding Tt values ​​(B) of fluorescent LAMP on milk samples with different concentrations of E. coli; from Figure 9 A shows that the range is from 1.92×10 to 1.92×10. 7 Within a CFU / mL concentration gradient of *E. coli*, the fluorescent LAMP system consistently produced specific amplification curves, while the negative control group showed no amplification signal and no false positives. Figure 9 As shown in Figure B, as the concentration of *E. coli* in the milk sample gradually decreased, the LAMP amplification initiation time gradually delayed, and the amplification threshold (Tt) value increased systematically, exhibiting a good concentration-response linear relationship, demonstrating the excellent quantitative detection potential of this detection system. Even when the *E. coli* concentration was as low as 19.2 CFU / mL, the system could still detect effective amplification signals, indicating that the fluorescent LAMP method combined with FS@U6N@PVBTAC nucleic acid extraction technology has extremely high sensitivity and can achieve accurate detection of low-abundance *E. coli* in milk. These results fully verify that the nucleic acid extracted from the composite material has high purity and low residual amplification inhibitors, perfectly adapting to high-sensitivity fluorescence isothermal amplification detection, and meeting the needs for rapid and accurate screening of trace pathogens in dairy products.

[0072] Figure 10 Actual photographs showing the color changes of samples with different bacterial concentrations under visual colorimetric LAMP. Figure 10 A represents different concentrations of E. coli (1.92 × 10⁻⁶). 7 A photograph of the sample color before the LAMP reaction (~1.92×10 CFU / mL). Figure 10 B is a photograph of the sample color after the LAMP reaction. (Combined with...) Figure 10 A and B allow for direct observation of different E. coli concentrations (1.92 × 10⁻⁶). 7 The colorimetric difference corresponding to ~1.92×10 CFU / mL was significant. High-concentration bacterial suspension samples, after LAMP amplification, completely changed from purple to blue, exhibiting a highly distinctive colorimetric phenomenon. As the bacterial concentration decreased, the amplification product content decreased, and the blue color gradually lightened, showing a purple-blue gradient transition. The negative control group remained purple throughout, showing no color change. Compared to quantitative fluorescence detection, visual colorimetric LAMP relies on color changes for qualitative discrimination, requiring no sophisticated fluorescence detection instruments; results can be interpreted visually, making it simple to operate and cost-effective. Although its detection sensitivity (1.92×10 CFU / mL) is relatively low... 3The CFU / mL level is slightly lower than that of the fluorescent LAMP system, but it fully meets the qualitative detection needs of grassroots field and rapid screening of fresh dairy products. It complements the aforementioned fluorescent detection system and greatly expands the practical application scenarios of this nucleic acid extraction and detection platform.

[0073] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing interface-modified magnetic MOF-polymer brush composite nanomaterials, characterized in that, Includes the following steps: (1) Preparation of FS magnetic core: Fe3O4 nanoparticles were prepared by coprecipitation method, and then coated with silica to obtain FS magnetic core; (2) Preparation of FS@U6NMOF composite material: UIO-66-NH2 layer was grown in situ on the surface of FS magnetic core by solvothermal method; (3) RAFT chain transfer agent grafting: RAFT chain transfer agent CPCP was grafted onto the surface of FS@U6N to obtain FS@U6N-CPCP intermediate; (4) PVBTAC polymer brush polymerization: Using VBTAC as monomer, polymer brushes were grafted through surface-initiated RAFT polymerization reaction, and FS@U6N@PVBTAC was obtained after purification.

2. The method for preparing the interface-modified magnetic MOF-polymer brush composite nanomaterial according to claim 1, characterized in that, The interface-modified magnetic MOF-polymer brush composite nanomaterial includes the following steps: (1) Preparation of FS magnetic core: Fe3O4 nanoparticles were prepared by coprecipitation method. Fe3O4 was dispersed in ethanol, and deionized water and ammonia were added in sequence to obtain a mixed system. Tetraethyl orthosilicate was dissolved in ethanol to obtain a tetraethyl orthosilicate solution, which was then added dropwise to the mixed system. After stirring at room temperature, the mixture was washed and dried to obtain the FS magnetic core. (2) Preparation of FS@U6NMOF composite material: The FS magnetic core, zirconium tetrachloride and 2-aminoterephthalic acid were uniformly dispersed in DMF; after adding acetic acid, a high-temperature solvothermal reaction was carried out. After the reaction was completed, the product was collected by magnetic separation, washed and vacuum dried to obtain FS@U6NMOF composite material. (3) RAFT chain transfer agent grafting: 4-cyano-4-(phenylcarbothio)valerate and N-hydroxysuccinimide were dissolved in dichloromethane (DCM), and then dicyclohexylcarbodiimide was added and stirred to complete CPCP activation, resulting in an activated CPCP solution; the FS@U6NMOF composite material was evenly dispersed in a solvent, and the activated CPCP solution was added, and the reaction was carried out at room temperature in the dark. back The product collected by magnetic separation was washed and vacuum dried to obtain FS@U6N-CPCP intermediate; (4) PVBTAC polymer brush polymerization: FS@U6N-CPCP, vinylbenzyltrimethylammonium chloride (VBTAC) monomer, azobis(4-cyanopentanoic acid) (ACVA) initiator, and CPCP were added to deionized water and mixed. After nitrogen gas was introduced to remove oxygen, the mixture was sealed and reacted. After the reaction was completed, the mixture was cooled to room temperature, washed, magnetically separated and collected, vacuum dried and dispersed in deionized water for storage, thus obtaining the interface-modified magnetic MOF-polymer brush composite nanomaterial FS@U6N@PVBTAC.

3. The method for preparing the interface-modified magnetic MOF-polymer brush composite nanomaterial according to claim 2, characterized in that, The method for preparing Fe3O4 nanoparticles by coprecipitation in step (1) includes the following steps: Under nitrogen protection, ferrous chloride tetrahydrate and ferric chloride hexahydrate were dissolved in deionized water, heated to react, and ammonia water was added dropwise to continue the reaction. After washing and drying, Fe3O4 was obtained. The mass-to-volume ratio of ferrous chloride tetrahydrate, ferric chloride hexahydrate, deionized water, and ammonia is 0.3-0.4 g : 0.9-1.0 g : 20-25 mL : 2-3 mL; the concentration of the ammonia is 20-25%. The heating reaction is carried out at a temperature of 50-60℃ for 30-35 minutes. The continued reaction is carried out at a temperature of 50-60℃ for 30-35 minutes.

4. The method for preparing the interface-modified magnetic MOF-polymer brush composite nanomaterial according to claim 2, characterized in that, In the mixed system described in step (1), the mass-to-volume ratio of Fe3O4, ethanol, deionized water, and ammonia is 0.1-0.2g: 100-120mL: 25-30mL: 1-2mL, and the concentration of the ammonia is 20-25%; the mass-to-volume ratio of tetraethyl orthosilicate solution to ethanol in the tetraethyl orthosilicate solution is 100-110μL: 5-7mL. The ratio of tetraethyl orthosilicate to Fe3O4 is 100-110 μL: 0.1-0.2 g.

5. The method for preparing the interface-modified magnetic MOF-polymer brush composite nanomaterial according to claim 2, characterized in that, In step (2), the ratio of FS magnetic core, zirconium tetrachloride, 2-aminoterephthalic acid, DMF and acetic acid is 30-35 mg: 0.45-0.5 mmol: 0.45-0.5 mmol: 30-35 mL: 70-75 mmol; the temperature of the high-temperature solvothermal reaction is 100-120℃ and the reaction time is 20-24 h.

6. The method for preparing interface-modified magnetic MOF-polymer brush composite nanomaterials according to claim 2, characterized in that, The ratio of CPCP, NHS, DCM, DCC and FS@U6NMOF composite material in step (3) is: 2.5-3 mmol: 2.5-3 mmol: 5-7 mL: 2.5-3 mmol: 10-12 mg; The reaction time at room temperature in the dark is 20-24 hours.

7. The method for preparing the interface-modified magnetic MOF-polymer brush composite nanomaterial according to claim 2, characterized in that, In step (4), the ratio of FS@U6N-CPCP, VBTAC monomer, ACVA initiator, CPCP, and deionized water is 10-12 mg: 17-18 mmol: 1.4 × 10⁻⁶ mg. -2 -1.5×10 -2 mmol: 1.3×10 -1 -1.4×10 -1 mmol: 10-12 mL; The nitrogen gas is introduced for 30-40 minutes; the temperature of the sealed reaction is 70°C and the time is 20-24 minutes.

8. An interface-modified magnetic MOF-polymer brush composite nanomaterial, characterized in that, The composite nanomaterial is prepared according to any one of claims 1-7. The composite nanomaterial has a multi-layer core-shell structure, consisting of an FS magnetic core, a UiO-66-NH2 metal-organic framework intermediate layer, and a cationic PVBTAC polymer brush outer layer, from the inside to the outside.

9. The application of the interface-modified magnetic MOF-polymer brush composite nanomaterial according to claim 8, characterized in that, The composite nanomaterials are used to extract Escherichia coli from milk; the detection method is either fluorescence LAMP or visual colorimetric LAMP.

10. The application of the interface-modified magnetic MOF-polymer brush composite nanomaterial according to claim 9, characterized in that, Includes the following steps: Nucleic acid was extracted from milk using a suspension of FS@U6N@PVBTAC magnetic particles, and the results were determined by fluorescence detection or visual colorimetry in a LAMP reaction system.

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