A cell membrane biomimetic nanorecognition material, a preparation method and application thereof

Abstract

The application provides a cell membrane biomimetic nanorecognition material and a preparation method and application thereof, adopts magnetic nanoparticles as a carrier, fixes cell membranes to the magnetic carrier through a biological orthogonal reaction, finally obtains the cell membrane-based biomimetic nanorecognition material, and is used for the identification and risk assessment of unknown pollutants in food; the application combines the high magnetic response of the magnetic nanoparticles and the characteristics of the cell membrane-mediated highly complex biological interface performance, and wraps the active cell membranes containing target receptors on the surface of the magnetic nanoparticles through a covalent fixation mode, maximally maintains the integrity of the cell membranes and the three-dimensional structure and biological activity of the membrane receptor proteins thereon, gives the biomimetic nanomaterial excellent targeting performance, realizes the specific screening and rapid magnetic separation of unknown chemical risk substances in a complex food matrix, improves the identification efficiency of unknown chemical risks in food, and provides a new idea of toxicity-oriented screening of potential pollutants in food.

Description

Technical Field

[0001] This invention belongs to the field of material detection technology, and relates to a cell membrane biomimetic nano-recognition material, its preparation method and application. Background Technology

[0002] With the continuous improvement of the food regulatory system, the focus of food safety attention has shifted from the detection of known contaminants to the identification of unknown risk substances. Over-processing of food, packaging contamination, and the use of additives can all lead to the generation of unpredictable risk substances. These compounds often have complex structures, pose unrecognized chemical risks, and lack relevant safety standards, thus posing a significant threat to public health. Therefore, accurately identifying potential risk compounds in food is crucial for food safety monitoring and risk assessment.

[0003] Currently, high-performance liquid chromatography-tandem high-resolution mass spectrometry (HPLC-MS / MS) is widely used to screen for the illegal addition of chemical contaminants such as pesticides, veterinary drugs, and their metabolites. However, identifying unknown compounds remains a challenging task, especially in the absence of relevant guidance. Therefore, the identification of potential contaminants in complex food samples requires a toxicity-guided screening strategy. In recent years, the toxicity assessment of contaminants has mainly relied on cell or biological experiments, which are traditional non-targeted screening methods. These methods involve a comprehensive analysis of the sample without pre-setting specific target compounds to discover various potentially present chemical substances. Because non-targeted screening does not pre-set target compounds, it is suitable for the comprehensive analysis of unknown or complex samples.

[0004] However, due to the complexity of intracellular processes, non-targeted screening struggles to accurately identify specific target compounds and their mechanisms of action, making it difficult to elucidate the specific targets and their mechanisms of action. Furthermore, non-targeted screening typically requires large amounts of samples and experimental resources, resulting in high costs and long processing times. The results can also be affected by various factors, such as experimental conditions and matrix interference, leading to lower accuracy. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides a cell membrane biomimetic nano-recognition material, its preparation method, and its application, thereby solving the technical problems of high cost, long time consumption, and low accuracy in the prior art when using non-targeted screening of potential risk substances in complex food matrices to accurately identify specific target compounds and their mechanisms of action.

[0006] The discovery of potential hazardous substances in complex food matrices is a technical challenge due to its high cost and time-consuming nature.

[0007] This invention is achieved through the following technical solution:

[0008] A method for preparing a cell membrane biomimetic nanorecognition material includes the following steps:

[0009] S1: Fe3O4 magnetic nanoparticles were prepared and coated with SiO2 to obtain Fe3O4@SiO2; then 3-aminopropyltriethoxysilane was added for amination modification to obtain Fe3O4@SiO2-NH2;

[0010] S2: The Fe3O4@SiO2-NH2 was modified with DBCO-NHS to obtain dibenzocyclooctylene-functionalized magnetic nanoparticles; HEK293 cells expressing human epidermal growth factor receptor were cultured, and azide groups were labeled on the cell surface through non-natural sugar metabolism glycoengineering during the process. Cells were collected in the logarithmic phase and obtained by ultrasonic centrifugation.

[0011] S3: The cell membrane biomimetic nanorecognition material is prepared by performing a bioorthogonal reaction between the azidated cell membrane and the dibenzocyclooctylene-functionalized magnetic nanoparticles.

[0012] Preferably, the preparation of the Fe3O4 magnetic nanoparticles specifically involves: adding ferric chloride hexahydrate, ammonium acetate, and sodium citrate to ethylene glycol, stirring the mixture at room temperature, and then heat-treating the reaction product to obtain the Fe3O4 magnetic nanoparticles; the mass ratio of ferric chloride hexahydrate to sodium citrate is (3~6):(1~2); the heat treatment temperature is 180~220℃, and the time is 16~17 h.

[0013] Preferably, the preparation of Fe3O4@SiO2 is specifically as follows: Fe3O4 magnetic nanoparticles are added to ethyl silicate, and ammonia, ethanol and water are added, and the mixture is stirred to react and obtain Fe3O4@SiO2; the ratio of Fe3O4 magnetic nanoparticles to ethyl silicate is (0.1~0.2)g:(0.8~1.6)mL.

[0014] Preferably, the preparation of Fe3O4@SiO2-NH2 is specifically as follows: Fe3O4@SiO2 is added to toluene, ultrasonically dispersed, and then 3-aminopropyltriethoxysilane is added and stirred to react, thereby obtaining Fe3O4@SiO2-NH2; the ratio of Fe3O4@SiO2 to 3-aminopropyltriethoxysilane is (0.1~0.2)g:(1~2)mL.

[0015] Preferably, the preparation of the dibenzocyclooctyne-functionalized magnetic nanoparticles specifically involves: adding Fe3O4@SiO2-NH2 to a DBCO-NHS solution and reacting at room temperature to obtain the dibenzocyclooctyne-functionalized magnetic nanoparticles; the ratio of Fe3O4@SiO2-NH2 to DBCO-NHS is (0.1~0.2) g:(3~6) mL, and the concentration of DBCO-NHS is 4.45 mM.

[0016] Preferably, the labeling of cell surface with azide groups by non-natural sugar metabolism sugar engineering is specifically as follows: Ac4ManNAz is added to a complete culture medium and thoroughly mixed for culturing EGFR-HEK293 cells; when the cells grow to the logarithmic growth phase, they are digested and collected using trypsin digestion solution, centrifuged and sonicated, and then resuspended in phosphate buffer to obtain the azide-modified cell membrane.

[0017] Preferably, the process of preparing a cell membrane biomimetic nanorecognition material by subjecting the azidated cell membrane and the dibenzocyclooctyne-functionalized magnetic nanoparticles to a bioorthogonal reaction specifically involves: ultrasonically dispersing the dibenzocyclooctyne-functionalized magnetic nanoparticles, adding them to a suspension of the azidated cell membrane, and mixing them at room temperature to obtain the cell membrane biomimetic nanorecognition material; the mass ratio of the dibenzocyclooctyne-functionalized magnetic nanoparticles to the azidated cell membrane is 1:(2.5~3).

[0018] A cell membrane biomimetic nanorecognition material was prepared by the above method.

[0019] The above-mentioned cell membrane biomimetic nano-recognition material is used in screening potential chemical risk substances in food samples. In application, the cell membrane biomimetic nano-recognition material is added to the food sample extract and vortexed. The material is collected and eluted by a magnet. Finally, the eluent is analyzed by high performance liquid chromatography-tandem time-of-flight mass spectrometry.

[0020] The above-mentioned application of a cell membrane biomimetic nano-recognition material in screening potential chemical risk substances in food samples; the ratio of the cell membrane biomimetic nano-recognition material to the food sample extract is (5~10) mg:(1~2) mL.

[0021] Compared with the prior art, the present invention has the following beneficial technical effects:

[0022] This invention discloses a method for preparing a cell membrane biomimetic nanorecognition material. The method uses Fe3O4 magnetic nanoparticles as a carrier, which exhibit high magnetic responsiveness (40.38 emu g). -1This allows the prepared biomimetic nanomaterials for cell membrane recognition to be rapidly and easily separated from complex food matrices using an external magnetic field. This characteristic greatly improves screening efficiency and reduces operational complexity. By combining azide-modified cell membranes with dibenzocyclooctyne-functionalized magnetic nanoparticles through a bioorthogonal reaction, this invention maximizes the preservation of the cell membrane's integrity and the three-dimensional structure and bioactivity of its membrane receptor proteins. This feature ensures that the biomimetic nanomaterials can mimic the function of real cell membranes, thereby achieving specific recognition and binding of target contaminants. Due to the high expression of human epidermal growth factor receptors (or other specific target receptors) on the cell membrane, the prepared biomimetic nanomaterials have excellent targeting properties. This targeting ability enables the materials to accurately recognize and bind to unknown chemical risk substances in complex food matrices, improving the accuracy and efficiency of recognition. Compared to traditional non-targeted screening methods, this invention directly identifies specific receptors through targeted screening, avoiding interference from numerous non-target compounds, thereby reducing screening costs and time. Furthermore, the use of magnetic separation technology further simplifies the operation process and improves efficiency. This invention combines the highly complex biological interface properties mediated by cell membranes, covalently immobilizing active cell membranes onto the surface of magnetic nanoparticles to ensure accurate and reliable identification. This cell membrane-based biorecognition method is more biologically relevant and realistic than traditional chemical or physical methods. In summary, the method for preparing cell membrane biomimetic nanorecognition materials proposed in this invention has significant advantages in solving the technical problems faced by existing technologies in non-targeted screening of potential risk substances in complex food matrices, providing a new, efficient, accurate, and low-cost method for screening potential contaminants in food.

[0023] Furthermore, the preparation of the Fe3O4 magnetic nanoparticles specifically involves: adding ferric chloride hexahydrate, ammonium acetate, and sodium citrate to ethylene glycol, stirring the mixture at room temperature, and then heat-treating the reaction product to obtain the Fe3O4 magnetic nanoparticles; the mass ratio of ferric chloride hexahydrate to sodium citrate is (3~6):(1~2), which effectively controls the size of the synthesized magnetic nanoparticles; the heat treatment temperature is 180~220℃, and the time is 16~17 h. This process effectively ensures the crystallinity, phase composition, and magnetic properties of the Fe3O4 magnetic nanoparticles. Heat treatment within the temperature range of 180~220℃ helps to promote the growth of Fe... 3 O 4 The formation and growth of crystals are achieved while avoiding particle agglomeration or phase transformation caused by excessively high temperatures. A heat treatment time of 16 to 17 hours ensures that the reaction proceeds fully, resulting in a product with good crystallinity and magnetic properties.

[0024] Furthermore, the preparation of Fe3O4@SiO2 specifically involves: adding Fe3O4 magnetic nanoparticles to tetraethyl orthosilicate, and then adding ammonia, ethanol, and water, followed by stirring and reaction to obtain Fe3O4@SiO2; the ratio of Fe3O4 magnetic nanoparticles to tetraethyl orthosilicate is (0.1~0.2) g:(0.8~1.6) mL. Here, the SiO2 coating layer can effectively shield the magnetic dipole interaction of the Fe3O4 magnetic nanoparticles, reduce particle aggregation, and improve particle dispersibility and stability. By controlling the ratio of Fe3O4 magnetic nanoparticles to tetraethyl orthosilicate, the thickness and uniformity of the SiO2 coating layer can be adjusted. A thicker SiO2 coating layer can provide better protection, but excessive thickness may affect the magnetic properties of the particles; a thinner SiO2 coating layer may not completely cover the surface of the Fe3O4 magnetic nanoparticles, leading to aggregation. Therefore, selecting an appropriate ratio is crucial. The Fe3O4@SiO2 prepared by the above method exhibits excellent magnetic responsiveness, dispersibility, and stability. The introduction of the SiO2 coating not only improves the chemical stability and biocompatibility of the particles, but also enables the particles to show better application effects in subsequent bioorthogonal reactions and the preparation of cell membrane biomimetic nanorecognition materials.

[0025] Furthermore, the preparation of Fe3O4@SiO2-NH2 specifically involves: adding Fe3O4@SiO2 to toluene, ultrasonically dispersing it, then adding 3-aminopropyltriethoxysilane, stirring the reaction mixture to obtain Fe3O4@SiO2-NH2. The ratio of Fe3O4@SiO2 to 3-aminopropyltriethoxysilane is (0.1~0.2) g:(1~2) mL. The introduction of the amino (-NH2) functional group gives the Fe3O4@SiO2 surface more active sites, which can interact with it. It reacts with molecules containing functional groups such as carboxyl and aldehyde groups, thus broadening its application range. Aminated Fe3O4@SiO2-NH2 exhibits better hydrophilicity and biocompatibility, which is beneficial for its application in biomedicine, environmental remediation, and other fields. By controlling the ratio of Fe3O4@SiO2 to 3-aminopropyltriethoxysilane, the degree of amination modification and the distribution of amino functional groups can be regulated. An appropriate ratio ensures that the amino functional groups are uniformly distributed on the surface of the SiO2 coating layer, avoiding agglomeration caused by over-modification. Aminated Fe3O4@SiO2-NH2 exhibits better magnetic responsiveness, dispersibility, and stability. These improved properties lead to better application effects in subsequent bioorthogonal reactions, the preparation of cell membrane biomimetic nanomaterials, and targeted drug delivery.

[0026] Furthermore, the preparation of the dibenzocyclooctyne (DBCO)-functionalized magnetic nanoparticles specifically involves: adding Fe3O4@SiO2-NH2 to a DBCO-NHS solution and reacting at room temperature to obtain the dibenzocyclooctyne-functionalized magnetic nanoparticles; the ratio of Fe3O4@SiO2-NH2 to DBCO-NHS is (0.1~0.2) g:(3~6) mL, and the concentration of DBCO-NHS is 4.45 mM. The introduction of the DBCO functional group gives the magnetic nanoparticles more reactivity and functional properties. DBCO can undergo efficient click chemistry reactions with molecules containing azide groups. This reaction has advantages such as fast reaction rate, high selectivity, and mild conditions, providing more possibilities for the further functionalization and application of magnetic nanoparticles. By controlling the ratio of Fe3O4@SiO2-NH2 to DBCO-NHS and the concentration of DBCO-NHS, the degree of functionalization modification and the distribution of DBCO functional groups can be regulated. Appropriate ratio and concentration can ensure that DBCO functional groups are uniformly distributed on the surface of magnetic nanoparticles, avoiding agglomeration caused by over-modification or functional loss caused by insufficient modification.

[0027] Furthermore, the labeling of azide groups on the cell surface via non-natural sugar metabolism glycoengineering specifically involves: adding Ac4ManNAz to a complete culture medium and mixing thoroughly, then culturing EGFR-HEK293 cells; when the cells reach the logarithmic growth phase, digesting and collecting the cells with trypsin digestion solution, centrifuging and sonicating them, and resuspending them in phosphate buffer to obtain the azide-modified cell membrane. Through non-natural sugar metabolism glycoengineering technology, Ac4ManNAz can be effectively taken up by EGFR-HEK293 cells and participate in metabolism, thereby efficiently introducing azide groups onto the cell surface. This method has high labeling efficiency and precise labeling location, which is beneficial for subsequent biological research and applications.

[0028] Furthermore, the process of preparing a cell membrane biomimetic nanorecognition material by performing a bioorthogonal reaction between the azidated cell membrane and the dibenzocyclooctyne-functionalized magnetic nanoparticles specifically involves: ultrasonically dispersing the dibenzocyclooctyne-functionalized magnetic nanoparticles, adding them to a suspension of the azidated cell membrane, and mixing them at room temperature to obtain the cell membrane biomimetic nanorecognition material; the mass ratio of the dibenzocyclooctyne-functionalized magnetic nanoparticles to the azidated cell membrane is 1:(2.5~3). The azidated cell membrane retains many important characteristics of the original cell membrane, such as membrane proteins and membrane lipids. By combining it with the magnetic nanoparticles through a bioorthogonal reaction, a nanorecognition material with cell membrane biomimetic properties can be prepared. The click chemistry reaction between the DBCO functional group and the azide group has the advantages of high efficiency, speed and high selectivity. By precisely controlling the mass ratio of the dibenzocyclooctyne-functionalized magnetic nanoparticles to the azide-modified cell membrane, effective connection between the cell membrane and the magnetic nanoparticles can be achieved, thereby producing high-quality cell membrane biomimetic nanorecognition materials. Controlling the mass ratio of the dibenzocyclooctyne-functionalized magnetic nanoparticles to the azide-modified cell membrane within the range of 1:(2.5~3) can ensure sufficient reaction and effective connection between the two. At the same time, this ratio can also avoid the problems of agglomeration caused by excessive addition of nanoparticles or insufficient connection caused by insufficient addition. Attached Figure Description

[0029] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0030] Figure 1 This is a flowchart illustrating the preparation of EGFR / MNPs cell membrane biomimetic nanorecognition materials based on bioorthogonal reactions in Example 1 of the present invention;

[0031] Figure 2 The images shown are transmission electron microscope (TEM) images of the EGFR / MNPs cell membrane biomimetic nanorecognition material in Example 1 of this invention; wherein, (a) is a TEM image of Fe3O4@SiO2, and (b) is a TEM image of EGFR / MNPs.

[0032] Figure 3 The graph shows the adsorption performance results of the EGFR / MNPs cell membrane biomimetic nanorecognition material in Example 2 of this invention; where A is the experimental result of the isothermal adsorption experiment, B is the experimental result of the adsorption kinetics experiment, and C is the experimental result of the adsorption selectivity experiment.

[0033] Figure 4 The results of high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) of roasted chicken samples after actual application in Example 3 of this invention and the time-of-flight mass spectrometry (TOF / MS) results of the screened compounds are shown.

[0034] Figure 5 The diagram shows the results of the toxicity effect verification of the compounds screened in Example 3 of this invention. In the diagram, A represents the cell viability detection results of EGFR-HEK293 cells treated with Harman and Norharman, B represents the expression of related proteins in EGFR-HEK293 cells treated with Harman and Norharman, C represents the relative level of p-EGFR / EGFR, D represents the relative level of p-ERK / ERK, E represents the results of in vitro reactive oxygen species detection, and F represents the results of mitochondrial membrane potential detection. Detailed Implementation

[0035] To enable those skilled in the art to understand the features and effects of the present invention, the terms and expressions used in the specification and claims are explained and defined in general below. Unless otherwise specified, all technical and scientific terms used herein have the ordinary meaning understood by those skilled in the art regarding the present invention, and in case of conflict, the definitions in this specification shall prevail.

[0036] The theories or mechanisms described and disclosed herein, whether right or wrong, should not in any way limit the scope of the invention, that is, the contents of the invention can be implemented without being limited by any particular theory or mechanism.

[0037] In this document, all features defined by numerical ranges or percentage ranges, such as numerical values, quantities, contents, and concentrations, are for the sake of brevity and convenience only. Accordingly, descriptions of numerical ranges or percentage ranges should be considered as covering and specifically disclosing all possible sub-ranges and individual numerical values ​​(including integers and fractions) within those ranges.

[0038] In this article, unless otherwise specified, the terms “contains,” “includes,” “containing,” “has,” or similar terms cover the meanings of “composed of” and “mainly composed of”. For example, “A contains a” covers the meanings of “A contains a and others” and “A contains only a”.

[0039] For the sake of brevity, not all possible combinations of the technical features in each implementation scheme or embodiment are described herein. Therefore, as long as there is no contradiction in the combination of these technical features, the technical features in each implementation scheme or embodiment can be combined arbitrarily, and all possible combinations should be considered within the scope of this specification.

[0040] like Figure 1As shown, this invention provides a method for preparing a cell membrane biomimetic nanorecognition material, comprising the following steps:

[0041] (1) Fe3O4 magnetic nanoparticles were prepared and coated with SiO2 to obtain Fe3O4@SiO2; then 3-aminopropyltriethoxysilyl (3-(Triethoxysilyl)propan-1-amine, APTES) was added for amination modification to obtain Fe3O4@SiO2-NH2;

[0042] (2) Fe3O4@SiO2-NH2 was modified with diphenylcyclooctyne-N-hydroxysuccinimide ester (DBCO-NHS) to obtain dibenzocyclooctyne-functionalized magnetic nanoparticles (DBCO-MNPs).

[0043] (3) HEK293 cells expressing high levels of human epidermal growth factor receptor (EGFR) were cultured. During the process, azide groups were labeled on the cell surface by non-natural sugar metabolism sugar engineering. Cells were collected in the logarithmic phase and obtained a suspension of azide-modified cell membranes by ultrasonic centrifugation.

[0044] (4) The cell membrane modified with the azide group is subjected to a bio-orthogonal reaction with dibenzocyclooctylene-functionalized magnetic nanoparticles (DBCO-MNPs) to obtain a cell membrane biomimetic nano-recognition material, which can be represented as EGFR / MNPs.

[0045] As a further explanation of the above scheme, the preparation method of Fe3O4 in step (1) is as follows: 3-6 g of ferric chloride hexahydrate, 9-12 g of ammonium acetate and 1-2 g of sodium citrate are added to 155-350 mL of ethylene glycol and stirred at room temperature for 1-1.5 hours. Subsequently, the mixture is transferred to a high-pressure reactor and reacted at 180-220℃ for 1-17 h. Fe3O4 is collected by magnet and washed three times repeatedly with 5 mL of anhydrous ethanol and 5 mL of deionized water, and then dried at 50-80℃.

[0046] The preferred method for preparing Fe3O4 in step (1) is as follows: 3.375 g of ferric chloride hexahydrate, 9.625 g of ammonium acetate, and 0.4 g of sodium citrate are added to 170 mL of ethylene glycol and stirred at room temperature for 2 hours. Subsequently, the mixture is transferred to a high-pressure reactor and reacted at 200°C for 16 h. Fe3O4 is collected using a magnet and washed three times repeatedly with 5 mL of anhydrous ethanol and 5 mL of deionized water, and then dried at 60°C.

[0047] As a further explanation of the above scheme, the preparation method of Fe3O4@SiO2 in step (1) is as follows: 0.1~0.2g of Fe3O4 is added to 0.8~1.6 mL of ethyl silicate, and 4~8 mL of 25% ammonia water, 80~160 mL of ethanol and 12~24 mL of deionized water are added. The mixture is stirred at 37~45℃ for 7~9 h. The obtained Fe3O4@SiO2 is washed three times with 5 mL of anhydrous ethanol and 5 mL of deionized water, and dried at 50~80℃.

[0048] Preferably, the preparation method of Fe3O4@SiO2 in step (1) is as follows: 0.2 g Fe3O4 is added to 1.6 mL of ethyl silicate, and 8 mL of 25% ammonia water, 160 mL of ethanol and 24 mL of deionized water are added. The mixture is stirred at 40°C for 8 h. The obtained Fe3O4@SiO2 is washed three times with 5 mL of anhydrous ethanol and 5 mL of deionized water, and then dried at 60°C.

[0049] As a further explanation of the above scheme, the preparation method of Fe3O4@SiO2-NH2 in step (1) is as follows: 0.1~0.2 g of Fe3O4@SiO2 is added to 12~25 mL of toluene, ultrasonically dispersed, and then 1~2 mL of 3-aminopropyltriethoxysilane is added. The mixture is stirred at 110~120℃ for 20~24 h. The obtained Fe3O4@SiO2-NH2 is washed three times with 5 mL of anhydrous ethanol and 5 mL of deionized water, and dried at 50~80℃.

[0050] Preferably, the preparation method of Fe3O4@SiO2-NH2 in step (1) is as follows: 0.2 g of Fe3O4@SiO2 is added to 25 mL of toluene, ultrasonically dispersed, and then 2 mL of 3-aminopropyltriethoxysilane is added. The mixture is stirred at 120 °C for 24 h. The obtained Fe3O4@SiO2-NH2 is washed three times with 5 mL of anhydrous ethanol and 5 mL of deionized water, and then dried at 60 °C.

[0051] As a further explanation of the above scheme, the preparation method of DBCO-MNPs in step (2) is as follows: 0.1~0.2g Fe3O4@SiO2-NH2 is added to 3~6 mL of DBCO-NHS (4.45 mM) solution, and the reaction is carried out at room temperature for 4~5 h. The obtained DBCO-MNPs are washed three times with 5 mL of anhydrous ethanol and 5 mL of deionized water, and dried at 50~80℃.

[0052] Preferably, the preparation method of DBCO-MNPs in step (2) is as follows: 0.1 g Fe3O4@SiO2-NH2 is added to 3 mL DBCO-NHS (4.45 mM) solution and reacted at room temperature for 4 h. The obtained DBCO-MNPs are washed three times with 5 mL anhydrous ethanol and 5 mL deionized water and dried at 60 °C.

[0053] As a further explanation of the above scheme, in step (3), HEK293 cells expressing high levels of human epidermal growth factor receptor (EGFR) are cultured. During this process, azide groups are labeled on the cell surface using non-natural sugar metabolism engineering. Specifically, 50 μM Ac4ManNAz is added to complete culture medium and thoroughly mixed to culture EGFR-HEK293 cells, which are then incubated at 37°C for 72 h. When the cells reach the logarithmic growth phase, they are digested and collected using trypsin digestion solution, centrifuged, and sonicated. After resuspending in phosphate buffer, a cell membrane suspension with azide-modified surface is obtained.

[0054] Ac4ManNAz is N-azidoacetylmannosamine-tetraacylated, a mannosamine derivative containing an azide group.

[0055] As a further explanation of the above scheme, the preparation method of the cell membrane biomimetic nano-recognition material in step (4) is as follows: DBCO-MNPs are ultrasonically dispersed and then added to a zizide-modified cell membrane suspension. The mixture is shaken and mixed at room temperature for 12 h, wherein the mass ratio of DBCO-MNPs to zizide-modified cell membrane is 1:(2.5~3). The concentration of the zizide-modified cell membrane suspension is 15 mg / mL.

[0056] In addition, the present invention also discloses the application of the above-mentioned cell membrane biomimetic nano-recognition material in screening potential chemical risk substances in food samples; in application, the cell membrane biomimetic nano-recognition material is added to the food sample extract and vortexed, the material is collected and eluted by a magnet, and finally the eluent is analyzed by high performance liquid chromatography-tandem time-of-flight mass spectrometry.

[0057] Specifically, the ratio of the cell membrane biomimetic nano-recognition material to the food sample extract is (5~10) mg:(1~2) mL.

[0058] Toxicological studies have shown that chemical substances can influence intracellular signaling pathways and release aberrant signals by binding to transmembrane receptors. Inspired by the binding capacity of receptor proteins, target-based biomimetic screening strategies should help effectively identify potential contaminants. Epidermal growth factor receptor (EGFR) is a transmembrane receptor widely distributed in various cells, controlling cell proliferation, growth, survival, migration, and differentiation by regulating complex intracellular signaling networks. Aberrant activation of EGFR may be closely related to the development of many chronic inflammatory diseases, such as metabolic syndrome, neurodegenerative diseases, and various cancers. By coating HEK293 cell membranes that highly express EGFR receptors onto the surface of magnetic carriers, these nanomaterials can directly replicate the biological interface properties of the source cells and completely maintain the three-dimensional conformation of the receptor protein on the membrane. Simultaneously, the specific transmembrane receptors on the biomimetic materials help to accurately reveal the interaction process between chemical substances and membrane receptors. Therefore, cell membrane-coated magnetic nanoparticles can serve as a biomimetic recognition platform for the discovery of potentially hazardous compounds in food matrices and provide a basis for further elucidating the mechanisms of toxic effects involved. By coating the HEK293 cell membrane overexpressing the EGFR receptor onto the surface of a magnetic carrier as a biomimetic recognition platform, the identification efficiency of unknown chemical risks in food is effectively improved, providing a new toxicity-oriented approach for screening potential contaminants in food.

[0059] This invention provides a cell membrane biomimetic nanomaterial for recognition, its preparation method, and its application. It utilizes magnetic nanoparticles as a carrier, immobilizing the cell membrane onto the magnetic carrier through a bioorthogonal reaction, ultimately yielding a cell membrane-based biomimetic nanomaterial for the identification and risk assessment of unknown contaminants in food. This invention combines the high specific surface area and high magnetic response of magnetic nanoparticles with the highly complex biological interface properties mediated by cell membranes. Furthermore, by covalently immobilizing the active cell membrane containing target receptors onto the surface of the magnetic nanoparticles, the integrity of the cell membrane and the three-dimensional structure and bioactivity of its membrane receptor proteins are preserved to the maximum extent. This endows the biomimetic nanomaterial with excellent targeting properties, enabling specific screening and rapid magnetic separation of unknown chemical risks in complex food matrices. This improves the identification efficiency of unknown chemical risks in food and provides a new toxicity-oriented approach for screening potential contaminants in food.

[0060] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0061] The following examples use instruments and equipment conventional in the art. Experimental methods in the following examples, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. All raw materials used in the following examples are conventional commercially available products with specifications conventional in the art. In this specification and the following examples, unless otherwise specified, "%" refers to weight percentage, "parts" refers to parts by weight, and "ratio" refers to weight proportion.

[0062] Example 1

[0063] This embodiment provides a method for preparing a cell membrane biomimetic nanorecognition material, specifically including the following steps:

[0064] (1) Preparation of Fe3O4 magnetic nanoparticles: A hydrothermal synthesis method was adopted. 3.375 g of ferric chloride hexahydrate, 9.625 g of ammonium acetate and 0.4 g of sodium citrate were added to 70 mL of ethylene glycol and stirred at room temperature for 2 hours. Subsequently, the mixture was transferred to a high-pressure reactor and reacted at 200 °C for 16 h. Fe3O4 was collected by magnet and washed three times with anhydrous ethanol and deionized water, and dried at 60 °C.

[0065] (2) Preparation of Fe3O4@SiO2 magnetic nanoparticles: 0.2 g Fe3O4 was added to 1.6 mL of tetraethyl orthosilicate, along with 8 mL of 25% ammonia, 160 mL of ethanol, and 24 mL of deionized water. The mixture was stirred at 40 °C for 8 h. The resulting Fe3O4@SiO2 was washed three times with anhydrous ethanol and deionized water and dried at 60 °C.

[0066] (3) Preparation of Fe3O4@SiO2-NH2 magnetic nanoparticles: 0.2 g of Fe3O4@SiO2 was added to 25 mL of toluene, ultrasonically dispersed, and then 2 mL of 3-aminopropyltriethoxysilane was added. The mixture was stirred at 120 °C for 24 h. The obtained Fe3O4@SiO2-NH2 was washed three times with anhydrous ethanol and deionized water and dried at 60 °C.

[0067] (4) Preparation of DBCO-MNPs magnetic nanoparticles: 0.1 g Fe3O4@SiO2-NH2 was added to 4.45 mMDBCO-NHS solution and reacted at room temperature for 4 h. The resulting DBCO-MNPs magnetic nanoparticles were washed three times with anhydrous ethanol and deionized water and dried at 60 °C.

[0068] (5) HEK293 cells expressing human epidermal growth factor receptor were cultured. During the process, azide groups were labeled on the cell surface by non-natural sugar metabolism sugar engineering. Cells were collected in the logarithmic phase and obtained by ultrasonic centrifugation. That is, 50 μM Ac4ManNAz was added to the complete culture medium and mixed thoroughly. The mixture was used to culture EGFR-HEK293 cells. The cells were incubated at 37℃ to express azide groups on the cell surface. When the cells grew to the logarithmic phase, they were digested and collected by trypsin digestion solution and centrifuged at 1000 rpm / min.

[0069] (6) The azide-labeled cells were resuspended in 50 mM Tris-HCl buffer and sonicated on ice for 3 s, with 3 s intervals, for 2 min each time. Then, the cells were centrifuged at 1000 rpm / min for 5 min, and the supernatant was collected. The cells were then centrifuged again at 12000 rpm / min at 4℃ for 20 min, and the precipitate was collected. The membrane precipitate was resuspended in PBS solution to obtain the azide-labeled cell membrane suspension.

[0070] (7) After ultrasonically dispersing DBCO-MNPs nanoparticles, they were added to a cell membrane suspension modified with azide groups and mixed by shaking at room temperature for 12 h to prepare a cell membrane biomimetic nanorecognition material. The mass ratio of cell membrane to DBCO / MNPs in the cell membrane suspension was 3:1. The prepared cell membrane biomimetic nanorecognition material was labeled as EGFR / MNPs.

[0071] The microstructure of the cell membrane biomimetic nanorecognition materials prepared by the above methods was characterized using transmission electron microscopy, such as... Figure 2 As shown, by Figure 2 It can be seen that both Fe3O4@SiO2 and EGFR / MNPs are nearly spherical with an average diameter of about 250 nm, and it can be clearly seen that a monolayer cell membrane is uniformly covered on Fe3O4@SiO2.

[0072] Comparative Example 1

[0073] The difference between this comparative example and Example 1 is that only steps (1) to (4) are performed, that is, only DBCO-MNPs magnetic nanoparticles are synthesized.

[0074] Comparative Example 2

[0075] The difference between this comparative example and Example 1 is step (5), in which HEK293 cell membranes without EGFR high expression are prepared. All other steps are the same, and the final product is represented as HEK / MNPs.

[0076] Furthermore, the adsorption performance of the cell membrane biomimetic nanorecognition material prepared in Example 1 and the materials prepared in Comparative Examples 1 and 2 were evaluated from three aspects: isothermal adsorption, adsorption kinetics, and adsorption selectivity. The results are shown in [the table below]. Figure 3 As shown in the figure, in the isothermal adsorption experiment, 5 mg of EGFR / MNPs, HEK / MNPs, and MNPs were added to 1 mL of gefitinib solutions of different concentrations (20–2500 mg / L) for adsorption. After the adsorption process, the adsorbents were magnetically separated, and the residual amount of gefitinib in the supernatant was determined by high-performance liquid chromatography. The results are as follows. Figure 3 As shown in Figure A, the adsorption capacity of EGFR / MNPs initially increases rapidly with increasing gefitinib concentration, then slows down, and eventually reaches saturation. Furthermore, the adsorption capacity of EGFR / MNPs for gefitinib is significantly higher than that of HEK / MNPs and MNPs.

[0077] In the adsorption kinetics experiment, 5 mg of adsorbent was added to 1 mL of gefitinib solution (200 mg / L) for adsorption. After a certain adsorption time (10 s–30 min), the residual amount of gefitinib in the supernatant was determined by high-performance liquid chromatography (HPLC). The results are as follows: Figure 3 As shown in Figure B, it can be seen that the adsorption amount of gefitinib gradually increases with increasing adsorption time, and then tends to stabilize, indicating that adsorption saturation has been reached.

[0078] In the adsorption selectivity experiment, one positive control drug (gefitinib) and three negative control drugs (gliclazide, nifedipine, and tamsulosin) were selected to investigate the selectivity of EGFR / MNPs. The results are as follows: Figure 3 As shown in Figure C, gefitinib was completely adsorbed by EGFR / MNPs, with a recovery rate significantly higher than that of the control group. Meanwhile, EGFR / MNPs did not show significant adsorption effects on the three negatively reacting drugs: gliclazide, nifedipine, and tamsulosin. These results indicate that EGFR / MNPs possess strong selectivity and affinity for drugs that can interact with EGFR.

[0079] Example 2

[0080] This embodiment provides a method for screening potential chemical risk substances in roasted chicken samples using a cell membrane biomimetic nanomaterial prepared in Example 1. The steps are as follows:

[0081] The first step involved pretreatment of the roasted chicken sample. The roasted chicken sample was cut into pieces and homogenized using a homogenizer. 6 g of the homogenized sample was weighed and placed in a 50 mL centrifuge tube. 10 mL of deionized water and 10 mL of acetonitrile were added, and the mixture was vortexed for 3 min, followed by sonication for 15 min. 4 g of anhydrous magnesium sulfate, 0.5 g of sodium chloride, and 5 mL of n-hexane were added to the centrifuge tube. The mixture was vortexed for another 10 min to ensure thorough interaction between the sample and the solvent. The sample was then centrifuged again at 2000 g for 10 min to achieve three-phase separation. The intermediate acetonitrile layer was collected, and hexane was added again to remove any remaining lipids. Finally, the defatted solution was dried by vacuum distillation and reconstituted with deionized water / methanol (1:1, v / v).

[0082] The second step, screening for potential risk substances, involved dispersing 5 mg of EGFR / MNPs in 1 mL of food sample extract and vortexing for 20 min. The magnetic composite material was collected using a magnet, first washed with isopropanol:water (9:1, v / v) to remove interference from non-specific interactions, then eluted with isopropanol:water (1:9, v / v). The eluent was further analyzed using HPLC-TOF-MS. Results are as follows: Figure 4 As shown, compared with the chromatogram of the roasted chicken sample extract, a large number of components were eluted during the screening process, ultimately identifying two potential contaminant components. Further analysis by TOF-MS revealed that the two peaks were identified as Harman (β-carboline) and Norharman (nor-β-carboline), respectively.

[0083] The third step involved screening and verifying the toxic effects of the compounds. To verify the inhibitory effect of Harman and Norharman compounds on the proliferation of EGFR-HEK293 cells, a CCK-8 assay was performed. Figure 5 As shown in Figure A, with increasing Harman and Norharman concentrations, the mean cell viability gradually decreased in a dose-dependent manner, with the half-maximal inhibitory concentration (IC50) decreasing. 50 The concentrations were 75 μM and 200 μM, respectively. This indicates that both screened compounds effectively inhibited the proliferation of EGFR-HEK293 cells. EGFR dimerization and phosphorylation can activate multiple downstream signaling pathways, controlling cell proliferation and growth. To further evaluate whether Harman and Norharman compounds can regulate the EGFR pathway, a western blot experiment was subsequently performed. Figure 5As shown in Figures B and C, compared with the control group, the phosphorylation levels of epidermal growth factor receptor (EGFR) and extracellular signal-regulated kinase (ERK) were significantly increased after treatment with Harman and Norharman. The results indicate that both screened compounds exert their effects by upregulating the EGFR and ERK phosphorylation pathways. Furthermore, EGFR activation leads to the generation of reactive oxygen species (ROS). Excessive ROS generation can further induce oxidative damage, even leading to apoptosis and necrosis. Therefore, the generation of ROS and its effects on mitochondrial membrane potential were investigated. The effects of Harman and Norharman on ROS levels were analyzed in EGFR-HEK293 cells. Figure 5 As shown in Figure E, the treated group exhibited brighter green fluorescence compared to the control group, indicating oxidative stress. To elucidate the impact of reactive oxygen species on mitochondrial integrity, mitochondrial membrane potential was measured using the JC-1 kit, and the results are shown below. Figure 5 As shown in F, red JC-1 aggregates indicate healthy polarized mitochondria, while green JC-1 monomers indicate abnormally depolarized mitochondria in apoptotic cells. The detection of green fluorescence indicating mitochondrial depolarization in both treatment groups suggests that elevated reactive oxygen species levels disrupt mitochondrial membrane potential and may induce early apoptosis.

[0084] Example 3

[0085] A method for preparing a cell membrane biomimetic nanorecognition material includes the following steps:

[0086] (1) The preparation method of Fe3O4 magnetic nanoparticles is as follows: 2 g of ferric chloride hexahydrate, 8 g of ammonium acetate and 0.1 g of sodium citrate were added to 50 mL of ethylene glycol and stirred at room temperature for 1 hour. Then, the mixture was transferred to a high-pressure reactor and reacted at 100 °C for 17 h. Fe3O4 was collected by magnet and washed three times with 5 mL of anhydrous ethanol and 5 mL of deionized water. It was dried at 50 °C to obtain Fe3O4 magnetic nanoparticles.

[0087] (2) Preparation method of Fe3O4@SiO2: 0.1 g Fe3O4 was added to 1.4 mL of ethyl silicate, along with 6 mL of 25% ammonia, 140 mL of ethanol and 20 mL of deionized water. The mixture was stirred at 30 °C for 9 h. The obtained Fe3O4@SiO2 was washed three times with 5 mL of anhydrous ethanol and 5 mL of deionized water and dried at 50 °C.

[0088] (3) The preparation method of Fe3O4@SiO2-NH2 is as follows: 0.1 g of Fe3O4@SiO2 is added to 20 mL of toluene, ultrasonically dispersed, and then 1 mL of 3-aminopropyltriethoxysilane is added. The mixture is stirred at 110 °C for 30 h. The obtained Fe3O4@SiO2-NH2 is washed three times with 5 mL of anhydrous ethanol and 5 mL of deionized water, and dried at 50 °C.

[0089] (4) The preparation method of DBCO-MNPs is as follows: 0.05 g Fe3O4@SiO2-NH2 is added to 5 mL DBCO-NHS (4.45 mM) solution and reacted at room temperature for 2 h. The obtained DBCO-MNPs are washed three times with 5 mL anhydrous ethanol and 5 mL deionized water and dried at 50 °C.

[0090] (5) Add 50 μM Ac4ManNAz to the complete culture medium and mix thoroughly. Use this mixture to culture EGFR-HEK293 cells and incubate at 37°C for 72 h. When the cells grow to the logarithmic growth phase, digest them with trypsin digestion solution, collect them, centrifuge and sonicate them. Then, resuspend them in phosphate buffer to obtain a cell membrane suspension with azide-modified surface.

[0091] (6) After ultrasonic dispersion, DBCO-MNPs were added to a suspension of cell membranes modified with azide groups and mixed by shaking at room temperature for 12 h. The mass ratio of DBCO-MNPs to cell membranes modified with azide groups was 1:2. The concentration of the cell membrane suspension modified with azide groups was 15 mg / mL, thus obtaining the cell membrane biomimetic nano-recognition material, namely EGFR / MNPs.

[0092] Example 4

[0093] A method for preparing a cell membrane biomimetic nanorecognition material includes the following steps:

[0094] (1) The preparation method of Fe3O4 magnetic nanoparticles is as follows: 3 g of ferric chloride hexahydrate, 9 g of ammonium acetate and 1 g of sodium citrate were added to 175 mL of ethylene glycol and stirred at room temperature for 4 hours. Then, the mixture was transferred to a high-pressure reactor and reacted at 200 °C for 16 h. Fe3O4 was collected by magnet and washed three times with 5 mL of anhydrous ethanol and 5 mL of deionized water. It was dried at 80 °C to obtain Fe3O4 magnetic nanoparticles.

[0095] (2) Preparation method of Fe3O4@SiO2: 0.2 g Fe3O4 was added to 1.6 mL of ethyl silicate, and 8 mL of 25% ammonia water, 160 mL of ethanol and 24 mL of deionized water were added. The mixture was stirred at 40 °C for 8 h. The obtained Fe3O4@SiO2 was washed three times with 5 mL of anhydrous ethanol and 5 mL of deionized water and dried at 80 °C.

[0096] (3) The preparation method of Fe3O4@SiO2-NH2 is as follows: 0.2 g of Fe3O4@SiO2 is added to 25 mL of toluene, ultrasonically dispersed, and then 2 mL of 3-aminopropyltriethoxysilane is added. The mixture is stirred at 110 °C for 20 h. The obtained Fe3O4@SiO2-NH2 is washed three times with 5 mL of anhydrous ethanol and 5 mL of deionized water, and dried at 80 °C.

[0097] (4) The preparation method of DBCO-MNPs is as follows: 0.2 g Fe3O4@SiO2-NH2 is added to 6 mL DBCO-NHS (4.45 mM) solution and reacted at room temperature for 5 h. The obtained DBCO-MNPs are washed three times with 5 mL anhydrous ethanol and 5 mL deionized water and dried at 80 °C.

[0098] (5) Add 50 μM Ac4ManNAz to the complete culture medium and mix thoroughly. Use this mixture to culture EGFR-HEK293 cells and incubate at 37°C for 72 h. When the cells grow to the logarithmic growth phase, digest them with trypsin digestion solution, collect them, centrifuge and sonicate them. Then, resuspend them in phosphate buffer to obtain a cell membrane suspension with azide-modified surface.

[0099] (6) After ultrasonic dispersion, DBCO-MNPs were added to a suspension of cell membranes modified with azide groups and mixed by shaking at room temperature for 12 h. The mass ratio of DBCO-MNPs to cell membranes modified with azide groups was 1:4. The concentration of the cell membrane suspension modified with azide groups was 15 mg / mL, thus obtaining the cell membrane biomimetic nano-recognition material, namely EGFR / MNPs.

[0100] Example 5

[0101] A method for preparing a cell membrane biomimetic nanorecognition material includes the following steps:

[0102] (1) The preparation method of Fe3O4 magnetic nanoparticles is as follows: 3 g of ferric chloride hexahydrate, 9 g of ammonium acetate and 1 g of sodium citrate were added to 170 mL of ethylene glycol and stirred at room temperature for 1.5 hours. Then, the mixture was transferred to a high-pressure reactor and reacted at 200 °C for 16 h. Fe3O4 was collected by magnet and washed three times with 5 mL of anhydrous ethanol and 5 mL of deionized water. It was then dried at 60 °C to obtain Fe3O4 magnetic nanoparticles.

[0103] (2) Preparation method of Fe3O4@SiO2: 0.2 g Fe3O4 was added to 1.6 mL of ethyl silicate, and 8 mL of 25% ammonia water, 160 mL of ethanol and 24 mL of deionized water were added. The mixture was stirred at 40 °C for 8 h. The obtained Fe3O4@SiO2 was washed three times with 5 mL of anhydrous ethanol and 5 mL of deionized water and dried at 60 °C.

[0104] (3) The preparation method of Fe3O4@SiO2-NH2 is as follows: 0.2 g of Fe3O4@SiO2 is added to 25 mL of toluene, ultrasonically dispersed, and then 2 mL of 3-aminopropyltriethoxysilane is added. The mixture is stirred at 120 °C for 25 h. The obtained Fe3O4@SiO2-NH2 is washed three times with 5 mL of anhydrous ethanol and 5 mL of deionized water, and dried at 60 °C.

[0105] (4) The preparation method of DBCO-MNPs is as follows: 0.1 g Fe3O4@SiO2-NH2 is added to 3 mL DBCO-NHS (4.45 mM) solution and reacted at room temperature for 2.5 h. The obtained DBCO-MNPs are washed three times with 5 mL anhydrous ethanol and 5 mL deionized water and dried at 60 °C.

[0106] (5) Add 50 μM Ac4ManNAz to the complete culture medium and mix thoroughly. Use this mixture to culture EGFR-HEK293 cells and incubate at 37°C for 72 h. When the cells grow to the logarithmic growth phase, digest them with trypsin digestion solution, collect them, centrifuge and sonicate them. Then, resuspend them in phosphate buffer to obtain a cell membrane suspension with azide-modified surface.

[0107] (6) After ultrasonic dispersion, DBCO-MNPs were added to a suspension of cell membranes modified with azide groups and mixed by shaking at room temperature for 12 h. The mass ratio of DBCO-MNPs to cell membranes modified with azide groups was 1:3. The concentration of the cell membrane suspension modified with azide groups was 15 mg / mL, thus obtaining the cell membrane biomimetic nano-recognition material, namely EGFR / MNPs.

[0108] This method uses magnetic nanoparticles as a carrier to immobilize cell membranes onto the magnetic carrier through a bioorthogonal reaction, ultimately producing a cell membrane-based biomimetic nanomaterial for the identification and risk assessment of unknown contaminants in food. This invention combines the high magnetic response (40.38 emu g) of magnetic nanoparticles... -1 The biomimetic nanomaterials possess the characteristics of highly complex biological interfaces mediated by cell membranes. Furthermore, by covalently immobilizing active cell membranes containing target receptors onto the surface of magnetic nanoparticles, the integrity of the cell membrane and the three-dimensional structure and bioactivity of its membrane receptor proteins are preserved to the maximum extent. This endows the biomimetic nanomaterials with excellent targeting properties, enabling the specific screening and rapid magnetic separation of unknown chemical risks in complex food matrices. This improves the identification efficiency of unknown chemical risks in food and provides a new toxicity-oriented approach for screening potential contaminants in food.

[0109] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. The application of a cell membrane biomimetic nanomaterial for screening potential chemical risk substances in food samples, characterized in that, Includes the following steps: The sample to be tested was pretreated to obtain a food sample extract; Then, the cell membrane biomimetic nano-recognition material was added to the food sample extract and vortexed. The material was collected by a magnet and eluted with a mixture of isopropanol and water. Finally, the eluent was analyzed by high-performance liquid chromatography-tandem time-of-flight mass spectrometry, and β-carboline and norβ-carboline were screened out. The pretreatment specifically involved: cutting the sample into pieces and homogenizing it; adding deionized water and acetonitrile to the homogenized sample and vortexing and sonicating; then adding anhydrous magnesium sulfate, sodium chloride, and n-hexane and continuing vortexing to ensure sufficient interaction between the sample and solvent; centrifuging again and collecting the intermediate acetonitrile layer; adding n-hexane again to remove residual lipids; finally, drying the defatted solution by vacuum distillation and reconstitution with deionized water and methanol to complete the pretreatment. The cell membrane biomimetic nanorecognition material was prepared by the following method: S1: Fe3O4 magnetic nanoparticles were prepared and coated with SiO2 to obtain Fe3O4@SiO2; then 3-aminopropyltriethoxysilane was added for amination modification to obtain Fe3O4@SiO2-NH2; S2: The Fe3O4@SiO2-NH2 was modified with DBCO-NHS to obtain dibenzocyclooctylene-functionalized magnetic nanoparticles; HEK293 cells expressing human epidermal growth factor receptor were cultured, and azide groups were labeled on the cell surface through non-natural sugar metabolism glycoengineering during the process. Cells were collected in the logarithmic phase and obtained by ultrasonic centrifugation. S3: The azide-modified cell membrane and the dibenzocyclooctylene-functionalized magnetic nanoparticles are subjected to a bioorthogonal reaction to obtain the cell membrane biomimetic nanorecognition material. The preparation of the Fe3O4 magnetic nanoparticles specifically involves: adding ferric chloride hexahydrate, ammonium acetate, and sodium citrate to ethylene glycol, stirring the mixture at room temperature, and then heat-treating the reaction product to obtain the Fe3O4 magnetic nanoparticles; the mass ratio of ferric chloride hexahydrate to sodium citrate is (3~6):(1~2); the heat treatment temperature is 180~220℃, and the time is 16~17 h; The preparation of Fe3O4@SiO2-NH2 is specifically as follows: Fe3O4@SiO2 is added to toluene, ultrasonically dispersed, and then 3-aminopropyltriethoxysilane is added. The mixture is stirred and reacted to obtain Fe3O4@SiO2-NH2. The ratio of Fe3O4@SiO2 to 3-aminopropyltriethoxysilane is (0.1~0.2) g: (1~2) mL.

2. The application of the cell membrane biomimetic nanorecognition material according to claim 1 in screening potential chemical risk substances in food samples, characterized in that, The preparation of Fe3O4@SiO2 is specifically as follows: Fe3O4 magnetic nanoparticles are added to ethyl silicate, and ammonia, ethanol and water are added, and the mixture is stirred to react and obtain Fe3O4@SiO2; the ratio of Fe3O4 magnetic nanoparticles to ethyl silicate is (0.1~0.2)g:(0.8~1.6)mL.

3. The application of the cell membrane biomimetic nanorecognition material according to claim 1 in screening potential chemical risk substances in food samples, characterized in that, The preparation of the dibenzocyclooctyne-functionalized magnetic nanoparticles specifically involves: adding Fe3O4@SiO2-NH2 to a DBCO-NHS solution and reacting at room temperature to obtain the dibenzocyclooctyne-functionalized magnetic nanoparticles; the ratio of Fe3O4@SiO2-NH2 to DBCO-NHS is (0.1~0.2) g:(3~6) mL, and the concentration of DBCO-NHS is 4.45 mM.

4. The application of the cell membrane biomimetic nanorecognition material according to claim 1 in screening potential chemical risk substances in food samples, characterized in that, The process of labeling cell surface with azide groups via non-natural sugar metabolism engineering is as follows: Ac4ManNAz is added to a complete culture medium and thoroughly mixed for culturing EGFR-HEK293 cells; when the cells grow to the logarithmic growth phase, they are digested and collected using trypsin digestion solution, centrifuged and sonicated, and then resuspended in phosphate buffer to obtain the azide-modified cell membrane.

5. The application of the cell membrane biomimetic nanorecognition material according to claim 1 in screening potential chemical risk substances in food samples, characterized in that, The method of preparing a cell membrane biomimetic nanorecognition material by performing a bioorthogonal reaction between the azidated cell membrane and the dibenzocyclooctyne-functionalized magnetic nanoparticles is as follows: the dibenzocyclooctyne-functionalized magnetic nanoparticles are ultrasonically dispersed and then added to a suspension of the azidated cell membrane. The mixture is shaken at room temperature to prepare the cell membrane biomimetic nanorecognition material. The mass ratio of the dibenzocyclooctyne-functionalized magnetic nanoparticles to the azidated cell membrane is 1:(2.5~3).

6. The application of the cell membrane biomimetic nanorecognition material according to claim 1 in screening potential chemical risk substances in food samples, characterized in that, The ratio of the cell membrane biomimetic nano-recognition material to the food sample extract is (5~10) mg:(1~2) mL.

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