Series of engineered magnetic nano materials for metabolic crown characterization and preparation method and application thereof

Abstract

The invention discloses a series of engineered magnetic nano materials for metabolic crown characterization, a preparation method of the engineered magnetic nano materials, an integrated method of metabolic crown construction and corresponding metabonomics characterization based on the engineered magnetic nano materials, and application of the engineered magnetic nano materials in disease screening and diagnosis. The series of engineered magnetic nano-materials have differentiated surface function modifications, and when the engineered magnetic nano-materials are exposed to biological fluid, a unique metabolic crown can be formed. Through the disclosed integrated method for constructing and characterizing the biological fluid metabolic crown based on the series of engineered magnetic nano-materials, in-situ characterization of the metabolic crown on the surface of the nano-material can be directly realized through laser desorption ionization mass spectrometry detection without additional separation steps; obtained biological fluid metabolic crown mass spectrum data can be successfully applied to accurate discrimination and metabolic marker screening of endometriosis-related ovarian cancer patients, which shows that the biological fluid metabolic crown mass spectrum has a wide application prospect in the aspect of early screening of malignant transformation of endometriosis.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of nanomaterials and biological detection, and particularly relates to a series of engineered magnetic nanomaterials for metabolic corona characterization and a preparation method and application thereof. BACKGROUND

[0002] The engineered construction of nanoparticles (NPs) is a transformative innovation in the field of nanomedicine, but due to the uncontrollable adsorption behavior of NPs in biological systems, it has seriously hindered the clinical application of engineered NPs in biological imaging, drug delivery, precision diagnosis and cancer immunotherapy, etc. When NPs are exposed to a biological environment, biomolecules (such as proteins, metabolites, lipids, etc.) will spontaneously adsorb on the surface of the nanoparticles to form a biomolecular corona. The biomolecular corona directly affects the fate of nanoparticles in the biological system, including cell uptake, toxicity, and biodistribution, etc. However, current research on biomolecular corona mainly focuses on interpreting the composition and behavior evolution of protein corona, largely ignoring other biomolecular components. Among them, metabolites (molecular weight <1000 Da) play a very key role in the signal cascade, and their species and abundance in biological systems are much higher than proteins. Similar to protein corona, nanoparticles exposed to a biological environment can also spontaneously form a metabolic corona and obtain a unique metabolic fingerprint. The lagging development of metabolic corona characterization technology has limited the development of metabolic corona research. Currently, the commonly used metabolic corona characterization technology is mainly based on mass spectrometry (MS) non-in situ characterization methods (such as liquid chromatography-mass spectrometry [LC-MS], gas chromatography-mass spectrometry [GC-MS], capillary electrophoresis-mass spectrometry [CE-MS]), which mainly rely on centrifugation and elution steps to separate the metabolic corona from the biological environment and the surface of NPs, respectively. This additional separation process inevitably damages the integrity of the corona in composition and structure, and the risk of contamination caused by the adsorption of exogenous biomacromolecules during the operation process will also increase. Therefore, developing in situ metabolic corona characterization methods with the principle of maximizing the integrity of the metabolic corona is the fundamental driving force for the rapid development of metabolic corona research, and is crucial for decoding biomolecular corona, interpreting the mechanism of biological-nanometer interface interaction and accelerating the transformation of nanomedicine.

[0003] Compared with LC-MS, GC-MS and CE-MS, matrix-assisted laser desorption / ionization mass spectrometry (MALDI MS) has the advantages of high sensitivity, large throughput, fast analysis speed, simple sample preparation process, low sample consumption, strong detection resistance to salt / protein interference, and is widely used in the development of large-scale clinical sample metabolomics analysis technology. The efficiency of the metabolomics analysis platform based on MALDI MS is mainly determined by the matrix material, and a large number of nanomaterials have been developed as matrices with high sensitivity and selectivity for detecting small molecule metabolites, including noble metal nanomaterials, metal oxide nanomaterials, carbon-based nanomaterials, silicon-based nanomaterials, metal-organic frameworks (MOFs) and covalent organic frameworks (COFs). Among them, Fe3O4 NPs stand out with their extremely high cost performance and are recognized as the most ideal matrix for small molecule metabolomics detection based on MALDI MS technology, and are widely used in disease screening, biomarker screening and metabolite qualitative and quantitative analysis. More attention should be paid to the fact that the inherent magnetism of Fe3O4 NPs facilitates the rapid separation of biomolecules (such as metabolites, proteins, etc.) from complex systems, that is, the target analyte is enriched with minimal sample loss. Therefore, by fully utilizing the advantages of Fe3O4 NPs as a small molecule metabolite detection matrix and the advantages of simple enrichment and separation operation, it is expected to develop a metabolome characterization platform based on MALDI MS, and to realize the in-situ characterization of metabolome on the surface of NPs. SUMMARY

[0004] In order to develop advanced, simple and efficient metabolome characterization technology, realize in-situ characterization of metabolome on the surface of NPs, and expand the application of metabolome in clinic, the purpose of the present application is to provide an engineered magnetic nanomaterial for metabolome characterization and a preparation method thereof, and to realize the construction of metabolome in biological fluids, in-situ characterization of metabolome on the surface of NPs and its application in clinical disease diagnosis and screening by using the method.

[0005] The present application provides a series of engineered magnetic nanomaterials for metabolome characterization, which includes Fe3O4 magnetic nanomaterial, Fe3O4 magnetic composite nanomaterial with core-shell structure taking Fe3O4 magnetic nanomaterial as core and surface coated with SiO2 or TiO2, Fe3O4 magnetic nanocomposite material with amino (-NH2) or carboxyl (-COOH) loaded on the surface, and mixed magnetic nanomaterials of the above materials; the engineered magnetic nanomaterial has the following chemical formula: Fe3O4, Fe3O4@SiO2, Fe3O4@TiO2, Fe3O4-NH2, Fe3O4-COOH, Fe3O 4（a） +Fe3O4@SiO 2（b）+Fe3O4@TiO 2（c） +Fe3O4-NH 2（d） +Fe3O4-COOH （e） MFNM (abbreviation), wherein a=0.0-1.0, b=0.0-1.0, c=0.0-1.0, d=0.0-1.0, e=0.0-1.0, a, b, c, d, e cannot be 0 at the same time, and a+b+c+d+e=1.

[0006] In the present application, the engineered magnetic nanomaterial is at least two or more of Fe3O4, Fe3O4@SiO2, Fe3O4@TiO2, Fe3O4-NH2, Fe3O4-COOH.

[0007] The present application proposes a series of preparation methods of engineered magnetic nanomaterials for metabolic coronary characterization, and the specific steps are as follows: (1) Preparation method of Fe3O4 (1.1) Dissolve ferric chloride hexahydrate (FeCl3·6H2O) in ethylene glycol solution, ultrasonic mixing, stirring at room temperature for 20-40 min, concentration 18-20 mg·mL -1 ; (1.2) Under magnetic stirring at room temperature, uniformly disperse sodium acetate (CH3COONa) into the solution obtained in step (1.1), and stir at room temperature for 20-40 min; (1.3) Transfer the solution obtained in step (1.2) to a reaction kettle, and react at 160-200℃ for 16-24 hours; (1.4) Under the action of an external magnetic field, the product obtained in step (1.3) is washed with deionized water and ethanol alternately, and the obtained solid product is vacuum dried at 50℃, then sealed and stored at room temperature, to obtain a magnetic nanomaterial of Fe3O4; (2) Preparation method of Fe3O4@SiO2 (2.1) Uniformly disperse the product obtained according to the preparation method of (1) Fe3O4 into an ethanol solution, ultrasonic mixing, stirring for 20-40 min, concentration 1.0-1.5 mg·mL -1 ; (2.2) Under magnetic stirring at room temperature, add 5% ammonium hydroxide (NH3·H2O) solution to the solution obtained in step (2.1); (2.3) Slowly add dropwise an ethanol solution of tetraethyl orthosilicate (Si(OC2H5)4) (concentration 460-480 mg·mL -1 ) to the solution obtained in step (2.2), and stir vigorously at room temperature for 12 hours; (2.4) Under the action of an external magnetic field, the solid product obtained in step (2.3) is washed alternately with deionized water and ethanol, dried at 50°C under vacuum, and then stored at room temperature in a sealed container to obtain a magnetic composite nanomaterial with a core-shell structure, in which a Fe3O4 magnetic nanomaterial is used as a core and silica (SiO2) is coated on the surface; (3) Preparation method of Fe3O4@TiO2 (3.1) The product obtained according to the preparation method of (1) Fe3O4 is uniformly dispersed in an ethanol solution and ultrasonically mixed, and stirred for 20-40 min, with a concentration of 0.40-0.45 mg·mL -1 ; (3.2) Under magnetic stirring at room temperature, an ammonium hydroxide solution is uniformly dispersed in the solution obtained in step (3.1); (3.3) Under vigorous stirring at room temperature, a 24% tetrabutyl titanate (Ti(OCH2CH2CH2CH3)4) ethanol solution is slowly added dropwise to the solution obtained in step (3.2), followed by reaction at 45°C for 20-24 hours, and finally the solid product is washed with ethanol under the action of an external magnetic field and dried at 50°C under vacuum; (3.4) The product obtained in step (3.3) is calcined at 300-500°C in an air atmosphere for 2-3 hours, with a heating rate of 1-2°C / min, to obtain a magnetic composite nanomaterial with a core-shell structure, in which a Fe3O4 magnetic nanomaterial is used as a core and titanium oxide (TiO2) is coated on the surface; (4) Preparation method of Fe3O4-NH2 (4.1) The product obtained according to the preparation method of (1) Fe3O4 is uniformly dispersed in a 2wt% (3-aminopropyl)triethoxysilane aqueous solution, and reacted under stirring at room temperature for 20-24 hours, with a concentration of 0.30-0.35 mg·mL -1 ; (4.2) Under the action of an external magnetic field, the solid product obtained in step (4.1) is washed alternately with deionized water and ethanol, dried at 50°C under vacuum, and then stored at room temperature in a sealed container to obtain a Fe3O4 magnetic nanocomposite material with a surface loaded and modified with amino groups (-NH2); (5) Preparation method of Fe3O4-COOH (5.1) Ferric chloride hexahydrate (FeCl3·6H2O) is dissolved in an ethylene glycol solution, ultrasonically mixed, and stirred at room temperature for 20-40 min, with a concentration of 18-20 mg·mL -1 ; (5.2) Under magnetic stirring at room temperature, sodium acetate (CH3COONa) and sodium citrate trihydrate (Na3C6H5O7·3H2O) are uniformly dispersed into the solution obtained in step (5.1), and stirred at room temperature for 20-40 min; (5.3) The solution obtained in step (5.2) is transferred into a reaction kettle, and reacted at 160-200℃ for 16-24 hours; (5.4) Under the action of an external magnetic field, the product obtained in step (5.3) is washed with deionized water and ethanol alternately, and the obtained solid product is vacuum dried at 50℃, and then stored at room temperature in a sealed state to obtain a four-iron oxide magnetic nanocomposite material with a carboxyl (-COOH) group on the surface; (6) Preparation method of MFNM The materials obtained in steps (1), (2), (3), (4) and (5) are uniformly mixed in any mass ratio, and stored at room temperature in a sealed state to obtain a MFNM magnetic nanocomposite material.

[0008] In the present application, the molar ratio of the ferric chloride hexahydrate (FeCl3·6H2O) added in step (1) to the sodium acetate (CH3COONa) is 1:8.5-8.8.

[0009] In the present application, the volume ratio of the solution in step (2.1), the ammonium hydroxide solution added in step (2.2) to the tetraethyl orthosilicate ethanol solution added in step (2.3) is 160:40:0.8-1.2.

[0010] In the present application, the volume ratio of the solution in step (3.1), the ammonium hydroxide solution added in step (3.2) to the tetrabutyl titanate ethanol solution added in step (3.3) is 269-271:1:9.0-9.2.

[0011] In the present application, the mass ratio of the ferric chloride hexahydrate (FeCl3·6H2O) added in step (5.1) to the sodium citrate trihydrate (Na3C6H5O7·3H2O) added in step (5.2) is 0.75-1.75:1.

[0012] The application of the engineered magnetic nanomaterial for metabolic crown characterization in the construction, characterization and related metabolomics analysis of biological fluid metabolic crown is as follows: (1) Exposing the engineered magnetic nanomaterial for metabolic crown characterization to biological fluid to form a metabolic crown: mixing the aqueous solution of the engineered magnetic nanomaterial with a biological fluid sample, incubating at 37℃ at 800-1200 rpm for 0.5-2.5 hours, washing with deionized water under the action of an external magnetic field, and then uniformly resuspending the engineered magnetic nanomaterial-metabolic crown complex in deionized water; (2) The engineered magnetic nanomaterial-metabonomics complex suspension obtained in step (1) is added dropwise to a target plate, and after natural drying, laser desorption ionization mass spectrometry analysis is performed to directly characterize the metabolites in the metabonomics on the surface of the nanomaterial, and a corresponding metabonomics fingerprint is obtained.

[0013] In the present application, the specific conditions for laser desorption ionization mass spectrometry analysis in step (2) are as follows: Bruker UltrafleXtreme MALDI-TOF / TOF mass spectrometer is used, 355 nm Nd:YAG laser light source is adopted, laser frequency is 1000-2000 Hz, acceleration voltage is 20 kV; the acquisition mode is cation reflector mode, and the mass-to-charge ratio range for acquisition is 100-1000 Da; metabonomics mass spectrum data is obtained from flexControl 3.4, and the data is exported in flexAnalysis 3.4.

[0014] The application of the engineered magnetic nanomaterial for metabonomics characterization in biological detection, the biological detection refers to metabolite detection in biological fluids, and the metabolite refers to endometriosis malignant transformation related metabolic markers.

[0015] Preferably, the above-mentioned metabolic markers are one or more of piperidine, L-cysteine, L-aspartic acid, urocanic acid, N-acetyl-L-aspartic acid, ascorbic acid, histidine or pyridoxal.

[0016] The use of the biological detection application includes the following steps: (I). The engineered magnetic nanomaterial for metabonomics characterization is obtained by the above-mentioned integrated method for biological fluid metabonomics construction and characterization, and the mass spectrum corresponding to the biological fluid metabolites is obtained; (II). The biological fluid metabolite mass spectrum obtained in step (I) is imported into an extreme gradient boosting-SHapley Additive exPlanations (Xgboost-SHAP) analysis model, and endometriosis malignant transformation related metabolic markers are screened.

[0017] Preferably, the Xgboost-SHAP analysis model in the above-mentioned step (II) is realized by Python 3.5.

[0018] In step (I), the biological fluid can be whole blood, plasma, serum, urine, saliva, tears, body fluid, gastric juice, feces, and preferably serum.

[0019] The conditions for screening the metabolic markers in step (II) are: mean |SHAP value| value is greater than 0.1, P value is less than 0.05, alignment with the human metabolome database, MALDI MS tandem mass spectrometry identification.

[0020] Compared with the prior art, the present application has the following beneficial effects: (1) A series of engineered magnetic nanomaterials developed by the present application take ferroferric oxide as the core, and a series of magnetic nanocomposites with different physical and chemical properties are constructed on the surface of the core through various chemical modifications (silica coating, titanium dioxide coating, amino functionalization, and carboxyl functionalization). When the NPs are exposed to biological fluids, metabolites are spontaneously and selectively adsorbed on the surface of the NPs to form a unique metabolic crown fingerprint. The inherent magnetism of the ferroferric oxide core allows the NPs-metabolic crown complex to be rapidly separated from the biological fluids while ensuring the integrity of the metabolic crown to the maximum extent. The ferroferric oxide core endows the engineered magnetic nanomaterials with the ability to serve as a matrix for MALDI MS metabolic crown detection. Therefore, the integrated method for metabolic crown construction and characterization based on the series of engineered magnetic nanomaterials can directly apply the NPs-metabolic crown complex obtained from the biological fluids to the MALDI MS metabolomics test without additional separation steps, thereby realizing the in-situ characterization of the metabolic crown on the surface of the NPs. The method based on the series of engineered magnetic nanomaterials in the present application can conveniently and efficiently integrate the engineered construction and in-situ characterization of the metabolic crown.

[0021] (2) The method developed by the present application can successfully analyze the expression differences of biological fluid metabolites between endometriosis-related ovarian cancer patients and control benign cyst patients in combination with machine learning algorithms, realize accurate discrimination of endometriosis-related ovarian cancer with high accuracy, and screen related metabolic markers, which have great potential in the early screening and diagnosis of endometriosis malignant transformation. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 It is a transmission electron microscope photo of a series of engineered magnetic nanomaterials of embodiment 1 of the present application.

[0023] Figure 2 It is an XPS graph of a series of engineered magnetic nanomaterials of embodiment 1 of the present application.

[0024] Figure 3 It is a transmission electron microscope photo of the metabolic crown formed by the MFNM of embodiment 2 of the present application in serum.

[0025] Figure 4 It is a representative metabolic crown mass spectrum of endometriosis-related ovarian cancer patients and control benign cyst patients of embodiment 2 of the present application.

[0026] Figure 5 Probability scatter plot output by the Xgboost-SHAP analysis model of the present application embodiment 3.

[0027] Figure 6 Rug plot of the differential expression of serum metabolic markers between endometriosis-related ovarian cancer and control benign cysts in the present application embodiment 3. DETAILED DESCRIPTION

[0028] The present application develops a convenient and efficient metabolic crown construction and characterization integrated method by using a series of engineered magnetic nanomaterials, and realizes mass spectrometric detection of serum metabolic crown.

[0029] In order to make the present application more obvious and easy to understand, the preferred embodiments are described in detail as follows. Those skilled in the art should understand that the following embodiments are only used to illustrate the present application, and are not limited to the present application.

[0030] The addition amount, content and concentration of various substances involved in the embodiments are described as "parts" unless otherwise specified; the percentage content is described as "mass percentage" unless otherwise specified.

[0031] In the embodiments herein, if no specific description is made for the operating temperature, the temperature generally refers to room temperature (15-35℃). Embodiment 1:

[0032] (1) The preparation method of Fe3O4 includes the following steps: (1.1) Dissolve 2.70 g of iron chloride hexahydrate (FeCl3·6H2O) in 150 mL of ethylene glycol solution, ultrasonic mixing, stirring at room temperature for 30 min; (1.2) Under magnetic stirring at room temperature, 7.20 g of sodium acetate (CH3COONa) is uniformly dispersed into the solution obtained in step (1.1), and stirred at room temperature for 30 min; (1.3) Transfer the solution obtained in step (1.2) to a reaction kettle, and react at 200℃ for 16 hours; (1.4) Under the action of an external magnetic field, the product obtained in step (1.3) is washed with deionized water and ethanol alternately, and the obtained solid product is vacuum dried at 50℃, then sealed and stored at room temperature, to obtain the Fe3O4 magnetic nanomaterial; (2) The preparation method of Fe3O4@SiO2 includes the following steps: (2.1) Disperse 50 mg of the Fe3O4 magnetic nanomaterial obtained according to the preparation method of (1) into 40 mL of ethanol solution, ultrasonic mixing, stirring for 30 min; (2.2) Under magnetic stirring at room temperature, 10 mL of 5% ammonium hydroxide (NH3H2O) solution was added to the solution obtained in step (2.1); (2.3) 250 μL of tetraethyl orthosilicate (Si(OC2H5)4) was dispersed in 0.50 mL of ethanol to form a mixed solution, which was slowly added dropwise to the solution obtained in step (2.2) under vigorous stirring at room temperature for 12 hours; (2.4) Under the action of an external magnetic field, the solid product obtained in step (2.3) was washed alternately with deionized water and ethanol, and dried under vacuum at 50°C, and then stored at room temperature in a sealed container to obtain a magnetic composite nanomaterial with a core-shell structure, in which the core was a Fe3O4 magnetic nanomaterial and the shell was silica (SiO2); (3) The preparation method of Fe3O4@TiO2 includes the following steps: (3.1) 80 mg of Fe3O4 magnetic nanomaterial obtained according to the preparation method of (1) was uniformly dispersed in 200 mL of ethanol solution and ultrasonically mixed, and stirred for 30 min; (3.2) Under magnetic stirring at room temperature, 0.75 mL of ammonium hydroxide solution was uniformly dispersed in the solution obtained in step (3.1); (3.3) Under vigorous stirring at room temperature, 6.60 mL of 24% tetrabutyl titanate (Ti(OCH2CH2CH2CH3)4) ethanol solution was slowly added dropwise to the solution obtained in step (3.2), followed by reaction at 45°C for 24 hours, and finally the solid product was washed with ethanol under the action of an external magnetic field and dried under vacuum at 50°C; (3.4) The product obtained in step (3.3) was calcined in an air atmosphere at 400°C for 2 hours, with a heating rate of 1°C / min, to obtain a magnetic composite nanomaterial with a core-shell structure, in which the core was a Fe3O4 magnetic nanomaterial and the shell was titanium oxide (TiO2); (4) The preparation method of Fe3O4-NH2 includes the following steps: (4.1) 100 mg of Fe3O4 magnetic nanomaterial obtained according to the preparation method of (1) was uniformly dispersed in 300 mL of 2wt% (3-aminopropyl) triethoxysilane aqueous solution, and reacted under stirring at room temperature for 24 hours; (4.2) Under the action of an external magnetic field, the solid product obtained in step (4.1) was washed alternately with deionized water and ethanol, and dried under vacuum at 50°C, and then stored at room temperature in a sealed container to obtain a Fe3O4 magnetic nanocomposite material with amino groups (-NH2) loaded and modified on the surface; (5) The preparation method of Fe3O4-COOH includes the following steps: (5.1) Dissolve 1.35 g of iron chloride hexahydrate (FeCl3-6H2O) in 75 mL of ethylene glycol solution, mix uniformly by ultrasonic, and stir at room temperature for 30 min; (5.2) Under magnetic stirring at room temperature, uniformly disperse 3.60 g of sodium acetate (CH3COONa) and 1.80 g of sodium citrate trihydrate (Na3C6H5O7-3H2O) into the solution obtained in step (5.1), and stir at room temperature for 30 min; (5.3) Transfer the solution obtained in step (5.2) into a reaction kettle, and react at 200°C for 16 hours; (5.4) Under the action of an external magnetic field, wash the product obtained in step (5.3) with deionized water and ethanol alternately, vacuum dry the obtained solid product at 50°C, and then seal and store at room temperature to obtain a ferroferric oxide magnetic nanocomposite material with carboxyl (-COOH) loaded on the surface; (6) Mix the engineered magnetic nanomaterials for metabolic crown characterization prepared according to the above preparation method uniformly at any mass ratio, and seal and store at room temperature to obtain a MFNM magnetic nanocomposite material.

[0033] Material characterization: Transmission electron microscope photos of a series of engineered magnetic nanomaterials are as shown in Figure 1 ; XPS graphs of a series of engineered magnetic nanomaterials are as shown in Figure 2 ; these graphs are significantly different from the magnetic nanomaterials of the prior art, indicating that they have unique characteristics in terms of physical and chemical properties. Example 2:

[0034] Using the MFNM magnetic nanocomposite material obtained in Example 1, the serum of 97 endometriosis-related ovarian cancer patients and 95 endometriosis benign cyst patients from the Obstetrics and Gynecology Hospital of Fudan University was subjected to metabolic crown mass spectrometry detection.

[0035] (1) Take 10 mg of the MFNM magnetic nanocomposite material prepared in Example 1, disperse into 1 mL of deionized water, mix uniformly by ultrasonic at room temperature, and obtain a uniformly dispersed engineered magnetic nanomaterial suspension; (2) Mix the engineered magnetic nanomaterial suspension obtained in step (1) with a serum sample of appropriate concentration, incubate at 37°C at 800-1200 rpm for 1 hour, wash with deionized water under the action of an external magnetic field, and then resuspend the MFNM-metabolic crown complex uniformly in deionized water to obtain an engineered magnetic nanomaterial-metabolic crown complex suspension; (3) 2 μL of the engineered magnetic nanomaterial-metabonomics complex suspension obtained in step (2) was added dropwise to a target plate, and after the sample points were naturally dried, laser desorption ionization mass spectrometry analysis was performed. Bruker UltrafleXtreme MALDI-TOF / TOF mass spectrometer was used, a 355 nm Nd:YAG laser light source was used, the laser frequency was 1000 Hz, the acceleration voltage was 20 kV; the acquisition mode was the positive ion reflector mode, and the mass-to-charge ratio range was 100-1000 Da; the metabonomics mass spectrum data were obtained from flexControl 3.4, and the data were exported in flexAnalysis 3.4, and the corresponding serum metabonomics fingerprint spectrum was obtained; The transmission electron micrograph of the metabonomics formed by MFNM in serum is shown in Figure 3 ; and the representative metabonomics mass spectrum of the endometriosis-related ovarian cancer patients and the control benign cyst patients is shown in Figure 4 . Example 3:

[0036] The serum metabonomics mass spectrum obtained in Example 2 was introduced into an extreme gradient boosting-SHapley Additive exPlanations (Xgboost-SHAP) analysis model, and endometriosis malignant transformation-related metabolic markers were screened. The steps are as follows: (1) The R language based on MALDIquant and MALDIquantForeign packages was used to pretreat the serum metabonomics mass spectrum, including peak intensity conversion and normalization, peak smoothing, baseline subtraction, peak alignment, peak identification and peak grouping; (2) Based on the serum metabonomics mass spectrum, an Xgboost-SHAP analysis model was constructed using Python 3.5 to select serum metabolic markers. Specifically, Python 3.5 and Metaboanalyst 6.0 were used to calculate the mean|SHAP value| and P value of each characteristic signal peak, and the characteristic signal peaks with a mean|SHAP value| value greater than 0.1 and a P value less than 0.05 were screened. The screened characteristics were matched with the human metabolome database, and MALDI MS tandem mass spectrometry identification was performed, and finally the serum metabolic markers were screened; The probability scatter plot output by the Xgboost-SHAP analysis model is shown in Figure 5 ; and the violin plot of the differential expression of serum metabolic markers between endometriosis-related ovarian cancer and control benign cyst is shown in Figure 6 ; Based on the analysis results of the Xgboost-SHAP analysis model, 8 metabolites are screened as potential endometrial endometriosis malignant transformation related metabolic markers, which are piperidine, L-cysteine, L-aspartic acid, urocanic acid, N-acetyl-L-aspartic acid, ascorbic acid, histidine, pyridoxal.

[0037] It should be noted that the above analysis model can also be suitable for screening of other disease related biomarkers.

[0038] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can also be made, which should be considered as the protection scope of the present application.

Claims

1. A series of engineered magnetic nanomaterials for characterizing metabolic coronaviruses, characterized in that... The term includes magnetic nanomaterials containing iron oxide (Fe3O4), magnetic composite nanomaterials with a core-shell structure consisting of iron oxide magnetic nanomaterials as the core and coated with silicon dioxide (SiO2) or titanium dioxide (TiO2), magnetic nanocomposites of iron oxide with surface-loaded and modified with amino (-NH2) or carboxyl (-COOH) groups, and mixed magnetic nanomaterials of the above materials; the engineered magnetic nanomaterials have the following chemical formula: Fe3O4, Fe3O4@SiO2, Fe3O4@TiO2, Fe3O4-NH2, Fe3O4-COOH, Fe3O 4（a） +Fe3O4@SiO 2（b） +Fe3O4@TiO 2（c） +Fe3O4-NH 2（d） +Fe3O4-COOH （e） (abbreviated as MFNM), where a=0.0-1.0, b=0.0-1.0, c=0.0-1.0, d=0.0-1.0, e=0.0-1.0, a, b, c, d, e cannot be 0 at the same time, and a+b+c+d+e=1.

2. The series of engineered magnetic nanomaterials for characterizing metabolic coronaviruses according to claim 1, characterized in that, The engineered magnetic nanomaterial is composed of at least two or more of Fe3O4, Fe3O4@SiO2, Fe3O4@TiO2, Fe3O4-NH2, and Fe3O4-COOH.

3. A method for preparing a series of engineered magnetic nanomaterials for characterizing metabolic coronaviruses as described in claim 1, characterized in that, The specific steps are as follows: (1) Preparation method of Fe3O4 (1.1) Dissolve ferric chloride hexahydrate (FeCl3·6H2O) in ethylene glycol solution, sonicate to mix, and stir at room temperature for 20-40 min to achieve a concentration of 18-20 mg·mL. -1 ; (1.2) Under magnetic stirring at room temperature, sodium acetate (CH3COONa) is uniformly dispersed into the solution obtained in step (1.1), and stirred at room temperature for 20-40 min; (1.3) Transfer the solution obtained in step (1.2) to a reaction vessel and react at 160-200℃ for 16-24 hours; (1.4) Under the action of an external magnetic field, the product obtained in step (1.3) is washed alternately with deionized water and ethanol. The resulting solid product is dried under vacuum at 50°C and then stored in a sealed container at room temperature to obtain magnetic nanomaterials of iron oxide. (2) Preparation method of Fe3O4@SiO2 (2.1) The product obtained according to the preparation method of Fe3O4 in (1) is uniformly dispersed in an ethanol solution, ultrasonically mixed, and stirred for 20-40 min to a concentration of 1.0-1.5 mg·mL. -1 ; (2.2) Add a 5% ammonium hydroxide (NH3·H2O) solution to the solution obtained in step (2.1) under magnetic stirring at room temperature; (2.3) Prepare an ethanol solution of tetraethyl orthosilicate (Si(OC2H5)4) (concentration 460-480 mg·mL). -1 Slowly add it dropwise to the solution obtained in step (2.2) and stir vigorously at room temperature for 12 hours; (2.4) Under the action of an external magnetic field, the solid product obtained in step (2.3) is washed alternately with deionized water and ethanol, dried under vacuum at 50°C, and then stored in a sealed container at room temperature to obtain a magnetic composite nanomaterial with a core-shell structure, consisting of iron oxide magnetic nanomaterial as the core and silicon oxide (SiO2) coated on the surface. (3) Preparation method of Fe3O4@TiO2 (3.1) The product obtained according to the preparation method of Fe3O4 in (1) is uniformly dispersed in an ethanol solution and ultrasonically mixed for 20-40 min. The concentration is 0.40-0.45 mg·mL. -1 ; (3.2) Under magnetic stirring at room temperature, the ammonium hydroxide solution is uniformly dispersed into the solution obtained in step (3.1); (3.3) Under vigorous stirring at room temperature, a 24% tetrabutyl titanate (Ti(OCH2CH2CH2CH3)4) ethanol solution was slowly added dropwise to the solution obtained in step (3.2), and then reacted at 45°C for 20-24 hours. Finally, the obtained solid product was washed with ethanol under an external magnetic field and dried under vacuum at 50°C. (3.4) The product obtained in step (3.3) is calcined in air at 300-500℃ for 2-3 hours with a heating rate of 1-2℃ / min to obtain a magnetic composite nanomaterial with a core-shell structure, consisting of iron oxide magnetic nanomaterial as the core and titanium oxide (TiO2) coated on the surface. (4) Preparation method of Fe3O4-NH2 (4.1) The product obtained according to the preparation method of Fe3O4 in (1) is uniformly dispersed in a 2wt% aqueous solution of (3-aminopropyl)triethoxysilane and reacted at room temperature with stirring for 20-24 hours, with a concentration of 0.30-0.35 mg·mL. -1 ; (4.2) Under the action of an external magnetic field, the solid product obtained in step (4.1) was washed alternately with deionized water and ethanol, dried under vacuum at 50°C, and then stored in a sealed container at room temperature to obtain a magnetic nanocomposite material of iron oxide with amino (-NH2) loaded on its surface. (5) Preparation method of Fe3O4-COOH (5.1) Dissolve ferric chloride hexahydrate (FeCl3·6H2O) in ethylene glycol solution, sonicate to mix, and stir at room temperature for 20-40 min to achieve a concentration of 18-20 mg·mL. -1 ; (5.2) Under magnetic stirring at room temperature, sodium acetate (CH3COONa) and sodium citrate trihydrate (Na3C6H5O7·3H2O) are uniformly dispersed into the solution obtained in step (5.1), and stirred at room temperature for 20-40 min. (5.3) Transfer the solution obtained in step (5.2) to a reaction vessel and react at 160-200℃ for 16-24 hours; (5.4) Under the action of an external magnetic field, the product obtained in step (5.3) was washed alternately with deionized water and ethanol. The resulting solid product was vacuum dried at 50°C and then stored in a sealed container at room temperature to obtain a magnetic nanocomposite material of iron oxide with carboxyl groups (-COOH) loaded on its surface. (6) Preparation method of MFNM The materials obtained in steps (1), (2), (3), (4) and (5) are mixed evenly in any mass ratio and stored in a sealed container at room temperature to obtain MFNM magnetic nanocomposite material.

4. The method according to claim 3, characterized in that, The molar ratio of ferric chloride hexahydrate (FeCl3·6H2O) to sodium acetate (CH3COONa) added in step (1) is 1:8.5-8.

8.

5. The method according to claim 3, characterized in that, The volume ratio of the solution in step (2.1), the ammonium hydroxide solution added in step (2.2), and the tetraethyl orthosilicate ethanol solution added in step (2.3) is 160:40:0.8-1.

2.

6. The method according to claim 3, characterized in that, The volume ratio of the solution in step (3.1), the ammonium hydroxide solution added in step (3.2), and the tetrabutyl titanate ethanol solution added in step (3.3) is 269-271:1:9.0-9.

2.

7. The method according to claim 3, characterized in that, The mass ratio of ferric chloride hexahydrate (FeCl3·6H2O) added in step (5.1) to sodium citrate trihydrate (Na3C6H5O7·3H2O) added in step (5.2) is 0.75-1.75:

1.

8. The application of the engineered magnetic nanomaterial for characterizing metabolic crowns as described in claim 1 in the construction, characterization, and related metabolomics analysis of biofluidic metabolic crowns, characterized in that, The specific steps are as follows: (1) The engineered magnetic nanomaterials used for characterizing the metabolic crown were exposed to a biological fluid to form a metabolic crown: the aqueous solution of the engineered magnetic nanomaterials was mixed with the biological fluid sample and incubated at 800-1200 rpm at 37°C for 0.5-2.5 hours. After washing with deionized water under the action of an external magnetic field, the engineered magnetic nanomaterials-metabolic crown complex was uniformly resuspended in deionized water. (2) The engineered magnetic nanomaterial-metabolism crown complex suspension obtained in step (1) is dropped onto the target plate and allowed to dry naturally before laser desorption / ionization mass spectrometry analysis is performed to directly characterize the metabolites in the metabolism crown on the surface of the nanomaterial and obtain the corresponding metabolism crown fingerprint spectrum.

9. The application according to claim 8, characterized in that, The specific conditions for laser desorption / ionization mass spectrometry analysis in step (2) are as follows: a Bruker UltrafleXtreme MALDI-TOF / TOF mass spectrometer is used, with a 355 nm Nd:YAG laser source, a laser frequency of 1000-2000 Hz, and an accelerating voltage of 20 kV; the acquisition mode is cation reflector mode, and the mass-to-charge ratio range is 100-1000 Da; metabolic crown mass spectrometry data are obtained from flexControl 3.4 and exported in flexAnalysis 3.

4.

10. The application of the engineered magnetic nanomaterial for characterizing metabolic coronary syndrome as described in claim 1 in biological detection, wherein the biological detection refers to the detection of metabolites in biological fluids, and the metabolites refer to metabolic markers related to the malignant transformation of endometriosis.

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