A heterogeneous composite for capturing exosomes and a preparation method and application thereof

Abstract

The present application belongs to the technical field of biological detection, and particularly relates to a heterogeneous composite material for capturing exosomes, a preparation method of the heterogeneous composite material, a method for specifically enriching exosomes and detecting exosome metabolites by using the heterogeneous composite material, and application of the method in disease screening and diagnosis. The heterogeneous composite material prepared by the present application has good magnetic response, excellent ultraviolet absorption capacity and electron-hole separation efficiency, can be successfully used for continuous capture of urine exosomes of healthy controls and bladder cancer patients and mass spectrum analysis of metabolites, and can screen 5 exosome metabolites as potential bladder cancer biomarkers, successfully realizes accurate discrimination of bladder cancer patients, and has great application prospect in large-scale population screening, early diagnosis and prognosis monitoring of bladder cancer.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to a heterogeneous composite material for capturing exosomes and its preparation method, as well as a method for specifically enriching exosomes and detecting exosome metabolites using this heterogeneous composite material and its application in disease screening and diagnosis. Specifically, it relates to a method for synthesizing novel magnetic transition metal oxide carbide materials, a method for continuously capturing exosomes and performing metabolite mass spectrometry analysis using magnetic transition metal oxide carbide materials, and a method for screening potential exosome metabolic biomarkers for bladder cancer. Background Technology

[0002] Exosomes are extracellular vesicles with a diameter of 30-150 nm and a phospholipid bilayer structure. They can be secreted by various cells and released into various body fluids, such as urine, blood, and tears. Exosomes contain various types of biomolecules, including proteins, nucleic acids, lipids, and metabolites. These can disrupt the dynamic balance between normal cells and the extracellular environment and mediate communication between cancer cells and surrounding normal cells. Therefore, exosomes are ideal for liquid biopsies. In recent years, the excellent performance of exosome-based liquid biopsies in early cancer diagnosis, monitoring, and prognosis has attracted widespread attention. Currently, researchers consider proteins and microRNAs in exosomes to be promising biomarkers. To date, there are few reports on the development and utilization of exosomal metabolites as biomarkers. However, metabolites are downstream products of transcriptional and translational biochemical reaction networks and can provide terminal information closer to the phenotype than the genome and proteome, showing great potential in directly reflecting an individual's physiological and pathological state. Therefore, it is necessary to develop detection and analysis methods for exosome metabolites to provide a new perspective for exosome-based liquid biopsy.

[0003] Pre-separation is a prerequisite for liquid biopsy via exosomes. Traditional exosome separation methods mainly include ultracentrifugation, size exclusion, and precipitation. However, these methods are time-consuming, costly, and yield products with limited purity, restricting their widespread application. Chromatography-tandem mass spectrometry is the most mature technique for exosome metabolite analysis, but its high cost and labor-intensive pretreatment steps, including quenching, lysis, metabolite extraction, concentration, and resolution, hinder its widespread application in clinical disease detection. In recent years, advances in nanoscience have provided new solutions for exosome separation and exosome metabolite analysis. Nanostructured adsorbents, with their high surface area, strong modification properties, abundant binding sites, and synergistic affinity effects, can achieve efficient exosome separation. Nanostructure-assisted laser desorption / ionization mass spectrometry utilizes the physical properties of nanostructured matrices, such as photothermal conversion, photoelectric effect, and surface plasmon resonance, to promote the desorption and ionization processes of metabolites and achieve selective adsorption of target analytes through specific structures. Compared with traditional mass spectrometry techniques, nanostructure-assisted laser desorption / ionization mass spectrometry offers simpler operation, faster analysis speed, higher throughput, and enhanced anti-interference capabilities, while ensuring low background interference, high sensitivity, and reproducibility. It has been widely used in various clinical scenarios to assist in medical decision-making.

[0004] Bladder cancer is a common malignant tumor of the urinary system, characterized by high incidence, high mutation rate, and high recurrence rate. In 2020, 573,278 new cases of bladder cancer were diagnosed globally, with 212,536 deaths. Based on the histopathological results of bladder muscle invasion, bladder cancer can be divided into non-muscle-invasive bladder cancer and muscle-invasive bladder cancer. These two subtypes of bladder cancer exhibit different molecular drivers, clinical outcomes, and treatment methods. Currently, cystoscopy and urine cytology are the gold standards for detecting bladder cancer. In fact, less than 30% of bladder cancer cases in suspected populations are diagnosed by invasive cystoscopy. Furthermore, the high recurrence rate of non-muscle-invasive bladder cancer necessitates frequent cystoscopy, and the low sensitivity of monitoring for rare exfoliated tumor cells in urine leads to high costs for bladder cancer screening and clinical management. The five-year survival rates for patients with early-stage bladder cancer, muscle-invasive bladder cancer, and metastatic bladder cancer have been reported to be 94%, 50%, and 20%, respectively. Under these circumstances, non-invasive liquid biopsy has great potential as a tool for bladder cancer detection. However, due to the high mutational heterogeneity during disease progression, no high-performance biomarkers have yet been identified that can be widely applied to liquid biopsy for bladder cancer. Therefore, there is an urgent need to develop advanced technologies for bladder cancer screening, early diagnosis, and treatment monitoring to provide comprehensive guidance for precision medicine. Summary of the Invention

[0005] Therefore, the present invention aims to provide a heterogeneous composite material for capturing exosomes and a method for preparing the same, as well as a method for specifically enriching exosomes and detecting exosome metabolites using the heterogeneous composite material and its application in disease screening and diagnosis.

[0006] To achieve the above objectives, the solution adopted by the present invention is as follows:

[0007] In a first aspect, the present invention provides a heterogeneous composite material for capturing exosomes, characterized in that it is a heterogeneous composite material of iron oxide modified with amino groups on the surface of an oxidized transition metal carbide material having a two-dimensional layered structure.

[0008] Preferably, the oxidized transition metal carbide material with a two-dimensional layered structure includes one or more of oxidized Ti3C2, Ti2C and Ti4C3 with a two-dimensional layered structure.

[0009] Preferably, the oxidized transition metal carbide material with a two-dimensional layered structure is titanium dioxide material with a two-dimensional layered structure.

[0010] Preferably, the method for preparing the oxidized transition metal carbide material with a two-dimensional layered structure includes the following steps: using hydrofluoric acid (HF) to peel off Al from titanium aluminum carbide (Ti3AlC2) to form a titanium carbide with a two-dimensional layered structure, and then oxidizing it at high temperature.

[0011] Secondly, the present invention also provides a method for preparing the heterogeneous composite material for capturing exosomes as described above, comprising the following steps:

[0012] Step (1), Preparation of oxidized transition metal carbide material with two-dimensional layered structure: Al in titanium aluminum carbide (Ti3AlC2) is stripped with hydrofluoric acid (HF) to form titanium carbide with two-dimensional layered structure, and then oxidized at high temperature to obtain the titanium dioxide material with two-dimensional layered structure.

[0013] Step (2) involves synthesizing the heterogeneous composite material as described above using a solvothermal method.

[0014] Preferably, step (1) includes the following steps:

[0015] Step (1.1): Slowly add titanium aluminum carbide (Ti3AlC2) to a 50% hydrofluoric acid (HF) solution and stir at room temperature for 16-24 hours. Wash the obtained solid repeatedly with deionized water by centrifugation until the pH value of the supernatant is greater than 6. Dry the product under vacuum at 50°C.

[0016] Step (1.2): Add the product obtained in step (1.1) to deionized water, and react the resulting suspension at 120-200℃ for 16-24h. Then wash with deionized water by centrifugation, and dry the resulting solid under vacuum at 50℃.

[0017] Step (1.3): Calcine the product obtained in step (1.2) at 200-500℃ for 2-3 hours in air, with a heating rate of 2-5℃ / min, and store the resulting solid at 4℃.

[0018] Preferably, the mass-to-volume ratio of the titanium aluminum carbide (Ti3AlC2) to the 50% hydrofluoric acid (HF) solution is 1:(10-40) g / mL, and more preferably 1:25 g / mL.

[0019] Preferably, step (2) includes the following steps: the product obtained in step (1) is placed in ethylene glycol to form a suspension, ferric chloride hexahydrate, anhydrous sodium acetate and hydrophilic amine compounds are added to the above suspension, the mixture is stirred until the solid is completely dissolved, and then the mixture is reacted at 200-300°C for 6-12 hours. Finally, the solid is washed with deionized water under the action of an external magnetic field, dried under vacuum at 50°C, and stored at 4°C.

[0020] Preferably, the hydrophilic amine compound is selected from 1,2-propanediamine, 1,3-butanediamine, 1,2-butanediamine, 1,3-pentanediamine, 1,4-pentanediamine, 4-methyl-1,3-pentanediamine, 2-methyl-1,3-pentanediamine, 2-methyl-1,4-pentanediamine, 3-methyl-1,4-pentanediamine, 2,4-pentanediamine, 2,5-pentanediamine, and 2,4-hexanediamine. 1,3-Hexanediamine, 1,4-Hexanediamine, 1,5-Hexanediamine, 5-methyl-1,3-Hexanediamine, 4-methyl-1,3-Hexanediamine, 3-methyl-1,3-Hexanediamine, 2-methyl-1,3-Hexanediamine, 2-methyl-1,4-Hexanediamine, 3-methyl-1,4-Hexanediamine, 5-methyl-1,4-Hexanediamine, 2-methyl-1,5-Hexanediamine, 3-methyl The hydrophilic amine compound is selected from one or more of 2,4-hexanediamine, 1,3-hexanediamine, 1,4-hepta-diamine, 1,5-hepta-diamine, 1,3-octanediamine, 1,4-octanediamine, and 1,5-octanediamine; preferably, the hydrophilic amine compound is selected from one or more of 2,4-hexanediamine, 1,3-hexanediamine, 1,4-hexanediamine, 1,5-hexanediamine, 5-methyl-1,3-hexanediamine, 4-methyl-1,3-hexanediamine, 3-methyl-1,3-hexanediamine, 2-methyl-1,3-hexanediamine, 2-methyl-1,4-hexanediamine, 3-methyl-1,4-hexanediamine, 5-methyl-1,4-hexanediamine, 2-methyl-1,5-hexanediamine, 3-methyl-1,4-hexanediamine, 5-methyl-1,4-hexanediamine, 2-methyl-1,5-hexanediamine, 3-methyl-1,5-hexanediamine, and 4-methyl-1,5-hexanediamine.

[0021] Preferably, the hydrophilic amine compound is selected from one or more of polyacrylamide, polyamide, polyimide, polyetherimide and polyamide; preferably, the hydrophilic amine compound is polyetherimide.

[0022] Preferably, the percentage of each atom in the two-dimensional heterogeneous composite material of amino-modified iron oxide obtained in step (2) is: C 9.99%, O 65.78%, Ti 17.89%, Fe 4.90%, N 1.43%.

[0023] Thirdly, the present invention also provides the application of the magnetic composite material for capturing exosomes as described above and the magnetic composite material for capturing exosomes prepared by the preparation method as described above in the separation and detection of exosomes.

[0024] Fourthly, the present invention also provides a method for screening cancer exosome metabolite biomarkers, comprising the following steps:

[0025] Step (1): Capture exosomes using the heterogeneous composite material for capturing exosomes as described above and the heterogeneous composite material for capturing exosomes prepared by the preparation method described above: Add the heterogeneous composite material to the target sample, incubate at room temperature, wash with phosphate buffer solution, add deionized water, and obtain a suspension of the heterogeneous composite material for capturing exosomes.

[0026] Step (II): The heterogeneous composite material suspension of the captured exosomes obtained in Step (I) is dropped onto the target plate, and after it dries naturally, laser desorption / ionization mass spectrometry analysis is performed to obtain the mass spectrum of the exosomes.

[0027] Step (3): Import the exosome mass spectrometry spectrum obtained in Step (2) into the orthogonal partial least squares discriminant analysis model to screen exosome metabolic biomarkers.

[0028] Preferably, the specific conditions for laser desorption / ionization mass spectrometry analysis in step (ii) are as follows: a Bruker UltrafleXtreme MALDI-TOF / TOF mass spectrometer is used, with a 355nm Nd:YAG laser source, a laser frequency of 2000Hz, and an accelerating voltage of 20kV; the acquisition mode is cation reflector mode, and the mass-to-charge ratio range is 100-1000Da; exosome mass spectrometry data are obtained from flexControl 3.4 and exported in flexAnalysis 3.4.

[0029] Preferably, the conditions for screening exosome metabolic markers in step (iii) are: variable importance projection value greater than 1, P value less than 0.05, absolute value of p(cov,x-axis) greater than 0.05, and absolute value of p(corr,y-axis) greater than 0.5.

[0030] Preferably, the target sample in step (i) is urine.

[0031] Fifthly, the present invention also provides an exosome metabolic marker for bladder cancer, obtained by a method for screening cancer exosome metabolic markers as described above. The obtained exosome metabolic marker for bladder cancer is ethanolamine, L-aspartic acid, L-acetylcarnitine, prostaglandin E2, and 5,6-dihydroxyprostaglandin F1a.

[0032] In a sixth aspect, the present invention also provides a kit for separating exosomes, comprising the magnetic composite material for capturing exosomes as described above and the magnetic composite material for capturing exosomes prepared by the preparation method described above.

[0033] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0034] (1) The heterogeneous composite material proposed in this invention contains two components: titanium dioxide and amino-modified iron oxide. It can chelate with phospholipids on the exosome membrane through titanium-oxygen clusters and iron-oxygen clusters, and bind with negatively charged exosomes through protonated amino groups. The material has good magnetic responsiveness. Therefore, the method of this invention can achieve specific enrichment and separation of exosomes in a simple and efficient manner.

[0035] (2) The heterogeneous composite material of the present invention has a two-dimensional layered stacked structure, which determines its high efficiency in photothermal conversion and ion transfer. The titanium dioxide and iron oxide components have high melting points and low thermal conductivity, which can significantly reduce background interference in laser desorption / ionization mass spectrometry analysis and achieve effective thermal desorption of metabolites. The surface of the material is rough, with strong ultraviolet absorption capacity and high electron-hole separation efficiency, which can achieve selective adsorption of small molecule metabolites in complex biological fluids and efficiently transfer the laser energy of the mass spectrometry ion source to the metabolites, promoting the ionization of metabolites. Therefore, the present invention can achieve effective mass spectrometry detection of exosome metabolites.

[0036] (3) This invention can analyze the expression differences of exosome metabolites between healthy controls and bladder cancer patients through machine learning models, thereby screening exosome metabolic markers and achieving accurate identification of bladder cancer.

[0037] (4) The heterogeneous composite material prepared by this invention has good magnetic responsiveness, excellent ultraviolet absorption capacity and electron-hole separation efficiency. It can be successfully used for continuous capture and metabolite mass spectrometry analysis of exosomes in the urine of healthy controls and bladder cancer patients. Five exosome metabolites were screened as potential biomarkers for bladder cancer, and accurate identification of bladder cancer patients was successfully achieved. This shows that it has great application prospects in large-scale population screening, early diagnosis and prognosis monitoring of bladder cancer. Attached Figure Description

[0038] Figure 1 This is a scanning electron microscope image of the magnetic oxide transition metal carbide material of Embodiment 1 of the present invention.

[0039] Figure 2 This is a transmission electron microscope image of the magnetic oxide transition metal carbide material of Embodiment 1 of the present invention.

[0040] Figure 3 This is a hysteresis loop diagram of the magnetic oxide transition metal carbide material of Embodiment 1 of the present invention.

[0041] Figure 4 This is the X-ray diffraction pattern of the magnetic oxide transition metal carbide material of Example 1 of the present invention.

[0042] Figure 5 The Zeta potential diagrams are for the products obtained in steps (1), (2), (3), and (4) of Embodiment 1 of the present invention.

[0043] Figure 6 The ultraviolet absorption spectra of the products obtained in steps (1), (2), (3), and (4) of Example 1 of the present invention are shown.

[0044] Figure 7 The transient photocurrent response curves of the products obtained in steps (1), (2), (3), and (4) of Embodiment 1 of the present invention are shown.

[0045] Figure 8 The images show the CD9, CD63, and HSP70 protein blots of the magnetic oxidized transition metal carbide material used to capture exosomes in Example 2 of this invention and the corresponding urine.

[0046] Figure 9 The images show representative physical spectra of exosomes from healthy controls and bladder cancer patients in Example 2 of this invention.

[0047] Figure 10 This is a graph showing the probability values ​​output by the orthogonal partial least squares discriminant analysis model in Embodiment 3 of the present invention.

[0048] Figure 11This is a violin plot of exosome metabolic markers in healthy controls and bladder cancer patients in Example 3 of the present invention. Detailed Implementation

[0049] This invention utilizes the specific binding of magnetic transition metal oxide carbide materials with exosomes to achieve continuous enrichment, separation, and metabolic mass spectrometry detection of urinary exosomes.

[0050] The technical solution of the present invention will be further described below with reference to specific embodiments; however, the scope of protection of the present invention is not limited to these embodiments. All changes or equivalent substitutions that do not depart from the concept of the present invention are included within the scope of protection of the present invention.

[0051] Example 1: Synthesis of Magnetic Oxidized Transition Metal Carbide Materials

[0052] (1) First, 2.00g of titanium aluminum carbide was slowly added to 50mL of 50% hydrofluoric acid solution and stirred at room temperature for 24 hours. The solid obtained was washed repeatedly by centrifugation with deionized water until the pH value of the supernatant was greater than 6. The product was then vacuum dried at 50℃.

[0053] (2) Take the product obtained in step (1), add 500 mL of deionized water, and stir for 30 minutes. Then, transfer the suspension to a stainless steel reactor lined with polytetrafluoroethylene and react at 120°C for 16 hours. Then, wash the obtained solid with deionized water by centrifugation and dry it under vacuum at 50°C.

[0054] (3) Calcine the product obtained in step (2) at 300°C for 3 hours in air at a heating rate of 5°C·min-1, and store the obtained solid at 4°C.

[0055] (4) First, take the product obtained in step (3), add 60 mL of ethylene glycol, and stir until homogeneous. Next, add 300 mg of ferric chloride hexahydrate, 1.08 mg of hexamethylenediamine, and 1.20 mg of anhydrous sodium acetate to the above suspension and stir until the solid is completely dissolved. Then, transfer the suspension to a stainless steel reactor lined with polytetrafluoroethylene and react at 200 °C for 6 hours. Finally, under the action of an external magnetic field, wash the obtained solid with deionized water, vacuum dry it at 50 °C, and store it at 4 °C to obtain the magnetic oxide transition metal carbide material.

[0056] Scanning electron microscope images of magnetic transition metal oxide carbide materials, such as Figure 1 Transmission electron microscopy images of magnetic transition metal oxide carbide materials, such as... Figure 2 The hysteresis loop diagram of magnetic oxide transition metal carbide materials is shown below. Figure 3 X-ray diffraction patterns of magnetic transition metal oxide carbide materials are as follows: Figure 4The Zeta potential diagrams of the products obtained in steps (1), (2), (3), and (4) are shown below. Figure 5 The ultraviolet absorption spectra of the products obtained in steps (1), (2), (3), and (4) are as follows: Figure 6 The transient photocurrent response curves of the products obtained in steps (1), (2), (3), and (4) are shown in the figure below. Figure 7 .

[0057] Analysis results: From Figures 1 to 3 It can be seen that magnetic oxide transition metal carbide materials exhibit a two-dimensional layered stacked structure, with a rough surface and strong magnetic responsiveness; from Figures 4 to 5 It can be seen that the aptamer-coupled polycrystalline carbon material contains titanium dioxide and iron oxide crystals, and the material exhibits positive charge due to amino modification; from Figures 6 to 7 It can be seen that the material's ultraviolet absorption capacity at 355 nm and photocurrent response are continuously enhanced during the synthesis process.

[0058] Example 2: The aptamer obtained in Example 1 was coupled with polycrystalline carbon material for the continuous capture and metabolomics analysis of urinary exosomes from 112 healthy controls and 113 bladder cancer patients.

[0059] (1) Take 0.1 mg of magnetic oxidized transition metal carbide material and add it to 25 μL of target sample. Incubate at room temperature for 20 minutes, wash with phosphate buffer solution, add 5 μL of deionized water to obtain magnetic oxidized transition metal carbide material suspension for capturing exosomes;

[0060] (2) 1 μL of the magnetic transition metal oxide carbide material suspension obtained in step (1) was dropped onto the target plate and allowed to dry naturally before laser desorption / ionization mass spectrometry analysis was performed. A Bruker UltrafleXtremeMALDI-TOF / TOF mass spectrometer was used with a 355 nm Nd:YAG laser source, a laser frequency of 2000 Hz, and an accelerating voltage of 20 kV. The acquisition mode was cation reflector mode, and the mass-to-charge ratio range was 100-1000 Da. Mass spectrometry data were obtained from flexControl 3.4 and exported from flexAnalysis 3.4 to obtain the exosome mass spectrum.

[0061] The magnetic oxidized transition metal carbide material capturing exosomes and the corresponding CD9, CD63, and HSP70 protein blots of urine are shown below. Figure 8 Representative property spectra of exosomes from healthy controls and bladder cancer patients are shown in [reference needed]. Figure 9 .

[0062] Analysis results: From Figure 8 It can be seen that magnetic transition metal oxide carbide materials can effectively enrich and separate exosomes in urine; from Figure 9 It can be seen that exosome mass spectra of healthy controls and bladder cancer patients can be successfully obtained using magnetic oxidized transition metal carbide materials.

[0063] Example 3: The exosome mass spectra obtained in Example 2 were imported into an orthogonal partial least squares discriminant analysis model to screen exosome metabolic biomarkers.

[0064] (1) The exosome mass spectra were preprocessed using R language based on the MALDIquant and MALDIquantForeign packages, including peak intensity conversion and normalization, peak smoothing, baseline subtraction, peak alignment, peak identification and peak grouping.

[0065] (2) Based on exosome mass spectrometry, an orthogonal partial least squares discriminant analysis model was constructed using SIMCA-P 14.1 to select exosome metabolic biomarkers. Specifically, SIMCA-P 14.1 was used to output the probability value of each sample belonging to the bladder cancer group, and the variable importance projection value, P value, p(cov,x-axis) value, and p(corr,y-axis) value of each peak signal in the exosome mass spectrometry were calculated. Peak signals with variable importance projection values ​​greater than 1, P values ​​less than 0.05, absolute values ​​of p(cov,x-axis) greater than 0.05, and absolute values ​​of p(corr,y-axis) greater than 0.5 were selected as exosome metabolic biomarkers.

[0066] The probability value graph output by the orthogonal partial least squares discriminant analysis model is shown below. Figure 10 .

[0067] Analysis results: Figure 10 It can be seen that the orthogonal partial least squares discriminant analysis model based on exosome mass spectrometry can accurately distinguish between healthy controls and bladder cancer patients. Figure 11 As can be seen, the five exosome metabolic markers of bladder cancer obtained through the above analysis model are ethanolamine, L-aspartate, L-acetylcarnitine, prostaglandin E2, and 5,6-dihydroxyprostaglandin F1a. They showed significant differences in expression between healthy controls and bladder cancer patients (P value < 0.0001), which played an important role in distinguishing between healthy controls and bladder cancer patients.

[0068] The above analytical model can also be applied to other cancers.

[0069] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A heterogeneous composite material for capturing exosomes, characterized in that, A magnetic composite material of iron(III) oxide modified with amino groups is prepared by loading the surface of an oxidized transition metal carbide material with a two-dimensional layered structure. The preparation method of the oxidized transition metal carbide material with a two-dimensional layered structure includes the following steps: exfoliating Al from titanium aluminum carbide (Ti3AlC2) with hydrofluoric acid (HF) to form titanium carbide with a two-dimensional layered structure, and then oxidizing it at high temperature to obtain titanium dioxide material with a two-dimensional layered structure.

2. A method for preparing a heterogeneous composite material for capturing exosomes as described in claim 1, characterized in that, Includes the following steps: Step (1), Preparation of oxidized transition metal carbide material with two-dimensional layered structure: Al in titanium aluminum carbide (Ti3AlC2) is stripped with hydrofluoric acid (HF) to form titanium carbide with two-dimensional layered structure, and then oxidized at high temperature to obtain the titanium dioxide material with two-dimensional layered structure. Step (2): The heterogeneous composite material as described in claim 1 is synthesized by solvothermal method: The product obtained in step (1) is placed in ethylene glycol to form a suspension. Ferric chloride hexahydrate, anhydrous sodium acetate and hydrophilic amine compounds are added to the above suspension and stirred until the solid is completely dissolved. Then, the reaction is carried out at 200-300°C for 6-12 hours. Finally, the obtained solid is washed with deionized water under the action of an external magnetic field, dried under vacuum at 50°C, and stored at 4°C.

3. The preparation method according to claim 2, characterized in that, Step (1) includes the following steps: Step (1.1): Slowly add titanium aluminum carbide (Ti3AlC2) to a 50% hydrofluoric acid (HF) solution and stir at room temperature for 16-24 hours. Wash the obtained solid repeatedly with deionized water by centrifugation until the pH value of the supernatant is greater than 6. Dry the product under vacuum at 50°C. Step (1.2): Add the product obtained in step (1.1) to deionized water, and react the resulting suspension at 120-200°C for 16-24 h. Then wash with deionized water by centrifugation, and dry the resulting solid under vacuum at 50°C. Step (1.3): Calcine the product obtained in step (1.2) at 200-500°C for 2-3 hours in air, with a heating rate of 2-5°C / min, and store the resulting solid at 4°C.

4. The preparation method according to claim 3, characterized in that, The mass-to-volume ratio of the titanium aluminum carbide (Ti3AlC2) and the 50% hydrofluoric acid (HF) solution is 1:(10~40) g / mL.

5. The preparation method according to claim 2, characterized in that, The hydrophilic amine compounds are selected from 1,2-propanediamine, 1,3-butanediamine, 1,2-butanediamine, 1,3-pentanediamine, 1,4-pentanediamine, 4-methyl-1,3-pentanediamine, 2-methyl-1,3-pentanediamine, 2-methyl-1,4-pentanediamine, 3-methyl-1,4-pentanediamine, 2,4-pentanediamine, 2,5-pentanediamine, 2,4-hexanediamine, 1,3-hexanediamine, 1,4-hexanediamine, 1,5-hexanediamine, 5-methyl-1,3-hexanediamine, 4-methyl-1,3-hexanediamine, 3-methyl-1,3-hexanediamine, 2-methyl-1,3-hexanediamine, 2-methyl-1,4-hexanediamine, 3-methyl-1,4-hexanediamine, etc. One or more of 5-methyl-1,4-hexanediamine, 2-methyl-1,5-hexanediamine, 3-methyl-1,5-hexanediamine, 4-methyl-1,5-hexanediamine, 1,3-heptanediamine, 1,4-heptanediamine, 1,5-heptanediamine, 1,3-octanediamine, 1,4-octanediamine, and 1,5-octanediamine.

6. The preparation method according to claim 2, characterized in that, The hydrophilic amine compound is selected from one or more of polyacrylamide, polyamide, polyimide, polyetherimide and polyamide.

7. The preparation method according to claim 2, characterized in that, The percentage of each atom in the two-dimensional heterogeneous composite material of amino-modified iron oxide obtained in step (2) is: C 9.99%, O 65.78%, Ti 17.89%, Fe 4.90%, N 1.43%.

8. A method for screening cancer exosome metabolite biomarkers, characterized in that, Includes the following steps: Step (I): Capture exosomes using the magnetic composite material for capturing exosomes as described in claim 1 or the magnetic composite material for capturing exosomes prepared by the preparation method described in any one of claims 2-7: Add the heterogeneous composite material to the target sample, incubate at room temperature, wash with phosphate buffer solution, add deionized water, and obtain a magnetic composite material suspension for capturing exosomes. Step (II): The heterogeneous composite material suspension of the captured exosomes obtained in Step (I) is dropped onto the target plate, and after it dries naturally, laser desorption / ionization mass spectrometry analysis is performed to obtain the mass spectrum of the exosomes. Step (3): Import the exosome mass spectrometry data obtained in Step (2) into the orthogonal partial least squares discriminant analysis model to screen exosome metabolic biomarkers.

9. The method according to claim 8, characterized in that, The specific conditions for laser desorption / ionization mass spectrometry analysis in step (II) 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 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; exosome mass spectrometry data are obtained from flexControl 3.4 and exported from flexAnalysis 3.

4.

10. The method according to claim 8, characterized in that, The criteria for screening exosome metabolic markers in step (iii) are: variable importance projection value greater than 1, P value less than 0.05, absolute value of p(cov, x-axis) greater than 0.05, and absolute value of p(corr, y-axis) greater than 0.

5.

11. The method according to claim 8, characterized in that, The target sample in step (1) is urine.

12. An exosome metabolic marker for bladder cancer, characterized in that, The exosome metabolic markers for bladder cancer are obtained by the method for screening cancer exosome metabolites as described in any one of claims 8-11. The obtained exosome metabolic markers for bladder cancer are ethanolamine, L-aspartic acid, L-acetylcarnitine, prostaglandin E2, and 5,6-dihydroxyprostaglandin F1a.

13. A kit for isolating exosomes, characterized in that, This includes the heterogeneous composite material for capturing exosomes as described in claim 1, or the heterogeneous composite material for capturing exosomes prepared by the preparation method described in any one of claims 2-7.

Images