Two-dimensional titanium-containing nanosheet decorated with nanoscale iron-containing particles and application of two-dimensional titanium-containing nanosheet in Crohn disease diagnosis

Abstract

The invention discloses a two-dimensional titanium-containing nanosheet decorated with nanoscale iron-containing particles and application of the two-dimensional titanium-containing nanosheet in plasma exosome separation and Crohn disease diagnosis, and belongs to the technical field of biological detection based on exosomes. The prepared two-dimensional titanium-containing nanosheet decorated with the nanoscale iron-containing particles has strong interaction with phospholipid on the surface of the exosome, and efficient adsorption and separation of the exosome in a plasma sample to be detected can be achieved. In addition, the two-dimensional titanium-containing nanosheet further has strong ultraviolet absorption and high conductivity, exosome metabolites can be ionized, a remarkable mass spectrum signal is generated, and rapid diagnosis of Crohn's disease is achieved through an LDI-TOF MS analysis and detection technology.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological detection based on exosomes, and specifically relates to a two-dimensional titanium-containing nanosheet decorated with nanoscale iron-containing particles and a preparation method thereof, as well as applications of the nanosheet in separation of plasma exosomes and diagnosis of Crohn's disease. Background Art

[0002] Exosomes are an important component of blood, which are released from various types of cells and circulate in the blood to promote cell communication, immune response, inflammation and tumor progression. Exosomes contain a large number of biomacromolecules that are highly correlated with the state of individual cells, making them a valuable resource for potential biomarkers. Currently, exosome-based liquid biopsy plays an increasingly important role in disease diagnosis, typing and monitoring. In particular, liquid metabolic biopsy shows broad potential in utilizing the overall metabolic characteristics of exosomes, because small molecule metabolites are downstream products of biochemical reactions and have many advantages such as high sensitivity and real-time performance. To date, metabolite-based disease diagnosis usually requires machine learning of data collected from large-scale clinical samples and multiple targets.

[0003] Traditional methods for isolating exosomes generally include ultracentrifugation, size-exclusive chromatography, and ultrafiltration. These methods are often cumbersome to operate, have high requirements for equipment and operators, and cannot meet the growing demand for detection. Efforts have been made to quickly and efficiently separate exosomes to support downstream metabolic analysis, including microfluidics, immunoaffinity, microelectrodes, and modular platforms, among which the use of nanomaterials is an important part. Recently, laser desorption ionization-time of flight mass spectrometry (LDI-TOF MS) based on inorganic nanomaterials has become a potential solution for detecting exosome metabolites. Compared with common techniques such as nuclear magnetic resonance and liquid chromatography-mass spectrometry, LDI-TOF MS can greatly increase throughput and significantly shorten detection time. Inorganic nanomaterials have the advantages of high absorption, high energy transfer capacity, low background interference, and functionalization, and are the most commonly used matrix materials for LDI-TOF MS. These properties make them very suitable for the desorption and ionization of metabolites in biological samples, thereby obtaining information-rich metabolic fingerprints. LDI-TOF MS excels at quickly acquiring large amounts of metabolic data, and combining it with machine learning algorithms shows great potential in promoting rapid disease diagnosis, monitoring, and prognosis. Summary of the invention

[0004] One object of the present invention is to provide a two-dimensional titanium-containing nanosheet decorated with nanoscale iron-containing particles, and a preparation method and use thereof; a second object of the present invention is to provide a reagent for isolating exosomes; and another object of the present invention is to provide a diagnostic system for diagnosing Crohn's disease and its application.

[0005] The purpose of the present invention is achieved through the following technical solutions:

[0006] In a first aspect, the present invention provides a method for preparing a two-dimensional titanium-containing nanosheet decorated with nanoscale iron-containing particles, characterized in that the preparation method comprises the following steps:

[0007] Step 1.1: Dissolve the template, glacial acetic acid and concentrated hydrochloric acid in the reaction solvent 1, add the titanium precursor after sufficient stirring and ultrasonication, mix well and then put into an oven to heat and volatilize the solvent to obtain a titanium-containing gel;

[0008] Step 1.2: dissolving the titanium-containing gel obtained in step 1.1 in reaction solvent 2, transferring the solution to a reactor for solvothermal reaction, cooling the reactor to room temperature after the reaction, collecting the solid product, washing it, and then drying it to obtain a two-dimensional titanium-containing nanosheet;

[0009] Step 1.3: The two-dimensional titanium-containing nanosheets, trivalent iron salt and anhydrous sodium acetate obtained in step 1.2 are dispersed in ethylene glycol, fully stirred and ultrasonicated, and then transferred to a reactor for solvothermal reaction. After the reaction is completed, the reactor is cooled to room temperature, the solid product is collected and washed, and then dried to obtain a two-dimensional titanium-containing nanosheet decorated with nanoscale iron-containing particles.

[0010] Preferably, the template in step 1.1 is a triblock polymer; the reaction solvent 1 is an organic solvent, most preferably tetrahydrofuran.

[0011] In a specific embodiment of the present invention, the triblock polymer is poloxamer.

[0012] In some embodiments of the present invention, the usage ratio of the poloxamer, glacial acetic acid and concentrated hydrochloric acid is 1.3-1.6 g: 1-3 mL: 2-4 mL.

[0013] Preferably, the titanium precursor in step 1.1 is a titanate, and the titanate includes but is not limited to tetrabutyl titanate and isopropyl titanate.

[0014] In the most preferred embodiment of the present invention, the titanate is tetrabutyl titanate.

[0015] Preferably, the temperature for heating the volatilized solvent in step 1.1 is 30 to 70° C. and the time is 15 to 30 hours.

[0016] Preferably, the reaction solvent 2 in step 1.2 is an organic solvent, most preferably ethylene glycol.

[0017] Preferably, the solvent thermal reaction temperature in step 1.2 is 80 to 150° C. and the reaction time is 15 to 30 hours.

[0018] In a preferred embodiment of the present invention, the drying conditions in step 1.2 and step 1.3 are vacuum drying at 40-60°C.

[0019] Preferably, the trivalent iron salt in step 1.3 is ferric chloride and / or its hydrate; the temperature of the solvothermal reaction is 150-250° C., and the time is 1-3 hours.

[0020] In a preferred embodiment of the present invention, the trivalent iron salt is ferric chloride hexahydrate, and the usage ratio of the two-dimensional titanium-containing nanosheets, ferric chloride hexahydrate, anhydrous sodium acetate and ethylene glycol is 50-100 mg: 250-300 mg: 1-2 g: 60-70 mL.

[0021] In a second aspect, the present invention provides a two-dimensional titanium-containing nanosheet decorated with nanoscale iron-containing particles, characterized in that the two-dimensional titanium-containing nanosheet decorated with nanoscale iron-containing particles is prepared by the method provided by the first aspect of the present invention.

[0022] In a third aspect, the present invention provides a use of a two-dimensional titanium-containing nanosheet decorated with nanoscale iron-containing particles in at least one of the following:

[0023] a1) Use in the preparation of a reagent for isolating exosomes;

[0024] a2) Use in the preparation of a diagnostic system for diagnosing or assisting in the diagnosis of Crohn's disease.

[0025] In a fourth aspect, the present invention provides a reagent for isolating exosomes, the reagent comprising the two-dimensional titanium-containing nanosheets decorated with nanoscale iron-containing particles according to the second aspect of the present invention.

[0026] In a fifth aspect, the present invention provides a diagnostic system, characterized in that the diagnostic system comprises an exosome separation device and a laser desorption ionization-time of flight mass spectrometer (LDI-TOF MS); the exosome separation device is provided with an array-arranged two-dimensional titanium-containing nanosheet decorated with nanoscale iron-containing particles; the laser desorption ionization time of flight mass spectrometer is used to obtain an LDI-TOF MS mass spectrum of exosome metabolites in the sample to be tested.

[0027] The sample to be tested described in the present invention is plasma of a subject.

[0028] The array-arranged two-dimensional titanium-containing nanosheets decorated with nano-sized iron-containing particles are prepared by the following method: the two-dimensional titanium-containing nanosheets decorated with nano-sized iron-containing particles described in the second aspect of the present invention are evenly dispersed in deionized water, and dropped onto a stainless steel target plate with array points to obtain the array-arranged two-dimensional titanium-containing nanosheets decorated with nano-sized iron-containing particles.

[0029] In a sixth aspect, the present invention provides a use of the diagnostic system described in the fifth aspect of the present invention in the preparation of a product for diagnosing or assisting in the diagnosis of Crohn's disease.

[0030] The application comprises the following steps:

[0031] Step 1: Isolate and detect exosomes from batch samples to obtain exosome metabolite fingerprints;

[0032] Step 2: Perform machine learning algorithm based on the fingerprint map;

[0033] Step 3: Isolate and detect exosomes from the sample to be tested, obtain an LDI-TOF MS mass spectrum of the exosome metabolites, and determine whether the subject has Crohn's disease based on the probability score output by the mass spectrum in the range of 0 to 1 in the machine learning algorithm.

[0034] The judgment rule is: if the mass spectrum probability score of the sample to be tested is between 0 and 0.5, it is judged that the subject to be tested does not suffer from Crohn's disease; if the mass spectrum probability score of the sample to be tested is between 0.5 and 1, it is judged that the subject to be tested has Crohn's disease.

[0035] The batch samples described in the present invention include a training set and a test set, and the training set includes Crohn's disease patients and healthy people.

[0036] The method for obtaining an exosome metabolite fingerprint of the present invention comprises: performing LDI-TOF MS analysis on a batch sample after exosome separation according to the method provided by the present invention, normalizing the mass spectrum peak intensity with the total ion current of the obtained mass spectrum, averaging the mass spectrum of each sample, aligning the peak coordinates with a tolerance of 0.4, and tabulating the mass spectrum peak intensity of each sample to obtain an exosome metabolite fingerprint.

[0037] The machine learning algorithm described in the present invention is a conventional technical means in the field, including: inputting training set sample data, training model, evaluating model, inputting test set sample data for blind testing, and evaluating test set blind testing results.

[0038] Preferably, the exosome separation and detection method of the present invention comprises the following steps:

[0039] Step 1: using an array of two-dimensional titanium-containing nanosheets decorated with nano-sized iron-containing particles to separate exosomes from the sample to be tested, to obtain two-dimensional titanium-containing nanosheets combined with exosomes;

[0040] Step 2: The two-dimensional titanium-containing nanosheets bound to exosomes obtained in step 1 are washed and dried naturally, and directly subjected to LDI-TOF MS analysis.

[0041] In a specific embodiment of the present invention, step 1 specifically includes: diluting the sample to be tested with physiological saline, dropping it on the two-dimensional titanium-containing nanosheets decorated with nano-scale iron-containing particles arranged in an array, letting it stand, and washing to obtain the two-dimensional titanium-containing nanosheets decorated with nano-scale iron-containing particles arranged in an array and combined with exosomes.

[0042] In a specific embodiment of the present invention, the specific conditions of the LDI-TOF MS analysis in step 2 are: using a 355nm Nd:YAG laser light source, a laser frequency of 2000Hz, an acceleration voltage of 20kV, wherein the voltage at ion source 1 is 20kV, and the voltage at ion source 2 is 17.6kV; the acquisition mode is a reflection positive ion mode, and the acquisition range m / z is 100-1000Da; each sample is tested 5 times; mass spectrometry data is obtained from flexControl 3.4, and the data is exported in flexAnalysis3.4.

[0043] The technical solution provided by the present invention has the following beneficial technical effects:

[0044] (1) The present invention creatively prepares two-dimensional titanium-containing nanosheets decorated with nano-scale iron-containing particles. The two-dimensional titanium-containing nanosheets have a strong interaction with the phospholipids on the surface of exosomes, and can achieve efficient adsorption of exosomes in the plasma sample to be tested, thereby separating the exosomes and performing in situ mass spectrometry detection on the separated exosomes. The array-like arrangement of the two-dimensional titanium-containing nanosheets greatly shortens the experimental time, achieving faster, more sensitive, and simpler separation and detection of exosomes.

[0045] (2) The two-dimensional titanium-containing nanosheets decorated with nano-sized iron-containing particles prepared by the present invention have the advantages of strong ultraviolet absorption and high conductivity, which are conducive to absorbing laser energy and ionizing exosome components, and can efficiently ionize the separated exosome metabolites to produce significant mass spectrometry signals;

[0046] (3) The present invention uses a machine learning algorithm to identify the differences in exosome metabolism between Crohn's disease patients and healthy people. It can distinguish plasma samples from patients and healthy people with high accuracy and sensitivity, successfully realizing rapid in vitro diagnosis of Crohn's disease, and has high clinical value and good application prospects for large-scale Crohn's disease diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 This is a scanning electron microscope image of the two-dimensional titanium-containing nanosheet decorated with nano-sized iron-containing particles of Example 1.

[0048] Figure 2 This is a transmission electron micrograph of the two-dimensional titanium-containing nanosheets decorated with nano-sized iron-containing particles of Example 1.

[0049] Figure 3 This is an X-ray powder diffraction spectrum of the two-dimensional titanium-containing nanosheets decorated with nano-sized iron-containing particles in Example 1.

[0050] Figure 4 This is the UV-visible absorption spectrum of the two-dimensional titanium-containing nanosheets decorated with nano-sized iron-containing particles in Example 1.

[0051] Figure 5 The plasma exosome metabolic fingerprint mass spectrum obtained for the array-arranged two-dimensional titanium-containing nanosheets decorated with nanoscale iron-containing particles in Example 2.

[0052] Figure 6 This is the probability score diagram of all plasma samples in Example 3.

[0053] Figure 7 This is the receiver characteristic curve based on the training set and the test set of Example 3.

[0054] Figure 8 This is the cluster analysis heat map of Example 4.

[0055] Fig. 9 Graph showing the probability scores of all plasma samples based on important features in Example 4.

[0056] Fig.10 This is the receiver characteristic curve based on the training set and the test set of Example 4. DETAILED DESCRIPTION

[0057] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only some embodiments of the present invention, not all. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0058] Example 1 Preparation of two-dimensional titanium-containing nanosheets decorated with nano-sized iron-containing particles arranged in an array

[0059] (1) 1.5 g of poloxamer, 2.4 mL of glacial acetic acid and 3.2 mL of concentrated hydrochloric acid were dissolved in 30 mL of tetrahydrofuran, and 3.4 g of tetrabutyl titanate was added after thorough stirring and ultrasonication. After mixing evenly, the mixture was placed in an oven and heated to 45° C. for 24 hours to evaporate the solvent, thereby obtaining a titanium-containing gel;

[0060] (2) dissolving the titanium-containing gel obtained in step (1) in 30 mL of ethylene glycol, transferring the solution to a reactor and reacting it at 100° C. for 24 hours, cooling the reactor to room temperature after the reaction, collecting the solid product, washing the product with deionized water and ethanol three times respectively, and drying the product under vacuum at 50° C. to obtain a two-dimensional titanium-containing nanosheet;

[0061] (3) dispersing 100 mg of the two-dimensional titanium-containing nanosheets obtained in step (2), 300 mg of ferric chloride hexahydrate and 1.2 g of anhydrous sodium acetate in 60 mL of ethylene glycol, stirring thoroughly and ultrasonicating the mixture, transferring the mixture to a reactor and reacting at 200° C. for 1 hour. After the reaction, the reactor was cooled to room temperature, the solid product was collected and washed with deionized water and ethanol for 3 times respectively, and dried under vacuum at 50° C. to obtain two-dimensional titanium-containing nanosheets decorated with nanoscale iron-containing particles;

[0062] (4) The two-dimensional titanium-containing nanosheets decorated with nano-sized iron-containing particles obtained in step (3) are uniformly dispersed in deionized water, and dropped onto a stainless steel target plate with 16×24 array points to obtain array-arranged two-dimensional titanium-containing nanosheets decorated with nano-sized iron-containing particles.

[0063] The scanning electron microscope image of the two-dimensional titanium-containing nanosheets decorated with nano-sized iron-containing particles prepared as described above is as follows: Figure 1 As shown; transmission electron microscopy images of two-dimensional titanium-containing nanosheets decorated with nanoscale iron-containing particles are shown Figure 2 As shown; the X-ray powder diffraction pattern of the two-dimensional titanium-containing nanosheets decorated with nano-sized iron-containing particles is shown Figure 3 The UV-visible absorption spectrum of the two-dimensional titanium-containing nanosheets decorated with nano-sized iron-containing particles is shown in Figure 4 shown.

[0064] Analysis results: From Figure 1 and Figure 2 It can be seen that the two-dimensional titanium-containing nanosheets decorated with nano-sized iron-containing particles present a well-defined sheet structure, and the nano-sized iron-containing particles have a uniform hemispherical morphology with a diameter of about 50 nm; Figure 3 It can be seen that the two-dimensional titanium-containing nanosheets decorated with nano-sized iron-containing particles have excellent crystallinity; Figure 4 It can be seen that the two-dimensional titanium-containing nanosheets decorated with nano-sized iron-containing particles have excellent absorption intensity at the wavelength of the laser light source.

[0065] Example 2 Isolation and detection of exosomes in plasma of batch samples and obtaining fingerprints

[0066] (1) 168 Crohn's disease plasma and 103 healthy human plasma were randomly divided into training and test sets in a ratio of 4:1, corresponding to 217 and 54 samples, respectively;

[0067] (2) diluting the sample 30 times with physiological saline, dripping 2.5 μL of each sample on an array spot of a two-dimensional titanium-containing nanosheet decorated with nano-sized iron-containing particles arranged in an array, standing at 25° C. for 10 minutes, washing the array spot with physiological saline, and allowing it to dry naturally;

[0068] (3) Mass spectrometry analysis: The stainless steel target plate with bound exosomes in step (2) was directly subjected to LDI-TOF MS analysis using a Bruker ultrafleXtreme mass spectrometer with a 355 nm Nd:YAG laser source, a laser frequency of 2000 Hz, an acceleration voltage of 20 kV, ion source 1 voltage of 20 kV, and ion source 2 voltage of 17.6 kV; the acquisition mode was reflectron cation mode, and the acquisition range m / z was 100-1000 Da; each sample was tested 5 times; mass spectrometry data were obtained from flexControl 3.4 and exported in flexAnalysis 3.4; representative mass spectra are shown in Figure 2. Figure 5 shown.

[0069] (4) Obtaining fingerprint: The mass spectrum obtained in step (3) is normalized by the total ion current to obtain the mass spectrum peak intensity, the mass spectrum of each sample is averaged, the peak coordinates are aligned with a tolerance of 0.4, the mass spectrum peak intensity of each sample is tabulated, and the exosome metabolite fingerprint is obtained. The fingerprint is as follows: Figure 5 shown.

[0070] Analysis results: From Figure 5 It can be seen that the exosome metabolite fingerprint obtained by separation from two-dimensional titanium-containing nanosheets decorated with nanoscale iron-containing particles contains rich information, and the metabolite fingerprints of disease and health have good discrimination and have the potential to be used as a basis for diagnosis.

[0071] Example 3: Using the metabolic fingerprint of plasma exosomes for Crohn's disease diagnosis using deep learning algorithms

[0072] (1) Use the MALDIquant package in R software to process the raw mass spectrometry data and export the mass spectrometry peak intensity table;

[0073] (2) dividing the mass spectrum peak intensity table into a training set and a test set according to the grouping method in Example 2, and using the training set data to train the deep learning model;

[0074] (3) The deep learning model is a neural network model consisting of 5 hidden layers with a learning rate of 0.0005. It was established using Python 3.11, and the loss function was calculated using binary cross entropy. An early stopping strategy was used to prevent overfitting.

[0075] (4) The trained deep learning model is used to predict the test set data, and the model outputs the probability scores of all samples;

[0076] (5) Use the probability scores of the training set and test set samples to draw the receiver characteristic curve and calculate the area under the curve (AUC). The probability scores of all samples are as follows: Figure 6 The receiver characteristic curve is shown as Figure 7shown.

[0077] Analysis results: From Figure 6 It can be seen that the machine learning algorithm can distinguish disease samples from healthy samples very well, outputting high probability scores for disease samples and low probability scores for healthy samples. Figure 7 It can be seen that the probability score output by the machine learning algorithm can effectively distinguish between disease and healthy samples, and has great potential for use in the in vitro diagnosis of Crohn's disease in clinical practice.

[0078] Example 4: Using the metabolic fingerprint of plasma exosomes to extract a potential biomarker panel using a feature selection algorithm, and combining the biomarker panel with a deep learning algorithm for Crohn's disease diagnosis

[0079] (1) Use the MALDIquant package in R software to process the raw mass spectrometry data and export the mass spectrometry peak intensity table;

[0080] (2) dividing the mass spectrum peak intensity table into a training set and a test set according to the grouping method in Example 2, and using the training set data for feature selection;

[0081] (3) Use the cross-validated recursive feature elimination algorithm to select important features. The clustering heat map of important features is as follows: Figure 8 As shown;

[0082] (4) Train a deep learning model based on the important features of the training set. The trained deep learning model is used to predict the test set data, and the model outputs the probability scores of all samples.

[0083] (5) Use the probability scores of the training set and test set samples to draw the receiver characteristic curve and calculate the area under the curve (AUC). The probability scores of all samples are as follows: Fig. 9 The receiver characteristic curve is shown as Fig.10 shown.

[0084] Analysis results: From Figure 8 It can be seen that the intensity distribution of disease and health is significantly different, which is suitable for disease diagnosis and analysis. Fig. 9 It can be seen that the deep learning algorithm based on important features can distinguish disease samples from healthy samples very well, outputting high probability scores for disease samples and low probability scores for healthy samples. Fig.10 It can be seen that the probability score output by the deep learning algorithm based on important features can effectively distinguish between disease and healthy samples, and has great potential for use in the in vitro diagnosis of Crohn's disease in clinical practice.

[0085] The array-arranged two-dimensional titanium-containing nanosheets decorated with nano-scale iron-containing particles prepared by the present invention can achieve efficient separation of exosomes through strong interaction and shortened experimental time. Moreover, due to strong ultraviolet absorption and high conductivity, exosome components can be efficiently ionized, and exosome metabolite fingerprints are successfully extracted. With the help of machine learning algorithms, rapid in vitro diagnosis of Crohn's disease is achieved with high sensitivity and high accuracy. The technical solution provided by the present invention has broad application prospects in large-scale disease diagnosis.

[0086] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a two-dimensional titanium-containing nanosheet decorated with nanoscale iron-containing particles, characterized in that: The preparation method comprises the following steps: Step 1.1: Dissolve the template, glacial acetic acid and concentrated hydrochloric acid in the reaction solvent 1, add the titanium precursor after sufficient stirring and ultrasonication, mix well and then put into an oven to heat and volatilize the solvent to obtain a titanium-containing gel; Step 1.2: dissolving the titanium-containing gel obtained in step 1.1 in reaction solvent 2, transferring the solution to a reactor for solvothermal reaction, cooling the reactor to room temperature after the reaction, collecting the solid product, washing it, and then drying it to obtain a two-dimensional titanium-containing nanosheet; Step 1.3: The two-dimensional titanium-containing nanosheets, trivalent iron salt and anhydrous sodium acetate obtained in step 1.2 are dispersed in ethylene glycol, and after sufficient stirring and ultrasonication, they are transferred to a reactor for solvothermal reaction. After the reaction is completed, the reactor is cooled to room temperature, and the solid product is collected, washed, and then dried to obtain a two-dimensional titanium-containing nanosheet decorated with nano-sized iron-containing particles; The template in step 1.1 is a triblock polymer, and the triblock polymer is poloxamer.

2. A two-dimensional titanium-containing nanosheet decorated with nanoscale iron-containing particles, characterized in that The two-dimensional titanium-containing nanosheets decorated with nanoscale iron-containing particles are prepared by the method of claim 1.

3. Use of the two-dimensional titanium-containing nanosheets decorated with nanoscale iron-containing particles according to claim 2 in at least one of the following: a1) Use in the preparation of a reagent for isolating exosomes; a2) Use in the preparation of a diagnostic system for diagnosing or assisting in the diagnosis of Crohn's disease.

4. A reagent for isolating exosomes, characterized in that The reagent comprises the two-dimensional titanium-containing nanosheets decorated with nano-sized iron-containing particles as described in claim 2.

5. A diagnostic system, characterized in that: The diagnostic system includes an exosome separation device and a laser desorption ionization-time of flight mass spectrometer; the exosome separation device is provided with an array-like arrangement of the two-dimensional titanium-containing nanosheets decorated with nanoscale iron-containing particles as described in claim 2; the laser desorption ionization-time of flight mass spectrometer is used to obtain an LDI-TOF MS mass spectrum of exosome metabolites in the sample to be tested.

6. Use of the diagnostic system according to claim 5 in preparing a product for diagnosing or assisting in the diagnosis of Crohn's disease, the use comprising the following steps: Step 1: Isolate and detect exosomes from batch samples to obtain exosome metabolite fingerprints; Step 2: Perform machine learning algorithm based on the fingerprint map; Step 3: Separate and detect exosomes from the sample to be tested, obtain an LDI-TOF MS mass spectrum of exosome metabolites, and determine whether the subject has Crohn's disease based on the probability score output by the mass spectrum in the range of 0 to 1 in the machine learning algorithm; The judgment rule is: if the mass spectrum probability score of the sample to be tested is between 0 and 0.5, it is judged that the subject to be tested does not suffer from Crohn's disease; if the mass spectrum probability score of the sample to be tested is between 0.5 and 1, it is judged that the subject to be tested has Crohn's disease.

7. The use according to claim 6, characterized in that: The method for obtaining an exosome metabolite fingerprint comprises: performing LDI-TOF MS analysis on a batch sample after exosome separation according to the method provided by the present invention, normalizing the mass spectrum peak intensity with the total ion current of the obtained mass spectrum, averaging the mass spectrum of each sample, aligning the peak coordinates with a tolerance of 0.4, and tabulating the mass spectrum peak intensity of each sample to obtain an exosome metabolite fingerprint; the batch sample comprises a training set and a test set, and the training set comprises Crohn's disease patients and healthy people.

8. The use according to claim 6, characterized in that: The exosome separation and detection method comprises the following steps: Step 1: using an array of two-dimensional titanium-containing nanosheets decorated with nano-sized iron-containing particles to separate exosomes from the sample to be tested, to obtain two-dimensional titanium-containing nanosheets combined with exosomes; Step 2: The two-dimensional titanium-containing nanosheets bound to exosomes obtained in step 1 are washed and dried naturally, and directly subjected to LDI-TOF MS analysis.

9. The use according to claim 8, characterized in that: The step 1 specifically comprises: diluting the sample to be tested with physiological saline, dripping it onto the array-arranged two-dimensional titanium-containing nanosheets decorated with nano-scale iron-containing particles, letting it stand, and washing, so as to obtain the array-arranged two-dimensional titanium-containing nanosheets decorated with nano-scale iron-containing particles combined with exosomes.

10. The use according to claim 8, characterized in that: The specific conditions of the LDI-TOF MS analysis in step 2 are: using a 355nm Nd:YAG laser light source, a laser frequency of 2000Hz, an acceleration voltage of 20kV, wherein the voltage at ion source 1 is 20kV, and the voltage at ion source 2 is 17.6kV; the acquisition mode is the reflectron cation mode, and the acquisition The range of m / z was 100-1000Da; each sample was tested in 5 replicates; mass spectrometry data were obtained from flexControl 3.

4. And export the data in flexAnalysis 3.4.

Images