A matrix material, its preparation and use

Abstract

The present application relates to the technical field of in vitro detection, in particular to a matrix material, a preparation method and application thereof. The present application provides a new matrix material for MALDI detection, which comprises MXene material and gold nanowires attached to the surface thereof. By using the high-sensitivity nanomaterial as the matrix material for MALDI detection, metabolites related to diseases (such as X-linked Alport syndrome) can be identified, which opens up a new way for biomarker discovery of diseases and may have important potential in future clinical applications.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of in vitro detection, and in particular to a matrix material and a preparation method and use thereof. BACKGROUND

[0002] Matrix-assisted laser desorption ionization mass spectrometry (MALDI) is a soft ionization technique with the characteristics of rapid scanning, high throughput and suitability for analysis of biological macromolecules. MALDI mass spectrometry is a powerful analytical method that can be used to detect analytes, especially biological-derived analytes such as proteins, cells, microorganisms, etc. In MALDI detection, analytes on the target plate are combined with matrix material, and downstream of the combination of analytes and matrix material, the sample is ionized using a laser, and the ionized components are detected by mass spectrometry.

[0003] In MALDI detection technology, the matrix material is crucial to the detection, and its selection directly determines the sensitivity, resolution, signal quality, etc. of the detection. In recent years, metabolomics, as a high-throughput technology for studying the changes of metabolites in biological systems, has shown great potential in the fields of disease diagnosis and biomarker discovery. Nanomaterial-based technologies have been widely used in the detection of trace metabolites in biological samples due to their high sensitivity and specificity.

[0004] However, the existing methods for metabolomics analysis using MALDI detection still need to be further improved in terms of detection sensitivity and resolution. SUMMARY

[0005] The present application aims to at least partially solve one of the technical problems in the related art. To this end, one object of the present application is to provide a new matrix material for MALDI detection, which comprises a MXene material and gold nanowires attached to the surface thereof. The use of this high-sensitivity nanomaterial as a matrix material for MALDI detection can identify metabolites related to diseases (such as X-linked Alport syndrome), which opens up new avenues for biomarker discovery for diseases and may have important potential in future clinical applications.

[0006] To this end, the present application provides a matrix material in the first aspect. According to an embodiment of the present application, the matrix material comprises a MXene material containing Mo and C and gold nanowires attached to the surface thereof.

[0007] In recent years, technologies based on nanomaterials have been widely used in the detection of trace metabolites in biological samples due to their high sensitivity and specificity. MXene materials have become ideal candidates for designing nanoplatforms due to their unique physical and chemical properties, such as high electrical conductivity, excellent surface modification ability, and biocompatibility. Based on this, the inventors designed the matrix material, which includes MXene material containing Mo and C and gold nanowires attached to the surface of the MXene material. Using this material as a matrix material for MALDI detection, metabolites related to diseases (such as X-linked Alport syndrome) can be identified, which opens up new avenues for biomarker discovery for diseases.

[0008] According to an embodiment of the present application, in the matrix material, the molar ratio of each of Mo, C, and Au is 3:2:(1-4).

[0009] According to an embodiment of the present application, the diameter of the gold nanowires is 30-60 nm.

[0010] The second aspect of the present application provides a method for preparing the matrix material of the first aspect. According to an embodiment of the present application, the method comprises:

[0011] (1) mixing a molybdenum aluminum carbon MAX phase material with an etchant for etching treatment to remove the aluminum layer to obtain a MXene material;

[0012] (2) preparing a suspension of the MXene material, mixing the suspension with a chloroauric acid solution and a reducing agent to undergo a reduction reaction to obtain the matrix material.

[0013] According to an embodiment of the present application, the molybdenum aluminum carbon MAX phase material is Mo3AlC2.

[0014] According to an embodiment of the present application, the etchant is an alkali metal hydroxide.

[0015] According to an embodiment of the present application, the etchant is potassium hydroxide, and in the etching treatment, the molar ratio of molybdenum aluminum carbon MAX phase material to potassium hydroxide is 1:(100-105), and under this ratio, potassium hydroxide is sufficient.

[0016] According to an embodiment of the present application, the reaction time of the etching treatment is 23-25 h, and the reaction temperature is 24-26℃.

[0017] According to an embodiment of the present application, the reducing agent is ascorbic acid.

[0018] According to an embodiment of the present application, the concentration of the chloroauric acid solution is 0.1M, and the concentration of the ascorbic acid is 0.1M,

[0019] The mass ratio of the MXene material, the chloroauric acid solution and the ascorbic acid in the reduction reaction is 1: (10-11) : (10-11).

[0020] According to an embodiment of the present application, the reaction time of the reduction reaction is 23-25 h, and the reaction temperature is 24-26℃.

[0021] The third aspect of the present application provides a use of the matrix material of the first aspect or the matrix material prepared by the method of the second aspect in MALDI detection.

[0022] The fourth aspect of the present application provides a use of the matrix material of the first aspect or the matrix material prepared by the method of the second aspect in metabolite detection or metabolomics analysis.

[0023] The fifth aspect of the present application provides a metabolite detection method. According to an embodiment of the present application, the detection method comprises performing MALDI detection on a sample to be tested by using the matrix material of the first aspect or the matrix material prepared by the method of the second aspect, so as to realize detection of a target metabolite.

[0024] The sixth aspect of the present application provides a metabolomics analysis method. According to an embodiment of the present application, the analysis method comprises performing MALDI detection on a sample by using the matrix material of the first aspect or the matrix material prepared by the method of the second aspect, and performing the metabolomics analysis based on the detection result.

[0025] According to an embodiment of the present application, the sample is a body fluid sample derived from a healthy and / or diseased patient.

[0026] Additional aspects and advantages of the present application will be in part apparent and in part pointed out hereinafter in the description of embodiments of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0027] The above and / or additional aspects and advantages of the present application will become apparent and be readily appreciated from the following description of embodiments, taken in conjunction with the accompanying drawings, in which:

[0028] Figure 1 The schematic diagram of MXene@AuNWs nano-platform for detecting metabolic characteristics of XLAS patients is shown, wherein a is a fine preparation flow chart of MXene@AuNWs; and b is a detection principle diagram of MXene@AuNWs;

[0029] Figure 2Characterization images of gold nanowires and MXene materials are shown, wherein a is a TEM image of original gold nanowires, showing a size of 35.9 ± 9.7 nm; b is a SEM image of gold nanowires, showing a size of about 42.2 ± 12.4 nm; c is an XRD pattern of synthesized MXene; d is a SEM morphology image of MXene; e is an elemental analysis result image of MXene material; f is a morphology analysis image of gold nanowire hybridized MXene;

[0030] Figure 3 LDI-MS standard metabolite analysis images based on MXene@AuNWs are shown, wherein a-d show the results of MXene@AuNWs assisted LDI-MS for detecting standard metabolites 0.1 mg / mL phenylalanine, 0.1 mg / mL glucose, 0.5 M sodium chloride and 5 mg / mL bovine serum albumin (BSA), respectively;

[0031] Figure 4 Urine sample disease-related metabolite identification analysis images are shown, wherein A is a principal component analysis (PCA) result image of 17 healthy control group and 17 disease group urine samples; B is an orthogonal partial least squares discriminant analysis (OPLS-DA) result image of 17 healthy control group and 17 disease group urine samples; C is a volcano plot of metabolite analysis of 17 healthy control group and 17 disease group urine samples (x-axis is fold change, y-axis is p value); D is an ROC curve image of the metabolite with mass-to-charge ratio m / z = 278.43;

[0032] Figure 5 Blood sample disease-related metabolite identification analysis images are shown, wherein A is a principal component analysis (PCA) result image of 25 healthy samples and 17 disease samples; B is an orthogonal projection learning dimensionality reduction analysis (OPLS-DA) result image of 25 healthy samples and 17 disease samples; C is a volcano plot of metabolite analysis of 25 healthy samples and 17 disease samples; D is an ROC curve image of the metabolite with mass-to-charge ratio m / z = 250.098. DETAILED DESCRIPTION

[0033] Embodiments of the present application are described in detail below. The embodiments described below are exemplary only, and are not to be construed as limiting the present application.

[0034] It should be noted that the terms "first", "second" are used only for descriptive purposes, and cannot be understood as indicating or implying relative importance or implying a number of the technical features indicated. Thus, the features defined with "first", "second" can include one or more of the features explicitly or implicitly. Further, in the description of the present application, unless otherwise stated, the meaning of "a plurality" is two or more.

[0035] The endpoints of the ranges and any values disclosed herein are not limited to the precise values stated. The ranges or values should be construed to be roughly about the ranges or values. For ranges, the endpoints are included as well as intervening points which are included in the ranges. For values, the values are included as well as intervening values which are included in the values.

[0036] In order that the application can be more readily understood, certain technical and scientific terms are specifically defined below. Unless specifically defined herein, all other technical and scientific terms used in this document concur in meaning with the general use of those terms by those skilled in the art to which the application pertains.

[0037] In this document, the terms "comprising" or "including" are open-ended, that is, the inclusion of unspecified features or steps is not precluded.

[0038] In this document, the terms "optionally", "optional" or "optional" generally mean that the subsequently described event or circumstance can or can not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0039] Alport syndrome (AS) is a rare genetic kidney disease caused by abnormal expression of COL4A3 / 4 / 5 genes. These genes encode type IV collagen in the glomerular basement membrane (GBM). The disease gradually worsens over time, usually with persistent hematuria symptoms in childhood, and can eventually lead to end-stage renal disease. In addition to kidney damage, patients may develop sensorineural hearing loss in childhood or adolescence, often accompanied by eye abnormalities in adulthood. Although the molecular mechanism of XLAS has been clearly defined, the lack of effective early diagnostic methods and specific biomarkers poses a major challenge to the management of patients. Existing drugs and therapies can only delay disease progression and cannot change the inevitable outcome of end-stage renal failure, which makes AS the main cause of end-stage renal disease. Therefore, finding early diagnostic molecular markers for Alport syndrome is of key significance for achieving precise intervention and delaying kidney function decline.

[0040] In the MALDI detection technology, the matrix material is crucial for detection, and its selection directly determines the sensitivity, resolution, signal quality and the like of the detection. In recent years, as a high-throughput technology for studying the changes of metabolites in biological systems, metabolomics has shown great potential in the fields of disease diagnosis and biomarker discovery. Due to its high sensitivity and specificity, the technology based on nanomaterials has been widely used in the detection of trace metabolites in biological samples. Based on this, the application provides a new matrix material for MALDI detection, which uses the high-sensitivity nanomaterial as the matrix material for MALDI detection, and can identify metabolites related to diseases (such as X-linked Alport syndrome). These metabolites open up new avenues for biomarker discovery for diseases and may have important potential in future clinical applications.

[0041] Based on this, the application provides a matrix material, which comprises a MXene material containing Mo and C and gold nanowires attached to the surface of the MXene material.

[0042] According to a specific embodiment of the application, in the matrix material, the molar ratio of Mo, C elements is 3:2, and Au is coated on the surface of the material. For example, in the matrix material, the molar ratio of each of Mo, C and Au can be 3:2:1, 3:2:2, 3:2:3, 3:2:4, etc.

[0043] According to a specific embodiment of the application, the diameter of the gold nanowires is 30-60 nm. For example, the diameter of the gold nanowires can be 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, etc.

[0044] According to a specific embodiment of the application, the application further provides a method for preparing the matrix material described above, which comprises:

[0045] (1) mixing a molybdenum aluminum carbon MAX phase material with an etchant to perform etching treatment to remove the aluminum layer so as to obtain a MXene material;

[0046] (2) preparing a suspension of the MXene material, mixing the suspension with a chloroauric acid solution and a reducing agent to perform a reduction reaction so as to obtain the matrix material.

[0047] According to a specific embodiment of the application, the molybdenum aluminum carbon MAX phase material is Mo3AlC2.

[0048] According to a specific embodiment of the application, the etchant is an alkali metal hydroxide. The alkali metal hydroxide can be, for example, sodium hydroxide or potassium hydroxide.

[0049] According to a specific embodiment of the present application, the etchant is potassium hydroxide, and the molar ratio of the molybdenum aluminum carbon MAX phase material to potassium hydroxide in the etching process is 1: (100-105), and the potassium hydroxide is sufficient at this ratio.

[0050] According to a specific embodiment of the present application, the etchant is potassium hydroxide, and the molar ratio of the molybdenum aluminum carbon MAX phase material to potassium hydroxide in the etching process is 1: (100-105), and the potassium hydroxide is sufficient at this ratio.

[0051] According to a specific embodiment of the present application, the reaction time of the etching process is 23-25 h, and the reaction temperature is 24-26℃.

[0052] According to a specific embodiment of the present application, the reaction time of the etching process is 24 h, and the reaction temperature is 25℃.

[0053] According to a specific embodiment of the present application, the reducing agent is ascorbic acid.

[0054] According to a specific embodiment of the present application, the concentration of the chloroauric acid solution is 0.1M, and the concentration of the ascorbic acid is 0.1M,

[0055] The mass ratio of the MXene material, the chloroauric acid solution, and the ascorbic acid in the reduction reaction is 1: (10-11) : (10-11).

[0056] According to a specific embodiment of the present application, the mass ratio of the MXene material, the 0.1M chloroauric acid solution, and the 0.1M ascorbic acid in the reduction reaction is 1: 10.33978: 10.17612.

[0057] According to an embodiment of the present application, the reaction time of the reduction reaction is 23-25 h, and the reaction temperature is 24-26℃.

[0058] According to a specific embodiment of the present application, the reaction time of the reduction reaction is 24 h, and the reaction temperature is 25℃.

[0059] The present application further Figure 1 The fine preparation process of the matrix material MXene@AuNWs is shown in a; Figure 1 The detection principle of MXene@AuNWs is shown in b, first, the matrix material MXene@AuNWs is dispersed on the target plate for MALDI detection, then the sample to be detected is added, and the metabolomics analysis of healthy samples and diseased samples is carried out.

[0060] According to a specific embodiment of the present application, the present application also provides use of the matrix material as described above or the matrix material prepared by the method as described above in MALDI detection.

[0061] According to a specific embodiment of the present application, the present application also provides use of the matrix material as described above or the matrix material prepared by the method as described above in metabolite detection or metabolomics analysis.

[0062] The present application takes X-linked Alport syndrome disease as an example, and uses the matrix material of the present application to perform metabolomics analysis for detecting metabolic characteristics of XLAS patients. By using high-sensitivity nanomaterial technology, the inventors identify metabolites significantly related to XLAS. It should be noted that the high-sensitivity nanomaterial MXene@AuNWs (MXene modified gold nanowires) provided by the present application can analyze metabolites significantly related to various diseases, and opens up a new way for biomarker discovery related to these diseases.

[0063] X-linked Alport syndrome (XLAS) is a genetic kidney disease mainly caused by mutations in the COL4A5 gene, and is clinically manifested as progressive nephritis, hearing loss and eye abnormalities. Early diagnosis and discovery of specific biomarkers are crucial for the treatment and management of XLAS. The present application provides a metabolomics analysis method based on the MXene@AuNWs (MXene modified gold nanowires) nanoplatform for detecting metabolic characteristics of XLAS patients. By using high-sensitivity nanomaterial technology, the present application successfully identifies multiple metabolites significantly related to XLAS. These metabolites open up a new way for biomarker discovery of XLAS and may have important potential in future clinical applications.

[0064] According to a specific embodiment of the present application, the present application also provides a metabolite detection method, which comprises using the matrix material as described above or the matrix material prepared by the method as described above to perform MALDI detection on a sample to be detected, so as to realize detection of target metabolites.

[0065] According to a specific embodiment of the present application, the present application provides a metabolomics analysis method, which comprises using the matrix material as described above or the matrix material prepared by the method as described above to perform MALDI detection on a sample, and performing the metabolomics analysis based on the detection result.

[0066] The present application develops a metabolomics analysis method based on the MXene@AuNWs nanoplatform for detecting and analyzing metabolic profiles of XLAS patients. Through the platform, the inventors can identify specific metabolites related to XLAS, thereby providing new insights and methods for early diagnosis and biomarker discovery of XLAS.

[0067] According to a specific embodiment of the present application, the sample is a body fluid sample derived from a healthy and / or diseased patient. The body fluid sample can be, for example, blood, urine, interstitial fluid, etc.

[0068] The solutions of the present disclosure will be explained below with reference to examples. Those skilled in the art will understand that the examples below are only for illustrating the present disclosure and should not be considered as limiting the scope of the present disclosure. If a specific technique or condition is not mentioned in the examples, the technique or condition described in the literature in the art or according to the product manual is used. If the manufacturer of the reagent or instrument is not mentioned, it is a conventional product that can be obtained commercially.

[0069] Sample collection

[0070] In this study, children with Alport syndrome diagnosed by the Department of Nephrology, Rheumatology and Immunology of Shanghai Children's Hospital were used as the test group, and healthy children at the same time were used as the control group. Urine and serum samples were collected from all subjects for metabolomics testing.

[0071] Example 1 Preparation of MXene@AuNWs nano-platform

[0072] 1. Synthesis of MXene

[0073] During the synthesis of MXene, 1 gram of molybdenum aluminum carbide (Mo3AlC2, a MAX phase material) powder was added to a polytetrafluoroethylene (PTFE) reaction container. Then 40 milliliters of 40% potassium hydroxide (KOH) solution was slowly added, which acts as an etchant in the reaction. The mixture was continuously stirred at room temperature (about 25°C) for 24 hours. During this period, potassium hydroxide reacts with molybdenum aluminum carbide, gradually etching away the aluminum layer, thereby generating MXene. After the reaction is complete, the mixture is vacuum filtered. The obtained precipitate is washed repeatedly with deionized water until the pH value approaches neutral (pH value between 6-7). The purified MXene is placed in deionized water for 30 minutes of ultrasonic dispersion treatment, and then vacuum dried at 60°C for 12 hours. Finally, dry MXene powder is obtained.

[0074] 2. Preparation and characterization of MXene@AuNWs

[0075] Selecting the appropriate matrix material is crucial for the effectiveness of MALDI detection. To this end, the inventors synthesized gold nanowire functionalized MXene to achieve high sensitivity metabolite detection.

[0076] The synthesis method of MXene@AuNWs composite material is as follows: First, 1 gram of MXene was dispersed in 100 mL of deionized water and sonicated for 30 minutes to obtain a homogeneous MXene suspension. Then, 10 mL of 0.1 M chloroauric acid (HAuCl4) solution was added under continuous stirring. Next, 10 mL of 0.1 M ascorbic acid (AA) solution was slowly injected as a reducing agent. After stirring the mixture at room temperature for 24 hours, gold nanowires (AuNWs) grew on the MXene surface. After the reaction was complete, the product was separated by centrifugation, washed repeatedly with deionized water, and finally dried under vacuum at 60 °C for 12 hours to obtain the MXene@AuNWs composite material.

[0077] TEM images of the gold nanowires show a size of 35.9 ± 9.7 nm. Figure 2 (a) The nanowires, with a diameter of approximately 35.9 nm, exhibited good size uniformity, with the overall size concentrated within a relatively narrow range, indicating good control of the synthesis process. This uniformity and controllability are of great significance for the application of nanowires in the field of biosensing. SEM analysis of gold nanowires ( Figure 2 Image b) shows excellent morphology, with a size of approximately 42.2 ± 12.4 nm and uniform distribution, indicating well-controlled synthesis conditions. The uniformity of size and distribution suggests that the synthesis conditions were properly regulated, making it suitable for subsequent detection and other applications. The product was verified by X-ray diffraction during the synthesis of MXene.

[0078] XRD analysis of synthesized MXene ( Figure 2 Image c) shows a significant peak at 39.0°, which is in excellent agreement with reported data, thus providing strong evidence for successful synthesis. Scanning electron microscopy analysis provides morphological images of MXene ( Figure 2 (d), which provides an effective way for metabolite enrichment. Furthermore, elemental analysis of MXene materials ( Figure 2 The results showed that it contained elements such as carbon, molybdenum, and aluminum. Finally, morphology analysis was performed on the gold nanowire-modified MXene. Figure 2 (f) It was discovered that gold nanowires have been successfully attached to the surface of layered MXene. This unique morphological feature will facilitate highly sensitive metabolite detection and lay a solid foundation for subsequent metabolomics analysis.

[0079] The sample preparation steps for scanning electron microscopy (SEM) and transmission electron microscopy (TEM) analysis methods are as follows:

[0080] For SEM analysis, the sample was fixed on a conductive tape and coated with a thin layer of gold to enhance conductivity. An accelerating voltage of 5-20 kV was typically used, with magnification ranging from 1000x to 100,000x, and high-resolution surface topography images were obtained by adjusting the working distance; TEM analysis steps: the sample was prepared into an ultrathin section with a thickness of less than 100 nanometers and placed on a copper mesh. TEM used an accelerating voltage of 200 kV, and the magnification was selected from 10,000x to 500,000x as needed to observe the internal structure.

[0081] X-ray diffraction (XRD) analysis: A Bruker D8 Advance high-performance X-ray diffractometer was used. The parameter settings are as follows: the scanning range is usually set to the interval of 5° to 80°, the scanning rate is accurately controlled at 2° / min, the tube voltage is stably maintained at 40 kV, and the tube current is maintained at 40 mA.

[0082] Example 2 Performance evaluation of MXene@AuNWs as LDI substrate

[0083] When using MXene@AuNWs composite material for laser desorption / ionization (LDI) analysis, first, 0.5 milligrams of MXene@AuNWs sample was dispersed in 1 milliliter of deionized water, and ultrasonic treatment was performed for 10 minutes to ensure uniform dispersion. Then 5 microliters of the dispersion was added to a pre-cleaned MALDI target plate, and air-dried at room temperature. The sample was analyzed by a MALDI-TOF mass spectrometer, and a laser with a wavelength of 355 nanometers was used to realize the desorption / ionization process. The laser energy was set to about 20 microjoules, and the mass spectrum was obtained by scanning the range of 100-1000 m / z in positive ion mode. The inventors evaluated the salt tolerance and protein tolerance of MXene@AuNWs assisted LDI-MS: first, 0.1 mg / mL phenylalanine and glucose metabolite solution was prepared, then sodium chloride was added to a concentration of 0.5 M for salt tolerance test, and BSA was added to a concentration of 5 mg / mL for protein tolerance evaluation. The LDI-MS signal was continuously monitored during the experiment to evaluate the performance of the matrix.

[0084] The inventors evaluated the standard metabolite analysis of MXene@AuNWs assisted LDI-MS in positive ion mode. Phenylalanine was detected in the matrix, and the Na peak and K peak of the standard could be distinguished ( Figure 3 a); glucose was detected in the matrix, and the Na and K peaks were obvious ( Figure 3 b); the salt tolerance of the matrix was not affected ( Figure 3 c); the protein resistance of the matrix was not affected ( Figure 3In d). The salt tolerance and protein tolerance test results of LDI technology showed that its salt tolerance concentration was 0.5 M sodium chloride, and the protein tolerance concentration was 5 mg / mL bovine serum albumin (BSA). At the same time, the test concentration of phenylalanine and glucose was 0.1 mg / mL. These parameters verify the stability and reliability of LDI technology in the analysis of complex biological samples, indicating that the technology can still maintain high detection performance in high salt and high protein environment.

[0085] Example 3 Metabolomics analysis

[0086] A total of 42 children were included in this study, 17 children with XLAS as the experimental group, and 25 healthy children as the control group at the same time. MXene@AuNWs assisted LDI-MS was used to extract urine and serum metabolite spectrum. Urine samples were collected and divided into positive (patient) and negative (healthy) groups, the positive group contained 17 urine cases, and the negative group contained 17 urine cases. After the researchers collected the serum samples, they were divided into a positive group (patient group) and a negative group (healthy control group), the positive group contained 17 serum samples, and the negative group contained 25 serum samples. It is especially noteworthy that in order to avoid possible bias in the cohort, the two groups were matched in terms of age and gender distribution, and the results showed that there was no significant difference in age or gender between the two groups. To maximize the use of clinical samples and improve data reliability, each urine or serum sample was tested independently three times under the same conditions, and three spectrum data were collected for each sample as raw analysis data.

[0087] In principal component analysis (PCA), first, the data needs to be collected and arranged in matrix form, where rows represent samples and columns represent variables. Then the data is standardized so that each variable has a mean of zero and a variance of one. Next, the covariance matrix is calculated to reflect the correlation between variables. By eigenvalue decomposition or singular value decomposition (SVD), the principal components and their corresponding eigenvalues are extracted, and the first few principal components (usually accounting for more than 70% of the cumulative variance) are selected for analysis. Finally, the original data is projected into the selected principal component space to generate score and loading plots, thereby interpreting the main variation patterns and the relative contribution of each variable.

[0088] 1. Urine sample metabolite analysis

[0089] By performing principal component analysis (PCA) on 17 control urine samples and 17 disease urine samples, it was found that the two groups could not be effectively distinguished in the principal component space, indicating that PCA has limitations in distinguishing these complex metabolic fingerprint data and is difficult to fully extract differential information. However, further analysis using orthogonal partial least squares discriminant analysis (OPLS-DA) successfully achieved effective separation of control and disease samples. OPLS-DA highlights the metabolic characteristics closely related to disease status by eliminating the interference of irrelevant variables, resulting in significant differences between the two groups in the model. This analysis method not only helps to identify potential metabolic biomarkers, but also provides important support for the application of urine metabolomics in disease diagnosis and personalized medicine Figure 4 Fig. 2A, B). The volcano plot shows the significant changes in metabolites, with the horizontal axis showing the fold change between the control and disease groups, and the vertical axis showing the statistical significance (p-value) of these differences. The significantly up-regulated and down-regulated metabolites are distributed on both sides of the volcano plot, and the key metabolites closely related to disease status are marked. This visualization method not only directly reveals the presence of potential markers, but also provides strong data support for further study of the role of these markers in disease mechanisms Figure 4 Fig. 2C). The area under the curve (AUC) value of the metabolite at m / z = 278.43 is 0.752, indicating that this metabolite has very high diagnostic accuracy in distinguishing control and disease samples. An AUC value close to 1.0 means that the metabolite at this m / z value exhibits very strong sensitivity and specificity in distinguishing between healthy and disease states. Such a high AUC value suggests that this metabolite may be a strong biomarker, providing important reference for early diagnosis of disease, laying the foundation for accurate diagnosis and personalized medical strategies in clinical applications Figure 4 Fig. 2D).

[0090] 2. Blood sample metabolite analysis

[0091] After performing principal component analysis (PCA) on 25 healthy blood samples and 17 disease blood samples, it was found that the two samples could not be effectively distinguished in the principal component space. This indicates that when dealing with such complex data, PCA fails to extract enough differential information to distinguish between healthy and disease states Figure 5 Fig. 3A). However, by further applying orthogonal partial least squares discriminant analysis (OPLS-DA) method for directional analysis of the data, the results showed that OPLS-DA successfully achieved effective separation of healthy and disease samples Figure 5The significant classification effect indicates that OPLS-DA can better capture the key variables and patterns related to the disease state, providing a more powerful tool for clinical diagnosis and biomarker discovery. The volcano plot clearly shows that multiple genes have significant expression differences between the diseased group and the healthy group. It is particularly noteworthy that some genes show significant up-regulation or down-regulation trends in the diseased samples (marked by red dots in the volcano plot), which may become potential biomarkers. Through further functional verification and clinical correlation analysis, it will help to confirm the practical application value of these genes in disease diagnosis or treatment Figure 5 The study found that there was a significant difference in the metabolite peak at m / z = 250.098 (AUC = 0.672) between the two groups of samples. Specifically, the metabolite level at this m / z value in the diseased samples was significantly lower than that in the healthy samples, suggesting that this metabolite may be closely related to the onset or progression of the disease. In addition, a negative feedback rule was observed: as the disease worsened, the content of this metabolite showed a reverse regulation trend, which may reflect certain metabolic or physiological compensation mechanisms in the body Figure 5

[0092] The above experimental results show that the metabolomics analysis platform based on MXene@AuNWs has great potential in the discovery of biomarkers for X-linked Alport syndrome. By in-depth analysis of the metabolic fingerprint, metabolites closely related to XLAS can be identified, providing a new research direction for early diagnosis and treatment.

[0093] In the description of the present specification, the description of the terms "one embodiment", "some embodiments", "example", "specific example", "some embodiments" or "some examples" and the like means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present application. In the present specification, the illustrative description of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any appropriate manner in any one or more embodiments or examples. In addition, different embodiments or examples described in the present specification and the features of different embodiments or examples can be combined and combined by those skilled in the art without contradiction.

[0094] Although the embodiments of the present application have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present application, and those skilled in the art can make changes, modifications, replacements and variations to the above embodiments within the scope of the present application.​

Claims

1. The application of matrix materials in MALDI detection, characterized in that, The matrix material includes MXene material and gold nanowires attached to its surface, wherein the MXene material contains Mo and C. In the matrix material, the molar ratio of Mo, C and Au is 3:2:(1-4). The diameter of the gold nanowires is 30-60 nm.

2. The use according to claim 1, characterized in that, The method for preparing the matrix material includes: (1) Mix the molybdenum-aluminum-carbon MAX phase material with an etchant and perform etching to remove the aluminum layer in order to obtain MXene material; (2) Prepare a suspension of the MXene material, mix the suspension with chloroauric acid solution and reducing agent to undergo a reduction reaction, so as to obtain the matrix material.

3. The use according to claim 2, characterized in that, The molybdenum-aluminum-carbon MAX phase material is Mo3AlC2.

4. The use according to claim 2, characterized in that, The etchant is an alkali metal hydroxide.

5. The use according to claim 2, characterized in that, The etchant is potassium hydroxide, and in the etching process, the molar ratio of molybdenum-aluminum-carbon MAX phase material to potassium hydroxide is 1:(100-105).

6. The use according to claim 2, characterized in that, The etching process takes 23-25 ​​hours and is carried out at a temperature of 24-26°C.

7. The use according to claim 2, characterized in that, The reducing agent is ascorbic acid.

8. The use according to claim 7, characterized in that, The concentration of the chloroauric acid solution is 0.1M, and the concentration of the ascorbic acid is 0.1M. In the reduction reaction, the mass ratio of MXene material, chloroauric acid solution and ascorbic acid is 1:(10-11):(10-11).

9. The use according to claim 2, characterized in that, The reduction reaction takes 23-25 ​​hours and is carried out at a temperature of 24-26 °C.

10. Applications of matrix materials in metabolite detection or metabolomics analysis. The matrix material includes MXene material and gold nanowires attached to its surface, wherein the MXene material contains Mo and C. In the matrix material, the molar ratio of Mo, C and Au is 3:2:(1-4). The diameter of the gold nanowires is 30-60 nm.

11. A method for detecting metabolites, characterized in that, This includes using matrix materials for MALDI detection of test samples to achieve the detection of target metabolites. The matrix material includes MXene material and gold nanowires attached to its surface, wherein the MXene material contains Mo and C. In the matrix material, the molar ratio of Mo, C and Au is 3:2:(1-4). The diameter of the gold nanowires is 30-60 nm.

12. A metabolomics analysis method, characterized in that, This includes performing MALDI assays on samples using matrix materials, and then conducting the aforementioned metabolomics analysis based on the assay results. The matrix material includes MXene material and gold nanowires attached to its surface, wherein the MXene material contains Mo and C. In the matrix material, the molar ratio of Mo, C and Au is 3:2:(1-4). The diameter of the gold nanowires is 30-60 nm.

13. The metabolomics analysis method according to claim 12, characterized in that, The samples are bodily fluid samples derived from healthy and / or sick patients.

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