Flower-like metal oxide heterojunction for mass spectrometric analysis and detection of glaucoma metabolites and preparation method and application of flower-like metal oxide heterojunction

Abstract

The invention relates to a flower-like metal oxide heterojunction and a preparation method and application thereof, the flower-like metal oxide heterojunction is composed of cobalt oxide and platinum, has a high specific surface area and a hierarchical porous structure, and is helpful for improving the enrichment ability of metabolites and the laser ionization efficiency, thereby significantly improving the detection sensitivity based on MALDI mass spectrometry. By constructing a heterojunction interface, the charge transfer efficiency and the local surface plasmon effect are enhanced, the signal intensity is improved, the background noise is effectively inhibited, and the signal reproducibility and the detection stability are improved.

Description

Technical Field

[0001] This invention belongs to the fields of materials chemistry and biomedical detection technology, specifically relating to a flower-shaped metal oxide heterojunction for mass spectrometry analysis and detection of glaucoma metabolites, its preparation method and application. Background Technology

[0002] Primary glaucoma (PG) is a leading cause of irreversible vision loss worldwide, accounting for a significant proportion of blindness cases in both developed and developing regions. Currently, approximately 76 million people worldwide have PG, with a prevalence of about 3.5% in the 40-80 age group. Recent estimates indicate that, driven by factors such as population aging and increased life expectancy, the number of people with PG globally is projected to exceed 112 million by 2040. Despite the increasing global burden of the disease, PG remains largely undiagnosed, particularly in low- and middle-income countries, where over 90% of cases go undetected.

[0003] Currently, standard diagnostic methods commonly used in clinical practice include intraocular pressure measurement, visual field testing, and optic nerve imaging for the initial detection, subtyping, and long-term monitoring of glaucoma (PG). However, these methods have limited sensitivity in detecting early or asymptomatic PG, potentially leading to delayed clinical intervention, by which time optic nerve damage is often irreversible. With the accelerating global aging population, the number of people suffering from glaucoma is expected to increase significantly. Therefore, there is an urgent need to establish more sensitive, scalable, and convenient screening strategies to achieve early identification and timely treatment intervention for PG, thereby minimizing the risk of avoidable visual impairment.

[0004] Given the current challenges and unmet clinical needs, metabolomics-based biomarker discovery offers a promising strategy for advancing the early detection and severity stratification of prostatitis (PG). As a comprehensive and high-throughput analytical approach, metabolomics can identify small-molecule metabolites reflecting physiological and pathological processes in real time. Among various biological samples, blood-derived samples (such as serum) have significant advantages, including being minimally invasive, clinically readily available, and capable of reflecting systemic metabolic changes associated with ocular diseases. Notably, serum metabolite profiling analysis shows increasingly broad application potential in revealing the molecular characterization of disease occurrence, progression, and treatment response.

[0005] For prostatitis (PG), early pathological changes often precede clinical manifestations. Therefore, serum-based metabolic biomarkers hold promise for providing strong support for non-invasive, low-cost, and scalable screening programs. The identification of these biomarkers not only helps improve diagnostic accuracy but may also reveal the underlying mechanisms of disease development, thus laying the foundation for precision medicine management of PG.

[0006] Matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MS) has been widely used in metabolomics research in recent years due to its fast analysis speed, ease of operation, and good compatibility with small molecule metabolites. Its soft ionization properties minimize molecular fragmentation, making it ideal for the detection and analysis of trace metabolites in serum.

[0007] However, existing MALDI MS matrices still face many technical bottlenecks when detecting low-abundance metabolites in complex biological samples. In particular, traditional organic matrices suffer from strong background interference, uneven crystallization, and poor reproducibility in the low-quality range, which severely limits their application in clinical metabolomics.

[0008] Although various novel high-performance matrix materials have been developed and applied in MALDI MS, achieving significant progress in improving ionization efficiency, reducing background interference, and enabling the detection of trace metabolites in complex samples, there is still room for further optimization in terms of structural uniformity, process controllability, batch stability, and suitability for high-throughput clinical applications. Therefore, it is still necessary to explore the construction of novel composite matrix materials that combine high charge transfer efficiency, excellent enrichment capacity, and good process reproducibility to meet the requirements for highly sensitive and reproducible detection of metabolites in complex biological samples, and further promote the application expansion and technological transformation of MALDI MS in clinical metabolomics. Summary of the Invention

[0009] The purpose of this invention is to solve the problem of how to obtain a flower-shaped metal oxide heterojunction for mass spectrometry analysis to detect glaucoma metabolites, its preparation method and application.

[0010] The present invention solves the above-mentioned technical problems through the following technical solutions:

[0011] In a first aspect, the present invention provides a flower-shaped metal oxide heterojunction for mass spectrometry analysis of glaucoma metabolites, the flower-shaped metal oxide heterojunction comprising cobalt oxide and Pt.

[0012] Preferably, the cobalt oxide is Co3O4.

[0013] Preferably, in the flower-like metal oxide heterostructure, the molar ratio of Pt to Co is 1:4 to 4:1.

[0014] Based on the same inventive concept, a second aspect of the present invention provides a method for preparing the flower-like metal oxide heterojunction described in the first aspect, comprising the following steps:

[0015] Step 1: Prepare raw materials, including hydrated cobalt nitrate (Co(NO3)2·6H2O), potassium tetracyanoplatin(II)ate (K2[Pt(CN)4]), organic ligands, methanol and deionized water;

[0016] Step 2: Add K2[Pt(CN)4] and the organic ligand to a solution of methanol and deionized water to form the first solution;

[0017] Step 3: Add Co(NO3)2·6H2O to the mixed solution of methanol and water, stir, and form a second solution;

[0018] Step 4: Add the second solution dropwise to the first solution, stir, centrifuge, wash with deionized water and methanol, and dry to obtain the intermediate;

[0019] Step 5: The intermediate is oxidized by heating in air to obtain a flower-like metal oxide heterojunction.

[0020] Preferably, in step 1, the organic ligand is at least one selected from 2-methylimidazole, pyrazine, thioacetamide, 1,3,5-benzenetricarboxylic acid and terephthalic acid; more preferably, the organic ligand is pyrazine.

[0021] Preferably, in step 2, the volume ratio of methanol to deionized water is 1:1.

[0022] Preferably, in step 2, the concentration of the organic ligand is 0.1 to 0.5 mmol / L, and more preferably 0.1 mmol / L.

[0023] Preferably, in step 4, the stirring time is 0.5 to 4 hours, and more preferably 1 hour.

[0024] Preferably, in step 5, the temperature of the heating oxidation is 200-800℃, more preferably 400℃; the heating oxidation time is preferably 0.5-4h, more preferably 1 hour.

[0025] Preferably, the molar ratio of Co(NO3)2·6H2O to K2[Pt(CN)4] is 1:4-4:1, and more preferably 1:1.

[0026] Based on the same inventive concept, a third aspect of the present invention provides an application of the flower-shaped metal oxide heterostructure described in any of the first aspects above, wherein the flower-shaped metal oxide heterostructure is used as a matrix in laser desorption / ionization mass spectrometry analysis.

[0027] Based on the same inventive concept, a fourth aspect of the present invention provides an application of the flower-shaped metal oxide heterojunction described in any of the first aspects above, wherein the flower-shaped metal oxide heterojunction is used in screening glaucoma metabolic markers.

[0028] Based on the same inventive concept, a fifth aspect of the present invention provides a method for applying the flower-like metal oxide heterostructure described in the first aspect to screening metabolic biomarkers of glaucoma, comprising the following steps:

[0029] Step 1: The biological fluid sample to be tested is dropped onto the target plate and allowed to air dry at room temperature; then, an aqueous solution of the flower-shaped metal oxide heterojunction provided by the present invention is dropped onto its surface and allowed to air dry at room temperature; after the sample is completely dry, it is analyzed by laser desorption / ionization mass spectrometry to obtain the mass spectrum of metabolites in the biological fluid sample to be tested.

[0030] Step 2: Based on the mass spectrometry spectrum, screen for glaucoma metabolic markers in the biological fluid samples to be tested.

[0031] Preferably, step 2 includes:

[0032] The mass spectrometry data were imported into a neural network analysis model to screen for glaucoma metabolic markers in the biological fluid samples to be tested.

[0033] Preferably, the biological fluid sample to be tested in step 1 is a human serum sample.

[0034] Preferably, the human serum stock solution is diluted 10-50 times to obtain the human serum sample, and more preferably diluted 30 times.

[0035] Preferably, the laser desorption / ionization mass spectrometry technique in step 1 is performed using a Bruker autoflex maXMALDI-TOF / TOF mass spectrometer. The laser source used is a 355nm Nd:YAG laser with a laser frequency of 2000Hz and an accelerating voltage of 20kV. The mass spectrometry acquisition mode is cation reflector mode, and the mass-to-charge ratio range is 100-1000Da. The laser intensity is set to 80%. The mass spectrometry data of metabolites in the biological fluid sample to be tested are acquired using flexControl 3.4 software and exported using flexAnalysis 3.4 software.

[0036] Preferably, the screening criteria for biofluid metabolic biomarkers in step 2 include: using the discriminative power of a neural network model, combining fold change analysis and significance testing, and referring to the human metabolome database for metabolite annotation and identification, thereby screening out candidate metabolic biomarkers with statistical significance and biological relevance.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] 1. This invention provides a novel flower-like metal oxide heterojunction that can be used as a matrix material. It possesses a high specific area and a hierarchical porous structure, significantly improving the enrichment capacity of metabolites and laser ionization efficiency, thereby effectively enhancing the detection sensitivity of MALDIMS. By constructing a Co3O4-Pt heterojunction interface, charge transfer and localized surface plasmon resonance effects are enhanced, significantly reducing background interference while maintaining signal strength, thus improving signal reproducibility and detection stability. The flower-like metal oxide heterojunction can be prepared through a one-step room temperature self-assembly and air calcination process. The process conditions are mild, suitable for large-scale production, and offer advantages in operability and cost control. Compared with traditional organic matrices, this invention avoids the problem of interference peaks in low-mass regions, making it particularly suitable for high-throughput detection and screening of low-abundance metabolic biomarkers in complex biological fluids such as serum.

[0039] 2. This invention combines neural network models, fold change analysis, significance testing, and annotation from a human metabolome database to achieve efficient screening of glaucoma-related metabolic biomarkers. The established metabolite fingerprint can be used for early diagnosis of primary glaucoma, differentiation of clinical subtypes, and monitoring of disease progression severity. It has the advantages of being non-invasive, high-throughput, and low-cost, and is expected to provide new technological support for precise screening and personalized treatment of glaucoma.

[0040] 3. The detection platform constructed in this invention has good versatility and scalability. It is not only suitable for glaucoma, but can also be widely applied to the screening and detection of metabolic biomarkers for other metabolic-related diseases (such as diabetes, liver disease, and neurodegenerative diseases). By introducing an artificial intelligence-assisted metabolomics data analysis process, it achieves rapid processing and feature extraction of large-scale samples, possessing strong clinical translational potential and providing a reliable technical means for the early screening, disease classification, and dynamic monitoring of various major diseases. Attached Figure Description

[0041] Figure 1 This is a scanning electron microscope image of the flower-shaped metal oxide heterojunction of Embodiment 1 of the present invention.

[0042] Figure 2 This is a transmission electron microscope image of the flower-shaped metal oxide heterojunction of Embodiment 1 of the present invention.

[0043] Figure 3 This is an XPS image of the flower-shaped metal oxide heterojunction of Embodiment 1 of the present invention.

[0044] Figure 4 The image shows the X-ray diffraction pattern of the flower-like metal oxide heterojunction of Example 1 of the present invention.

[0045] Figure 5 This is the Zeta potential diagram of the product obtained in step (2) of Embodiment 1 of the present invention.

[0046] Figure 6 The image shows the ultraviolet absorption spectrum of the product obtained in step (2) of Example 1 of this invention.

[0047] Figure 7 This is the background image of the mass spectrometry spectrum of Embodiment 2 of the present invention.

[0048] Figure 8 This is a representative mass spectrometry spectrum of serum from healthy controls and patients with primary glaucoma in Example 2 of the present invention.

[0049] Figure 9 This is a confusion matrix diagram output by the neural network analysis model of Embodiment 3 of the present invention.

[0050] Figure 10 This is a heatmap showing the expression differences of serum metabolic markers of primary glaucoma screened in Example 3 of the present invention between healthy controls and patients with primary glaucoma. Detailed Implementation

[0051] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments.

[0052] It should be noted that, unless otherwise defined, the technical terms used in the following embodiments have the same meaning as commonly understood by those skilled in the art. Unless otherwise specified, the experimental reagents used in the following embodiments are conventional biochemical reagents; and the experimental methods described are conventional methods.

[0053] This invention provides a flower-shaped metal oxide heterojunction for mass spectrometry analysis to detect glaucoma metabolites, wherein the flower-shaped metal oxide heterojunction comprises cobalt oxide and Pt.

[0054] Specifically, the flower-shaped metal oxide heterojunction may consist only of cobalt oxide and Pt. The cobalt oxide is Co3O. 4， Flower-like metal oxide heterojunctions have a flower-like structure.

[0055] The flower-like metal oxide heterojunction provided by this invention can be used as a matrix material in laser desorption / ionization mass spectrometry (MALDI-MS). Its flower-like structure, with its high specific area and hierarchical pore structure, significantly improves the enrichment capacity of metabolites and laser ionization efficiency, effectively enhancing the detection sensitivity of MALDI-MS. Furthermore, by constructing a Co3O4-Pt heterojunction interface, charge transfer and localized surface plasmon resonance effects are enhanced, significantly reducing background interference while maintaining signal strength, thus improving signal reproducibility and detection stability.

[0056] In some embodiments, the molar ratio of Pt to Co in the flower-like metal oxide heterostructure is 1:4 to 4:1.

[0057] Specifically, when the molar ratio of Pt to Co is within the range of 1:4 to 4:1, the contents of Pt and Co are moderate, resulting in a uniform structure, high specific area, and multi-level channel structure in the final flower-like metal oxide heterojunction; and an effective heterojunction interface can be formed between Co3O4 and Pt.

[0058] When the molar ratio of Pt to Co is less than 1:4 or greater than 4:1, the content of Pt and Co differs too much, so that an effective heterojunction interface cannot be formed between Co3O4 and Pt, which is not conducive to practical use.

[0059] Furthermore, with a molar ratio of Pt to Co of 1:1, the resulting flower-like metal oxide heterojunction has a very uniform structure, a very high specific area, and a hierarchical pore structure. In addition, a good and effective heterojunction interface can be formed between Co3O4 and Pt.

[0060] Based on the same inventive concept, the present invention also provides a method for preparing a flower-like metal oxide heterojunction, comprising the following steps:

[0061] Step 1: Prepare raw materials, including hydrated cobalt nitrate (Co(NO3)2·6H2O), potassium tetracyanoplatin(II)ate (K2[Pt(CN)4]), organic ligands, methanol and deionized water;

[0062] Step 2: Add K2[Pt(CN)4] and the organic ligand to a solution of methanol and deionized water to form the first solution;

[0063] Step 3: Add Co(NO3)2·6H2O to the mixed solution of methanol and water, stir, and form a second solution;

[0064] Step 4: Add the second solution dropwise to the first solution, stir, centrifuge, wash with deionized water and methanol, and dry to obtain the intermediate;

[0065] Step 5: The intermediate is oxidized by heating in air to obtain a flower-like metal oxide heterojunction.

[0066] Specifically, the flower-like metal oxide heterojunction can be prepared by a one-step room temperature self-assembly and air calcination process. The process conditions are mild, suitable for large-scale production, and have good operability and cost control advantages. Compared with traditional organic matrices, this invention avoids the problem of interference peaks in low-quality regions, and is particularly suitable for high-throughput detection and screening of low-abundance metabolic biomarkers in complex biological fluids such as serum.

[0067] In some embodiments, in step 1, the organic ligand is at least one selected from 2-methylimidazolium, pyrazine, thioacetamide, 1,3,5-benzenetricarboxylic acid and terephthalic acid; preferably, the organic ligand is pyrazine.

[0068] In some embodiments, in step 2, the volume ratio of methanol to deionized water is 1:1.

[0069] In some embodiments, in step 2, the concentration of the organic ligand is 0.1–0.5 mmol / L, preferably 0.1 mmol / L.

[0070] Specifically, the concentration of organic ligands significantly affects the particle size of the resulting flower-like metal oxide heterojunctions. The higher the concentration of organic ligands, the larger the particle size of the resulting flower-like metal oxide heterojunctions, and the lower the concentration of organic ligands, the smaller the particle size of the resulting flower-like metal oxide heterojunctions.

[0071] When the concentration of organic ligands is 0.1–0.5 mmol / L, the concentration is suitable, resulting in a moderate particle size of the final flower-like metal oxide heterojunction, which can be used as a matrix material.

[0072] When the concentration of organic ligands is less than 0.1 mmol / L or greater than 0.5 mmol / L, the concentration of organic ligands is too high or too low, which makes the particle size of the final flower-like metal oxide heterojunction too high or too low. Particles that are too high or too low are not suitable for use as matrix materials.

[0073] In some embodiments, in step 4, the stirring time is 0.5 to 4 hours, more preferably 1 hour.

[0074] In some embodiments, in step 5, the temperature of the heating oxidation is 200-800°C, more preferably 400°C; the heating oxidation time is preferably 0.5-4 hours, more preferably 1 hour.

[0075] Specifically, when the heating oxidation temperature is 200–800°C, the heating oxidation temperature is suitable, and flower-shaped metal oxide heterojunctions with uniform morphology can be formed.

[0076] When the heating oxidation temperature is below 200℃, metal oxide heterojunctions cannot be formed; when the heating oxidation temperature is above 800℃, the morphology of the generated metal oxide heterojunctions collapses and cannot form a good flower-like structure.

[0077] In some embodiments, the molar ratio of Co(NO3)2·6H2O to K2[Pt(CN)4] is 1:4-4:1, more preferably 1:1.

[0078] Specifically, the reaction of platinum salts with a special coordination structure, such as K2[Pt(CN)4], with Co(NO3)2·6H2O can form effective coordination bonds, ensuring that the resulting metal oxide heterojunction has a flower-like structure. Experiments have shown that if other types of platinum salts are used to react with Co(NO3)2·6H2O, only spherical products can be formed, and a flower-like structure cannot be formed.

[0079] This invention also provides an application of a flower-shaped metal oxide heterostructure, which serves as a matrix in laser desorption / ionization mass spectrometry (MALDI-MS). The MALDI-MS is specifically the matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MS).

[0080] The present invention also provides an application of flower-shaped metal oxide heterojunctions in screening glaucoma metabolic markers.

[0081] This invention also provides a method for using flower-like metal oxide heterojunctions in screening glaucoma metabolic markers, comprising the following steps:

[0082] Step 1: The biological fluid sample to be tested is dropped onto the target plate and allowed to air dry at room temperature; then, an aqueous solution of the flower-shaped metal oxide heterojunction provided by the present invention is dropped onto its surface and allowed to air dry at room temperature; after the sample is completely dry, it is analyzed by laser desorption / ionization mass spectrometry to obtain the mass spectrum of metabolites in the biological fluid sample to be tested.

[0083] Step 2: Based on the mass spectrometry spectrum, screen for glaucoma metabolic markers in the biological fluid samples to be tested.

[0084] In some embodiments, step 2 includes: importing the mass spectrometry spectrum into a neural network analysis model to screen for glaucoma metabolic biomarkers in the biological fluid sample to be tested.

[0085] In some embodiments, the biological fluid sample to be tested in step 1 is a human serum sample.

[0086] In some embodiments, the human serum stock solution is diluted 10-50 times to obtain the human serum sample, and more preferably diluted 30 times.

[0087] In some embodiments, the laser desorption / ionization mass spectrometry technique in step 1 is performed using a Bruker autoflexmaX MALDI-TOF / TOF mass spectrometer. The laser source used is a 355nm Nd:YAG laser with a laser frequency of 2000Hz and an accelerating voltage of 20kV. The mass spectrometry acquisition mode is cation reflector mode, and the mass-to-charge ratio range is 100-1000Da. The laser intensity is set to 80%. The mass spectrometry data of metabolites in the biological fluid sample to be tested are acquired using flexControl 3.4 software and exported using flexAnalysis 3.4 software.

[0088] In some embodiments, the screening criteria for biofluid metabolic biomarkers in step 2 include: based on the discriminative power of a neural network model, combined with fold change analysis and significance test, and with reference to the human metabolome database for metabolite annotation and identification, thereby screening out candidate metabolic biomarkers with statistical significance and biological relevance.

[0089] In this invention, the application of flower-shaped metal oxide heterostructures in screening biofluid metabolic biomarkers, combined with neural network models, fold change analysis, significance tests, and annotation from human metabolome databases, achieves highly efficient screening of glaucoma-related metabolic biomarkers. The established metabolite fingerprint can be used for early diagnosis of primary glaucoma, differentiation of clinical subtypes, and monitoring of disease progression severity. It possesses advantages such as non-invasiveness, high throughput, and low cost, and is expected to provide new technological support for precise glaucoma screening and personalized treatment.

[0090] The detection platform constructed in this invention has good versatility and scalability. It is not only suitable for glaucoma but can also be widely applied to the screening and detection of metabolic biomarkers for other metabolic-related diseases (such as diabetes, liver disease, and neurodegenerative diseases). By introducing an artificial intelligence-assisted metabolomics data analysis workflow, it enables rapid processing and feature extraction of large-scale samples, possessing strong clinical translational potential and providing a reliable technical means for the early screening, disease classification, and dynamic monitoring of various major diseases.

[0091] This invention utilizes flower-shaped metal oxide heterostructures to achieve serum metabolite mass spectrometry detection in primary glaucoma.

[0092] 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.

[0093] Example 1

[0094] Preparation method of flower-like metal oxide heterojunctions:

[0095] (1) First, dissolve 0.1 mmol of K₂[Pt(CN)₄] and 0.1 mmol of pyrazine in a mixture of 35 mL of methanol and 35 mL of deionized water to form a homogeneous solution A. Then, dissolve 0.1 mmol of Co(NO₃)₂·6H₂O(Co)₂... 2+ Dissolve the sample in another 35 mL mixture of methanol and 35 mL of deionized water to form a homogeneous solution B. Then, add solution B dropwise to solution A while stirring continuously. After stirring for 1 hour, wash three times with deionized water and ethanol, and dry under vacuum at 50°C overnight.

[0096] (2) The product obtained in step (1) is calcined in air at 400°C for 1 hour, with a heating rate of 5°C·min. -1 A flower-like metal oxide heterojunction solid was obtained, and the solid was stored at room temperature.

[0097] The flower-like metal oxide heterojunction prepared in this embodiment was subjected to scanning electron microscopy (SEM) and transmission electron microscopy (TEM) tests. The SEM and TEM images of the obtained flower-like metal oxide heterojunction are shown below. Figure 1 and Figure 2 As shown, Figure 1 and Figure 2 In this study, the microflowers are composed of two-dimensional nanosheets that self-assemble and interconnect to form a three-dimensional flower-like hierarchical structure with abundant open spaces, with a particle size of approximately 4 micrometers. The two-dimensional nanosheets have a rough surface composed of uniformly distributed ultrafine nanoparticles. Their surface is covered with a dense porous structure, which facilitates the full exposure of active sites to small molecule metabolites.

[0098] The flower-like metal oxide heterojunction prepared in this embodiment was subjected to X-ray photoelectron spectroscopy (XPS) to investigate its surface composition and bonding state. The XPS pattern of the obtained flower-like metal oxide heterojunction is shown in the figure. Figure 3 As shown, Figure 3 The scanning spectroscopy confirmed the presence of cobalt, oxygen, and platinum.

[0099] X-ray diffraction (XRD) was performed on the flower-like metal oxide heterostructure prepared in this embodiment. The XRD pattern of the flower-like metal oxide heterostructure is shown below. Figure 4 As shown, Figure 4In the diffraction, specific diffraction peaks such as 19.1°, 31.3°, 36.8°, 59.4°, 65.2°, 40.0°, 46.5°, 67.8°, 81.5° and 86.1° correspond to the (111), (220), (311), (511) and (440) crystal planes of spinel-structured Co3O4, and the (111), (200), (311) and (222) crystal planes of face-centered cubic Pt.

[0100] The Zeta potential of the flower-like metal oxide heterojunction prepared in this embodiment was measured, and the Zeta potential diagram is shown below. Figure 5 As shown, Figure 5 In the MALDIMS positive ion mode, the zeta potential of the flower-like metal oxide heterojunction is -17.4 mV. The presence of negative surface charge is conducive to the enrichment of metal cations, thereby promoting the generation of cation addition peaks.

[0101] The ultraviolet absorption spectrum of the flower-like metal oxide heterojunction prepared in this embodiment was measured, and the obtained ultraviolet absorption spectrum is shown below. Figure 6 As shown, Figure 6 In this study, the flower-like metal oxide heterojunction exhibits strong absorption characteristics at 355 nm, demonstrating its potential as a high-efficiency matrix material for MALDI MS.

[0102] Example 2

[0103] The flower-like metal oxide heterojunction material obtained in Example 1 and α-cyano-4-hydroxycinnamic acid C were respectively compared. 10 Mass spectrometry analysis was performed on H7NO3 (hereinafter referred to as "conventional matrix 1") and 2,5-dihydroxybenzoic acid C7H6O4 (hereinafter referred to as "conventional matrix 2"). The background image of the obtained mass spectra is shown below. Figure 7 As shown.

[0104] Wherein, conventional matrix 1 and conventional matrix 2 are organic matrices commonly used in existing MALDIMS. The background image refers to the background peaks generated in the range of m / z < 1000 Da produced by flower-like metal oxide heterojunction materials, α-cyano-4-hydroxycinnamic acid and 2,5-dihydroxybenzoic acid as blank matrices.

[0105] Depend on Figure 7 It is known that traditional matrix 1 and traditional matrix 2 have strong interference from impurity peaks in the low molecular weight range, which can suppress and mask the signals of metabolites in the sample to be tested. However, the flower-shaped metal oxide heterojunction provided in this application has almost no interference from impurity peaks in the low molecular weight range. This fully demonstrates that compared with traditional organic matrices, the flower-shaped metal oxide heterojunction provided in this application can avoid the problem of interference peaks in the low-mass region, and is very suitable for high-throughput detection and screening of low-abundance metabolic markers in complex biological fluids such as serum.

[0106] Example 3

[0107] The flower-shaped metal oxide heterojunction material obtained in Example 1 was used for serum metabolite profile analysis of 273 healthy controls and 318 patients with primary glaucoma from two independent hospitals.

[0108] (1) Take 1.0 mg of flower-shaped metal oxide heterojunction material and add it to 1 mL of deionized water. Under room temperature conditions, it is ultrasonically treated until a uniformly dispersed flower-shaped metal oxide heterojunction material suspension is formed.

[0109] (2) Take 1 μL of serum sample that has been diluted 30 times and drop it onto a target plate for mass spectrometry analysis. After air drying at room temperature, drop the flower-shaped metal oxide heterojunction material suspension obtained in step (1) onto the target plate. After air drying, perform laser desorption / ionization mass spectrometry analysis using a Bruker autoflex maX MALDI-TOF / TOF mass spectrometer 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 acquisition range is 100-1000Da. Mass spectrometry data are acquired using flexControl 3.4 software and processed and exported using flexAnalysis 3.4 software to obtain mass spectra of serum metabolites. Figure 8 Representative spectrograms of serum from healthy controls and patients with primary glaucoma.

[0110] Example 4

[0111] The serum metabolic mass spectra obtained in Example 3 were imported into a neural network analysis model to screen for serum metabolic biomarkers.

[0112] (1) The obtained serum mass spectrometry data were preprocessed using the MALDIquant and MALDIquantForeign packages in R. The processing steps included: signal intensity conversion and normalization, spectrum smoothing, baseline correction, peak alignment, peak identification, and characteristic peak grouping, thereby obtaining a standardized metabolite peak intensity data matrix;

[0113] (2) Based on the preprocessed serum mass spectrometry data, a neural network analysis model is constructed using Python. This model is used to screen metabolic features with discriminative power from high-dimensional metabolic data for the screening of potential biomarkers. Specifically, an initial neural network model is first constructed and trained. Based on the feature weights or importance indices obtained during training, irrelevant features with weights below a preset threshold are removed. Then, the neural network model is reconstructed and trained based on the screened feature set. This feature screening and model reconstruction process can be terminated according to a set number of iterations or until the number of features reaches a preset lower limit, ultimately resulting in a trained neural network model.

[0114] The preprocessed serum mass spectrometry data is input into the trained neural network model, and the final output is a confusion matrix diagram (e.g., Figure 9 (As shown). Figure 9 In the study, 157 healthy controls were correctly diagnosed, 2 out of 185 glaucoma patients were misdiagnosed as healthy controls, and the remaining 183 were accurately diagnosed with glaucoma.

[0115] Combining the thresholds of fold change >1.5 or <0.67 with statistical significance (p<0.05), 52 metabolites with significant differences were screened. These candidate metabolites were cross-referenced with the Human Metabolite Database (HMDB) to identify 9 potential biomarkers with high discriminative power, such as... Figure 10 As shown, these nine metabolites exhibit significantly different expression patterns between the healthy control group and glaucoma patients, highlighting their strong discriminative potential and suggesting that they can be preliminarily identified as metabolic biomarkers for glaucoma.

Claims

1. A flower-shaped metal oxide heterojunction for mass spectrometry analysis of glaucoma metabolites, characterized in that, This includes cobalt oxides and platinum.

2. The flower-like metal oxide heterojunction as described in claim 1, characterized in that, The oxide of cobalt is Co3O4.

3. The flower-like metal oxide heterojunction as described in claim 1, characterized in that, The molar ratio of Pt to Co is 1:4 to 4:

1.

4. A method for preparing a flower-like metal oxide heterojunction according to any one of claims 1 to 3, characterized in that, include: K2[Pt(CN)4] and an organic ligand are added to a mixed solution of methanol and water to form the first solution; Co(NO3)2·6H2O is added to a mixed solution of methanol and water to form a second solution; The second solution was added dropwise to the first solution, and the mixture was stirred, centrifuged, washed, and dried to obtain the intermediate. The intermediate was oxidized by heating in air to obtain a flower-like metal oxide heterojunction.

5. The preparation method according to claim 4, characterized in that, The organic ligand is at least one selected from 2-methylimidazolium, pyrazine, thioacetamide, 1,3,5-benzenetricarboxylic acid and terephthalic acid; the concentration of the organic ligand is 0.1 to 0.5 mmol / L.

6. The preparation method according to claim 4, characterized in that, The heating oxidation temperature is 200-800℃, and the heating oxidation time is 0.5-4h.

7. The preparation method according to claim 4, characterized in that, The molar ratio of Co(NO3)2·6H2O and K2[Pt(CN)4] is 1:4 to 4:

1.

8. The application of a flower-like metal oxide heterojunction according to any one of claims 1 to 3, characterized in that, The flower-shaped metal oxide heterostructure is used as a matrix in laser desorption / ionization mass spectrometry analysis.

9. The application of a flower-like metal oxide heterojunction according to any one of claims 1 to 3, characterized in that, Application of the flower-like metal oxide heterojunction in screening glaucoma metabolic biomarkers.

10. A method for applying a flower-like metal oxide heterojunction according to any one of claims 1 to 3, characterized in that, include: The biological fluid sample to be tested was dropped onto a target plate, allowed to dry naturally, then an aqueous solution of flower-shaped metal oxide heterojunction was added, allowed to dry naturally, and laser desorption / ionization mass spectrometry was performed to obtain the mass spectrum of metabolites in the biological fluid sample to be tested. Based on the mass spectrometry spectrum, glaucoma metabolic biomarkers in the biological fluid samples to be tested were screened.

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