Nanometer bimetallic multi-shell hollow oxide and preparation method and application thereof

Abstract

This invention discloses a nano-bimetallic multi-shell hollow oxide, its preparation method, and its applications. Using divalent manganese nitrate, divalent nickel nitrate, and organic ligands as raw materials, a nano-shell hollow oxide is prepared through heating reaction and oxidative calcination. Containing two components, manganese and nickel oxides, the nanomaterial has a rough surface, strong ultraviolet absorption capacity, and good crystal structure, enabling selective adsorption of small-molecule metabolites in complex biofluids. It also efficiently absorbs laser energy from an ion source and transfers it to the metabolites, promoting efficient ionization. Based on this nanomaterial, this invention utilizes laser desorption / ionization mass spectrometry (LDI-MS) to extract metabolic fingerprints of biofluid metabolites from membranous nephropathy patients. Furthermore, a machine learning model is used to screen metabolic markers in serum and urine of patients with membranous nephropathy. This nanomaterial shows promising application prospects as a matrix for LDI-MS analysis of complex biofluids.

Description

Technical Field

[0001] This invention relates to a nano-bimetallic multi-shell hollow oxide, its preparation method and application, belonging to the field of biomedical detection technology. Background Technology

[0002] Metabolomics is an emerging omics field that studies metabolic substances within organisms. Biofluids, including bodily fluids such as blood and urine, carry a wealth of biomarkers. Analyzing these metabolites provides a comprehensive understanding of an organism's physiological state, the processes of disease occurrence and development, promotes a holistic understanding of biological systems at the molecular level, and provides data-driven, objective insights, laying a solid foundation for precise decision-making in medicine. Metabolites in biofluids reflect systemic physiological and pathological processes; therefore, joint analysis of serum and urine helps obtain more comprehensive metabolic information. By jointly analyzing metabolites in serum and urine, a more comprehensive understanding of an organism's metabolic state can be achieved, enabling early diagnosis of complex diseases, monitoring of disease progression, and evaluation of treatment effectiveness. This integrated diagnostic approach offers a new direction for personalized medicine and is expected to promote precision in disease diagnosis and treatment. Therefore, attempting to develop a diagnostic tool based on combined biofluids is a highly meaningful and challenging endeavor.

[0003] Currently, the main methods for detecting metabolic molecules in serum and urine are GC / LC MS and biochemical methods (such as immunofluorescence). Biochemical methods typically detect only one substance at a time, are easily affected by background signals, and have low specificity and sensitivity. GC / LC MS requires complex sample pretreatment, leading to long detection times and high costs. These limitations make it difficult to achieve low-cost, high-throughput detection of urine and serum samples, hindering large-scale clinical application.

[0004] Mass spectrometry (MS) plays a crucial role in metabolomics research. Laser desorption / ionization mass spectrometry (LDI MS), with its advantages of speed, high sensitivity, and high resolution, shows remarkable promise in generating metabolic fingerprints and is considered a powerful platform for large-scale analysis of clinical data. The rational design of the matrix is ​​essential for the formation of metabolic fingerprints. In real-world biological sample systems, urine and serum samples contain various biomolecules, and different pH levels and high salinity can hinder the detection of small molecules. Traditional organic matrices are even less suitable for small molecule detection. While inorganic nanomaterials (such as carbon-based, silicon-based, and noble metal materials) can be used for the detection of small molecule metabolites, they still have limitations when detecting complex biological samples. Therefore, there is an urgent need to develop new nanomaterials for the detection of metabolites in complex biological samples (urine and serum). The high melting and boiling points of metal oxides contribute to their thermal stability under laser irradiation, thus greatly reducing background interference. On the other hand, due to the synergistic effects between various metals, mixed metal oxides generally have a richer number of redox active sites than single metal oxides. The presence of multiple oxidation states and cations may facilitate electron transfer. Coordination polymers (CPs), with their flexible structures and ease of design and control, are excellent precursors for preparing metal oxides with specific morphologies and compositions. They can be converted into metal oxides with similar morphologies through a one-step heat treatment.

[0005] Therefore, developing a metal oxide matrix suitable for laser desorption / ionization mass spectrometry analysis of body fluid samples (such as urine and serum) will help extract metabolic fingerprints from body fluid samples. Furthermore, based on the metabolic fingerprints, metabolic biomarkers in body fluid samples that are relevant to the diagnosis and treatment of related diseases can be screened. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of existing technologies by providing a nano-bimetallic hollow multishell oxide, its preparation method, and its applications. The nano-bimetallic hollow multishell oxide provided by this invention can selectively adsorb small molecule metabolites in complex biological fluids. Therefore, it can serve as a matrix for extracting metabolite fingerprints using laser desorption / ionization mass spectrometry (LDI MS) analysis of small molecule metabolites in complex biological fluids. This invention utilizes this nano-bimetallic hollow multishell oxide to detect the metabolic mass spectra of serum and urine in patients with membranous nephropathy and to screen for metabolic markers in serum and urine of patients with membranous nephropathy.

[0007] To achieve this objective, the present invention provides a method for preparing nano-bimetallic multi-shell hollow oxides, comprising the following steps:

[0008] Step 1: Dissolve the divalent manganese salt, divalent nickel salt, and organic ligand in a polar organic solvent, and then heat and stir to react; the divalent manganese salt is manganese nitrate and / or manganese nitrate hydrate, the divalent nickel salt is nickel nitrate and / or nickel nitrate hydrate; the organic ligand is an aromatic carboxylic acid;

[0009] Step 2: After the reaction is complete, centrifuge, wash, and dry to obtain the intermediate product;

[0010] Step 3: The intermediate product obtained in Step 3 is oxidized and calcined in an air or oxygen atmosphere to obtain a nano-multi-shell hollow oxide. This nano-multi-shell hollow oxide contains two transition metal oxides, namely oxides of manganese and nickel.

[0011] Preferably, the aromatic carboxylic acid in step 1 is a phenyl carboxylic acid.

[0012] Preferably, the polar organic solvent in step 1 is selected from at least one of acetone, N,N-dimethylformamide (DMF), and N-methylpyrrolidone (NMP).

[0013] Preferably, the organic ligand in step 1 is selected from at least one of isophthalic acid, terephthalic acid, phthalic acid, benzoic acid, 1,2,3-benzenetricarboxylic acid, 1,2,4-benzenetricarboxylic acid, and 1,3,5-benzenetricarboxylic acid;

[0014] And / or, the polar organic solvent in step 1 is a mixed solvent composed of DMF and acetone.

[0015] Preferably, in step 1, the molar ratio of divalent manganese salt, divalent nickel salt, and organic ligand is 0.5–1:0.5–1:1–2.

[0016] Preferably, the heating and stirring reaction in step 1 is carried out at a temperature of 120–180°C for 3–5 hours.

[0017] Preferably, the oxidation and calcination temperature in step 3 is 400-600℃, and the time is 15-60 minutes.

[0018] The present invention also provides nano-bimetallic multi-shell hollow oxides prepared by the above preparation method.

[0019] This invention also provides applications of the nano-bimetallic multi-shell hollow oxide prepared by the above method, wherein the applications are selected from any one of the following 1)-3):

[0020] 1) Application in the analysis and detection of metabolites in biological samples for non-diagnostic and non-therapeutic purposes;

[0021] 2) Applications in the preparation of reagents or kits for the analysis and detection of metabolites in biological samples;

[0022] 3) Application in screening metabolic biomarkers for metabolic diseases. The metabolic biomarkers may be used for disease diagnosis, or they may be used for disease treatment and disease mechanism research.

[0023] Preferably, the biological sample includes a bodily fluid sample from a human or animal;

[0024] And / or, the metabolic diseases include membranous nephropathy.

[0025] More preferably, the body fluid sample includes serum and urine.

[0026] This invention also provides a method for screening metabolite markers of membranous nephropathy, comprising the following steps:

[0027] Step 1: Add the biofluid sample to the target plate and allow it to dry naturally. Then, add an aqueous solution of nano-bimetallic hollow oxide and allow it to dry naturally. Perform laser desorption / ionization mass spectrometry analysis to obtain the mass spectrum of the biofluid metabolite. The nano-bimetallic hollow oxide is prepared by the above method.

[0028] Step 2: Import the biofluid metabolism mass spectrometry spectrum obtained in Step 1 into the orthogonal partial least squares discriminant analysis model to screen biofluid metabolism biomarkers for membranous nephropathy.

[0029] Preferably, the biofluid samples in step 1 include body fluid samples from patients with membranous nephropathy and healthy individuals.

[0030] Preferably, the body fluid sample includes serum and urine samples.

[0031] Preferably, the specific conditions for laser desorption / ionization mass spectrometry analysis in step 1 are as follows: a Bruker UltrafleXtreme MALDI-TOF / TOF mass spectrometer is used, employing a 355nm Nd:YAG laser source with a laser frequency of 2000Hz and an accelerating voltage of 20kV; the acquisition mode is cation reflector mode, and the mass-to-charge ratio range is 100-1000Da; biofluid mass spectrometry data are obtained from flexControl 3.4 and exported from flexAnalysis 3.4.

[0032] And / or, the conditions for screening biofluid metabolite biomarkers in step 2 are as follows: 20 items with the largest projected importance values ​​of the selected variables in serum, a P-value less than 0.05, and a fold difference greater than 1.33 or less than 0.75 are selected as serum biomarkers; 50 items with the largest projected importance values ​​of the selected variables in urine, a P-value less than 0.05, and a fold difference greater than 1.33 or less than 0.75 are selected as urinary biomarkers.

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

[0034] 1. The nano-bimetallic hollow multishell oxide provided by this invention contains two components: manganese and nickel oxide. The nanomaterial has a rough surface, strong ultraviolet absorption capacity, and good crystal form, which can achieve selective adsorption of small molecule metabolites in complex biological fluids. It can also efficiently absorb the laser energy of the ion source and transfer it to the metabolites, promoting the efficient ionization of the metabolites. Based on this nano-bimetallic hollow multishell oxide, the metabolic fingerprint of metabolites in biological fluids of metabolic diseases such as membranous nephropathy can be extracted using laser desorption / ionization mass spectrometry (LDI MS) analysis technology.

[0035] 2. This invention optimizes eight machine learning models for serum and urine samples and ultimately selects the OPLS-DA algorithm; then, it analyzes the expression differences of metabolites between healthy controls and patients with membranous nephropathy through the metabolic fingerprinting of biological fluids, and then screens out metabolic biomarkers in serum and urine respectively. Attached Figure Description

[0036] Figure 1 This is a scanning electron microscope image of the nano-bimetallic multi-shell hollow oxide of Example 1 of the present invention.

[0037] Figure 2 This is a transmission electron microscope image of the nano-bimetallic multi-shell hollow oxide of Example 1 of the present invention.

[0038] Figure 3 This is the Zeta potential diagram of the nano-bimetallic multi-shell hollow oxide of Example 1 of the present invention.

[0039] Figure 4 The image shows the X-ray diffraction pattern of the nano-bimetallic multi-shell hollow oxide of Example 1 of this invention.

[0040] Figure 5 This is a particle size distribution diagram of the nano-bimetallic multi-shell hollow oxide of Example 1 of the present invention.

[0041] Figure 6 The images show the ultraviolet absorption spectra of product 1 and nano-bimetallic multi-shell hollow oxide (product 2) obtained in Example 1 of this invention.

[0042] Figure 7 Representative chromatograms of urine from healthy individuals, patients with membranous nephropathy, patients with poor prognosis, and patients with minimal change disease, as shown in Embodiment 2 of the present invention.

[0043] Figure 8 Representative mass spectra of serum from healthy individuals, patients with membranous nephropathy, patients with prognostic conditions, and patients with minimal change disease, as shown in Embodiment 2 of the present invention.

[0044] Figure 9 This is a metabolic pathway analysis diagram after the orthogonal partial least squares discriminant analysis model of Embodiment 3 of the present invention screened out serum and urine biomarkers. Detailed Implementation

[0045] To make the present invention more apparent and understandable, preferred embodiments are described in detail below with reference to the accompanying drawings.

[0046] Example 1

[0047] Synthesis of nano-bimetallic multi-shell hollow oxide materials:

[0048] (1) First, add 25 mL of DMF and 25 mL of acetone solution to a flask, then add 0.1 mmol of manganese nitrate tetrahydrate (Mn(NO3)2·4H2O), 0.1 mmol of nickel nitrate hexahydrate (Ni(NO3)2·6H2O), and 0.2 mmol of isophthalic acid in sequence, and stir for 4 hours to form a homogeneous solution 1. Transfer solution 1 to a Teflon-lined stainless steel autoclave and react at 160 °C for 4 hours to form solution 2. Centrifuge solution 2, wash it alternately with DMF and acetone, and vacuum dry it at 60 °C for 12 hours to obtain intermediate product 1;

[0049] (2) The product obtained in step (1) is oxidized and calcined in air at 500°C for 15 minutes, with a heating rate of 5°C·min. -1 The obtained solid was stored at room temperature to obtain nano-bimetallic hollow multishell oxide (product 2). The microstructure, structure, particle size distribution, and charge distribution of this nano-bimetallic hollow multishell oxide were characterized, and the results are as follows: Figures 1-6 As shown, Figure 1 The scanning electron microscope image of this nanobimetallic multi-shell hollow oxide shows that the material has a rough surface and a round shape. Figure 2 The image is a transmission electron microscope image of the nanobimetallic multi-shell hollow oxide, which clearly shows that the material exhibits a multi-shell hollow state. Figure 3 The Zeta potential diagram of this nano-bimetallic multi-shell hollow oxide is shown, with an average potential of -12.6 mV. The negatively charged surface is an important factor in enhancing metabolite detection. Figure 4The X-ray diffraction pattern of the nano-bimetallic multi-shell hollow oxide was obtained, which determined the crystal structure of the material. Its characteristic peaks correspond to the standard card patterns of NiMn2O4 (JCPDS:84-0542) and NiO (JCPDS:01-1239). Figure 5 The particle size distribution of the nano-bimetallic multi-shell hollow oxide is shown in the figure. The average particle size of the calcined oxide is 430 nm, which is reduced compared to product 1 (550 nm). Figure 6 The UV absorption spectra of product 1 and nano-bimetallic hollow shell oxide (product 2) are shown. The nano-bimetallic hollow shell oxide has a higher absorbance at 355 nm, indicating stronger energy absorption, which is beneficial for LDI MS analysis and detection.

[0050] Example 2

[0051] The nano-bimetallic multi-shell hollow oxide material obtained in Example 1 was used for laser desorption / ionization mass spectrometry (LDI) of serum and urine metabolites. MS) testing was performed on samples divided into: ① Serum testing group: serum metabolite profiles of 102 healthy individuals, 93 patients with membranous nephropathy, 34 patients with minimal change disease, and 67 prognostic patients (membranous nephropathy patients who have fully or partially recovered after clinical treatment, collectively referred to as the prognostic group); ② Urine testing group: urine metabolite profiles of 100 healthy individuals, 47 patients with membranous nephropathy, 21 patients with minimal change disease, and 34 prognostic patients; ③ Serum + Urine testing group: serum and urine metabolite profiles of 35 patients with membranous nephropathy, 19 patients with minimal change disease, and 33 prognostic patients; samples in this group were derived from samples from groups ① and ② that underwent both serum and urine metabolite profile testing; ④ Blindly tested serum + urine metabolite profiles of 16 patients with membranous nephropathy and 8 prognostic patients (the 24 samples in the blindly tested group were not duplicates of those in the previous group). The process and results are as follows:

[0052] (1) Take 1 mg of nano-bimetallic hollow oxide material and add it to 1 mL of deionized water. Dissolve it by sonication at room temperature to obtain a uniformly mixed suspension of nano-bimetallic hollow oxide material.

[0053] (2) Add 10 μL of serum sample to 290 μL of deionized water to obtain a diluted serum solution;

[0054] (3) Drop 1 μL of urine or diluted serum sample onto the target plate and allow it to air dry. Then, drop the nano-bimetallic multi-shell hollow oxide material suspension obtained in step (1) onto the target plate and allow it to air dry. Perform laser desorption / ionization mass spectrometry analysis using a Bruker UltrafleXtreme MALDI-TOF / TOF mass spectrometer with a 355 nm Nd:YAG laser source, a laser frequency of 2000 Hz, and an accelerating voltage of 20 kV. The acquisition mode is cation reflector mode, and the mass-to-charge ratio range is 100-1000 Da. Obtain mass spectrometry data from flexControl 3.4 and export the data from flexAnalysis 3.4 to obtain the metabolite mass spectra of urine and serum. Representative mass spectra are shown below. Figures 7-8 As shown.

[0055] Example 3

[0056] The urinary and serum metabolic mass spectra obtained in Example 2 were imported into an orthogonal partial least squares discriminant analysis model to screen for serum and urinary metabolic biomarkers:

[0057] (1) The urine mass spectra were preprocessed using R language based on the MALDIquant and MALDIquantForeign packages, including peak intensity conversion and normalization, peak smoothing, baseline subtraction, peak alignment, peak identification and peak grouping.

[0058] (2) Based on urine mass spectrometry, an orthogonal partial least squares discriminant analysis model was constructed using SIMCA 14.1 to select urine and serum metabolic biomarkers. Specifically, SIMCA-P 14.1 was used to calculate the variable importance projection value, P-value, and fold change of each peak signal in the serum and urine mass spectrometry. The top 20 variable importance projection values, P-values ​​less than 0.05, and fold changes greater than 1.33 or less than 0.75 in serum were selected as serum metabolic biomarkers, forming a serum biomarker panel (m / z). 122.83, 133.92, 134.91, 161.94, 176.04, 177.05, 187.00, 191.06, 193.02, 207.01, 209.01, 238.84, 244.88, 254.79, 268.87, 298.64, 300.67, 306.54, 553.78); The top 50 variables in urine with the highest projected importance values, p-values ​​less than 0.05, and fold differences greater than 1.33 or less than 0.75 were selected as urinary metabolic biomarkers, forming a urinary biomarker panel (m / z). 104.68, 106.67, 120.76, 122.81, 162.97, 164.92, 180.92, 196.90, 228.90, 345.39, 399.38, 405.38, 411.11, 414.61, 418.09, 421.09, 422.55, 430.78, 466.56, 472.52, 543.22); the combined panel is a combination of the serum biomarker panel and the urine biomarker panel.

[0059] (3) The m / z signals of the biomarker panels were matched using the Human Metabolome Database (HMDB, http: / / www.hmdb.ca / ) to obtain refined serum panels (m / z 133.92, 161.94, 176.04, 177.05, 187.00, 191.06, 193.02, 207.01, 209.01), refined urine panels (m / z 162.97, 164.92, 180.92, 196.90, 399.38, 418.09, 421.09, 543.22), and a refined combined panel composed of the two panels.

[0060] Figure 9 This is a metabolic pathway analysis diagram after the orthogonal partial least squares discriminant analysis model of this embodiment screened out serum and urine biomarkers.

[0061] The above embodiments are merely preferred embodiments of the present invention and are not intended to limit the present invention in any form or substance. It should be noted that those skilled in the art can make several improvements and additions without departing from the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention.

Claims

1. The application of a nano-bimetallic multi-shell hollow oxide in screening metabolic biomarkers for membranous nephropathy, characterized in that, The preparation method of the nano-bimetallic multi-shell hollow oxide includes the following steps: Step 1: Dissolve the divalent manganese salt, divalent nickel salt, and organic ligand in a polar organic solvent, and then heat and stir to react; the divalent manganese salt is manganese nitrate and / or manganese nitrate hydrate, the divalent nickel salt is nickel nitrate and / or nickel nitrate hydrate; the organic ligand is an aromatic carboxylic acid; Step 2: After the reaction is complete, centrifuge, wash, and dry to obtain the intermediate product; Step 3: The intermediate product obtained in Step 3 is oxidized and calcined in an air or oxygen atmosphere to obtain nano-shell hollow oxide.

2. The application as described in claim 1, characterized in that, The organic ligand in step 1 is selected from at least one of isophthalic acid, terephthalic acid, phthalic acid, benzoic acid, 1,2,3-benzenetricarboxylic acid, 1,2,4-benzenetricarboxylic acid, and 1,3,5-benzenetricarboxylic acid. And / or, the polar organic solvent in step 1 is a mixed solvent composed of DMF and acetone.

3. The application as described in claim 1, characterized in that, In step 1, the molar ratio of divalent manganese salt, divalent nickel salt, and organic ligand is 0.5~1:0.5~1:1~2.

4. The application as described in claim 1, characterized in that, In step 1, the heating and stirring reaction is carried out at a temperature of 120~180℃ for 3~5 hours.

5. The application as described in claim 1, characterized in that, In step 3, the oxidation and calcination temperature is 400-600℃ and the time is 15-60 minutes.

6. A method for screening metabolite biomarkers for membranous nephropathy, characterized in that, Includes the following steps: Step 1: Add the biofluid sample to the target plate and let it dry naturally. Then add an aqueous solution of nano-bimetallic multi-shell hollow oxide and let it dry naturally. Perform laser desorption / ionization mass spectrometry analysis to obtain the mass spectrum of the biofluid metabolite. Step 2: Import the biofluid metabolism mass spectrometry spectrum obtained in Step 1 into the orthogonal partial least squares discriminant analysis model to screen biofluid metabolism biomarkers for membranous nephropathy. The preparation method of the nano-bimetallic multi-shell hollow oxide includes the following steps: Step S1: Dissolve the divalent manganese salt, divalent nickel salt, and organic ligand in a polar organic solvent, and then heat and stir to react; the divalent manganese salt is manganese nitrate and / or manganese nitrate hydrate, the divalent nickel salt is nickel nitrate and / or nickel nitrate hydrate; the organic ligand is an aromatic carboxylic acid; Step S2: After the reaction is complete, centrifuge, wash, and dry to obtain the intermediate product; Step S3: The intermediate product obtained in step 3 is oxidized and calcined in an air or oxygen atmosphere to obtain nano-shell hollow oxide.

7. The method as described in claim 6, characterized in that, The specific conditions for laser desorption / ionization mass spectrometry analysis in step 1 are as follows: a Bruker UltrafleXtreme MALDI-TOF / TOF mass spectrometer is used, with a 355 nm Nd:YAG laser source, a laser frequency of 2000 Hz, and an accelerating voltage of 20 kV; the acquisition mode is cation reflector mode, and the mass-to-charge ratio range is 100-1000 Da; biofluid mass spectrometry data are obtained from flexControl 3.4 and exported from flexAnalysis 3.

4. And / or, the conditions for screening biofluid metabolite biomarkers in step 2 are as follows: 20 items with the largest projected importance values ​​of the selected variables in serum, a P-value less than 0.05, and a fold difference greater than 1.33 or less than 0.75 are selected as serum biomarkers; 50 items with the largest projected importance values ​​of the selected variables in urine, a P-value less than 0.05, and a fold difference greater than 1.33 or less than 0.75 are selected as urinary biomarkers.

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