Preparation method of cerium oxide micro-nano particles with a multi-level structure and its application in detection and identification of small molecule metabolites

Abstract

The present invention discloses a method for preparing cerium oxide micro-nano particles with a multi-level structure, comprising the following steps: Step 1, preparation of a template of 3-aminophenol-formaldehyde spheres; the extended #imgabs0# method is applicable to the preparation of spherical APF resins with uniform particle sizes; Step 2, growing cerium oxide on APF to obtain APF-CeO2; Step 3, removing the APF template to obtain multi-level structured cerium oxide micro-nano particles. The present invention also provides the application of the above-mentioned cerium oxide micro-nano particles in the detection and identification of small molecule metabolites. The detection method of the present invention is non-invasive, has a high detection throughput, high sensitivity, and low cost, meets the clinical requirements for obtaining metabolic fingerprint maps, realizes clinical applications such as diagnosis, prognosis, and biomarker screening, and has great application potential in the in-depth interpretation of the processes of organism growth and development and pathogenic mechanisms at the metabolomics level.

Description

Technical Field

[0001] The present invention relates to the technical fields of material synthesis and analysis and detection, and particularly relates to a preparation method of cerium oxide micro-nano particles with a multi-level structure and its application in the detection and identification of small molecule metabolites. Background Art

[0002] Metabolic analysis techniques, especially mass spectrometry techniques, play an irreplaceable role in clinical applications, including but not limited to diagnosis and prognosis. It is worth noting that traditional mass spectrometry techniques require strict treatment of samples through complex sample pretreatment processes (for example, desalting, removing high-abundance proteins, chromatographic methods) to achieve efficient analysis of metabolites. Laser desorption ionization mass spectrometry provides a promising solution, featuring high throughput and low cost. In this solution, customized matrix materials as nano-reactors are crucial for realizing an effective laser desorption ionization process to achieve metabolic analysis and biomedical applications. Currently, in order to achieve state-of-the-art metabolic analysis, researchers have been striving to construct multifunctional inorganic nanoparticle nano-reactors. For example, Wu et al. developed multifunctional immunomagnetic materials to combine rare cell capture in circulating tumor cell separation and metabolic analysis based on laser desorption ionization mass spectrometry. In addition, it has been reported that multifunctional platinum nano-reactors are used for visual biomarker detection and metabolic fingerprinting based on laser desorption ionization mass spectrometry. However, due to the current lack of advanced laser desorption ionization mass spectrometry platforms, solving multi-scenario clinical problems remains a challenge in this field, especially in prognosis prediction and biomarker screening. Summary of the Invention

[0003] In view of the above-mentioned defects of the prior art, the technical problem to be solved by the present invention is to provide a preparation method of cerium oxide micro-nano particles with a multi-level structure and its application in the detection and identification of small molecule metabolites.

[0004] To achieve the above object, the present invention provides a preparation method of cerium oxide micro-nano particles with a multi-level structure, including the following steps:

[0005] Step 1, preparation of template 3-aminophenol-formaldehyde spheres; The extended method is applicable to the preparation of spherical APF resins with uniform particle sizes:

[0006] 3-aminophenol and formaldehyde are added to a mixed solution of ammonia water, deionized water and ethanol, and stirred to obtain a mixture; then, the mixture is transferred to a high-pressure reaction kettle for hydrothermal reaction; after naturally cooling to room temperature, the spherical APF resin is obtained by centrifugation, washed with water and ethanol respectively, and dried in an oven for standby;

[0007] Step 2, growth of cerium oxide on APF to obtain APF-CeO2:

[0008] Disperse the synthesized spherical APF resin in ethanol and perform ultrasonic dispersion to obtain a well-dispersed APF solution; dissolve cerium nitrate hexahydrate and hexamethylenetetramine in deionized water, and then mix with the APF solution; stir the mixed solution; terminate the reaction by centrifugation and wash with ethanol and water respectively, and then dry in an oven to obtain the APF-CeO2 for standby;

[0009] Step 3: Remove the APF template to obtain multi-level structured cerium oxide micro-nano particles:

[0010] Heat the prepared APF-CeO2 in a muffle furnace to obtain the cerium oxide micro-nano particles.

[0011] Furthermore, the cerium oxide micro-nano particles are of multi-level structure and have a hollow structure with surface wrinkles, and the diameter is about 350 nanometers.

[0012] Furthermore, in Step 2, stir the mixed solution at room temperature for 2 hours, and then stir at 75 °C for another 4 hours.

[0013] The present invention provides the application of the above-mentioned cerium oxide micro-nano particles in the detection of small molecule metabolites, including the following steps:

[0014] Step A: Dilute the urine sample with deionized water, then spot it on the target plate and let it dry naturally;

[0015] Step B: Disperse the cerium oxide micro-nano particles in deionized water to form a matrix solution, and cover and spot it on the target plate where the urine sample is spotted in Step A to obtain the sample to be detected;

[0016] Step C: Perform MALDI mass spectrometry detection on the fingerprint spectrum of the sample to be detected;

[0017] Step D: Analyze the MALDI mass spectrometry detection results to draw a conclusion.

[0018] Furthermore, the biological small molecules in the urine sample include sugars and amino acids.

[0019] Furthermore, in Step C, the MALDI mass spectrometry detection uses the reflection mode, positive ion detection, and the detection range is set to 100 - 400 Da.

[0020] Furthermore, in Step A, the urine sample needs to be diluted 5 times with the deionized water, and 1 μL is taken and spotted on the target plate.

[0021] Furthermore, the concentration of the matrix solution in Step B is 1 mg / mL.

[0022] The present invention also provides the application of the above-mentioned cerium oxide micro-nano particles in the identification of small molecule metabolites, including the following steps:

[0023] Step a: Mix 1 mg / mL cysteine and hydroquinone in an equimolar ratio;

[0024] Step b: Disperse the cerium oxide micro-nano particles in deionized water to form a matrix solution with a concentration of 1 mg / mL;

[0025] Step c: For sample preparation, use sandwich spotting. Spot 1 μL of the 1 mg / mL matrix solution and let it dry naturally; then cover and spot 1 μL of the mixed sample in step a and let it dry naturally; finally, cover and spot 1 μL of the 1 mg / mL matrix solution again and let it dry naturally to obtain the sample to be identified in-source;

[0026] Step d: Perform MALDI mass spectrometry detection on the fingerprint spectrum of the sample to be identified in-source;

[0027] Step e: Analyze the MALDI mass spectrometry detection results to draw a conclusion.

[0028] Further, in step d, the MALDI mass spectrometry detection uses the reflection mode, positive ion detection, and the detection range is set to 100 - 400 Da.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] The cerium oxide micro-nano particle matrix synthesized in the present invention has a low preparation cost and simple synthesis steps. Using this micro-nano particle as the matrix material in mass spectrometry can solve the problems existing in traditional organic matrices, such as background interference and hot spot effect in the small molecule segment. In addition, in the present invention, urine samples only need to be diluted without complex sample pretreatment processes. The detection method of the present invention is non-invasive, has a high detection throughput, high sensitivity, and low cost, meets the clinical requirements for obtaining metabolic fingerprint spectra, realizes clinical applications such as diagnosis, prognosis, and biomarker screening, and has great application potential in the in-depth interpretation of processes such as the growth and development of organisms and the pathogenic mechanism at the metabolomics level.

[0031] The following will further illustrate the concept, specific structure, and technical effects of the present invention with reference to the accompanying drawings to fully understand the purpose, features, and effects of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 is the SEM characterization picture of the cerium oxide micro-nano particles prepared in the present invention;

[0033] Figure 2 is the mass spectrometry diagram of the present invention for matrix-assisted laser desorption / ionization time-of-flight mass spectrometry detection of the small molecular weight end of urine;

[0034] Figure 3 This is a schematic diagram of the results of the present invention for realizing the clinical prognosis prediction of medulloblastoma patients by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry;

[0035] Figure 4 This is a schematic diagram of the screening and identification of small molecules containing sulfhydryl groups by the present invention for matrix-assisted laser desorption / ionization time-of-flight mass spectrometry detection. Detailed implementation manners

[0036] The following introduces multiple preferred embodiments of the present invention with reference to the accompanying drawings of the specification to make its technical content clearer and easier to understand. The present invention can be embodied in many different forms of embodiments, and the protection scope of the present invention is not limited to the embodiments mentioned in the text.

[0037] Example 1 Preparation of cerium oxide micro / nano particle matrix

[0038] The preparation method of the cerium oxide micro / nano particle matrix includes the following steps:

[0039] Step 1. Preparation of the template 3-aminophenol-formaldehyde (APF) spheres; Extended The method is applicable to the preparation of spherical APF resins with uniform particle sizes:

[0040] Specifically, 2 g of 3-aminophenol and 2.8 mL of formaldehyde are added to a mixed solution of ammonia water solution (0.5 mL), deionized water (100 mL) and ethanol (40 mL), and stirred at 30 °C for 24 hours; then, the mixture is transferred to a high-pressure reaction kettle, the hydrothermal temperature is 100 °C, and the reaction is carried out for 24 hours; after naturally cooling to room temperature, the required APF is obtained by centrifugation, washed 3 times with water and ethanol respectively, and dried in an oven at 60 °C for standby.

[0041] Step 2. Growth of cerium oxide on APF (APF-CeO2):

[0042] Disperse 200 mg of the APF resin spheres synthesized in Step 1.1 in 30 mL of ethanol, and ultrasonically disperse for at least 30 minutes to obtain a well-dispersed APF solution; dissolve 0.065 g of cerium nitrate hexahydrate and 0.065 g of hexamethylenetetramine (HMT) in 30 mL of deionized water, and then mix with the APF solution; further stir the mixed solution at room temperature for 2 hours, and then stir at 75 °C for 4 hours; terminate the reaction by centrifugation and wash 3 times with ethanol and water respectively, and then obtain the APF-CeO2 sample by drying in an oven at 80 °C for standby;

[0043] Step 3. Remove the APF template to obtain a multi-level structure of cerium oxide micro / nano particles:

[0044] The prepared APF-CeO2 sample was heated to 450 °C in a muffle furnace at a heating rate of 5 °C / min and placed at 450 °C for 2 hours to obtain multi-structured cerium oxide micro-nano particles.

[0045] For the prepared cerium oxide micro-nano particle matrix, scanning electron microscope results were obtained using a NERCN-TC-006 field emission scanning electron microscope, as Figure 1 shown.

[0046] As Figure 1 can be seen, the diameter of the prepared cerium oxide micro-nano particles is about 350 nm. From the Figure 1 scanning electron microscope results, it can be seen that the size of the synthesized cerium oxide micro-nano particle matrix is uniform and the surface is rough.

[0047] Example 2: Detection of small molecule metabolites in urine based on multi-structured cerium oxide micro-nano particles

[0048] The steps for detecting small molecule metabolites in urine based on multi-structured cerium oxide micro-nano particles are as follows:

[0049] Step A: The urine sample needs to be diluted 5 times with deionized water. Take 1 μL and spot it on the target plate, and let it dry naturally;

[0050] Step B: Disperse the cerium oxide micro-nano particles in deionized water to form a matrix solution with a concentration of 1 mg / mL, cover the spot coating, and prepare the sample to be detected;

[0051] Step C: Perform metabolic fingerprint detection based on laser desorption ionization mass spectrometry on the sample to be detected; among them, the mass spectrometry detection uses the reflection mode, positive ion detection, and the detection range is set to 100 - 400 Da. The specific parameters are: laser wavelength 355 nm, laser frequency 2 kHz; acceleration voltage is 20 kV, and the repetition rate of delayed extraction is 1 kHz; the delay time is 150 ns; each analysis is superimposed with 2000 laser irradiations.

[0052] Step D: Analyze the mass spectrometry detection results to draw a conclusion, and the detection results are as Figure 2 shown.

[0053] As Figure 2 can be seen, the present invention can efficiently and rapidly detect and analyze small molecule substances in urine samples. This detection method is completely non-invasive, highly sensitive, low-cost, and has a high detection throughput, meeting the need for obtaining high-throughput fingerprints of small molecule substances in metabolomics in clinical practice.

[0054] Example 3: Prognosis prediction analysis of medulloblastoma patients based on multi-structured cerium oxide micro-nano particles

[0055] The prognostic prediction analysis steps for patients with medulloblastoma using cerium oxide micro-nano particles based on a multi-level structure are as follows:

[0056] Step A): According to the steps in Example 2, collect the metabolic fingerprints of the urine samples of the patients to be analyzed;

[0057] Step B): Divide the patients into a training set and a validation set;

[0058] Step C): Use the proportional hazards regression model on SPSS to perform multivariate regression analysis on the metabolic fingerprint signals in the training set, and set the threshold of the p-value parameter to 0.05 to obtain the corresponding coefficients of all metabolic features. The metabolic prognosis score is formed by summing the products of each feature and its coefficient, which serves as the metabolic risk score for each sample;

[0059] Step D): Using the median of the metabolic prognosis scores of the training set samples as the threshold, divide all samples into a high-risk group and a low-risk group;

[0060] Step E): Perform survival analysis on the Orange 3.25.0 software to study the difference in progression-free survival between the high-risk and low-risk groups of the training set and the validation set. The analysis results are as Figure 3 shown.

[0061] As Figure 3 can be seen, the cerium oxide micro-nano particles based on the multi-level structure invented by us can achieve urine metabolic fingerprint analysis based on laser desorption ionization mass spectrometry and can perform prognostic prediction analysis on patients with medulloblastoma according to metabolic features, which helps to achieve metabolic risk stratification for patients and guide personalized medicine.

[0062] Example 4: Application of cerium oxide micro-nano particles based on a multi-level structure in the identification of thiol-containing small molecule metabolites

[0063] Step a: Mix cysteine at 1 mg / mL with hydroquinone in an equimolar ratio;

[0064] Step b: Disperse the cerium oxide micro-nano particles in deionized water to form a matrix solution with a concentration of 1 mg / mL;

[0065] Step c: For sample preparation, use sandwich spotting. After spotting 1 μL of the 1 mg / mL matrix solution, let it dry naturally; then cover and spot 1 μL of the mixed sample in step a and let it dry naturally; finally, cover and spot 1 μL of the 1 mg / mL matrix solution again and let it dry naturally to obtain the sample to be identified in-source;

[0066] Step d: Perform metabolic fingerprint detection on the in-source identification sample based on laser desorption ionization mass spectrometry; in which the mass spectrometry detection uses the reflection mode, positive ion detection, and the detection range is set to 100 - 400 Da. The specific parameters are: laser wavelength 355 nm, laser frequency 2 kHz; acceleration voltage 20 kV, repetition rate of delayed extraction 1 kHz; delay time 150 ns; 2000 laser irradiations are superimposed for each analysis.

[0067] Step e: Analyze the mass spectrometry detection results and draw a conclusion. The detection results are as Figure 4 shown.

[0068] As can be seen from Figure 4 , the present invention can achieve specific recognition and identification of thiol small molecules through in-source addition reaction. The existence of specific migration peaks of thiol small molecules can achieve the identification of thiol small molecules in complex samples, which is crucial for the accurate identification of metabolic small molecules.

[0069] The preferred specific embodiments of the present invention have been described in detail above. It should be understood that those of ordinary skill in the art can make many modifications and variations according to the concept of the present invention without creative work. Therefore, all technical solutions that can be obtained by those skilled in the art in the technical field based on the concept of the present invention through logical analysis, reasoning, or limited experiments on the basis of the prior art should be within the protection scope determined by the claims.

Claims

1. Application of cerium oxide micro-nano particles in the detection of small molecule metabolites, characterized in that, It includes the following steps: Step A: After diluting the urine sample with deionized water, spot it on the target plate and let it dry naturally. Step B: Disperse the cerium oxide micro-nano particles in deionized water to form a matrix solution, and cover and spot it on the target plate where the urine sample was spotted in Step A to obtain the sample to be detected. Step C: Perform MALDI mass spectrometry detection on the fingerprint spectrum of the sample to be detected. Step D: Analyze the MALDI mass spectrometry detection results to draw a conclusion. The preparation method of cerium oxide micro-nano particles includes the following steps: Step 1, Preparation of template 3-aminophenol-formaldehyde spheres; Extended The method is applicable to the preparation of spherical APF resins with uniform particle sizes: Add 3-aminophenol and formaldehyde to the mixed solution of ammonia water solution, deionized water and ethanol, and stir to obtain a mixture; then transfer the mixture to a high-pressure reaction kettle for hydrothermal reaction; after naturally cooling to room temperature, obtain the spherical APF resin by centrifugation, wash it with water and ethanol respectively, and dry it in an oven for standby. Step 2: Cerium oxide grows on APF to obtain APF-CeO2: Disperse the synthesized spherical APF resin in ethanol and ultrasonically disperse it to obtain a well-dispersed APF solution; dissolve cerium nitrate hexahydrate and hexamethylenetetramine in deionized water, and then mix it with the APF solution. Stir the mixed solution; terminate the reaction by centrifugation and wash it with ethanol and water respectively, and then dry it in an oven to obtain the APF-CeO2 for standby. Step 3: Remove the APF template to obtain multi-level structured cerium oxide micro-nano particles: Heat the prepared APF-CeO2 in a muffle furnace to obtain the cerium oxide micro-nano particles.

2. The application of the cerium oxide micro-nano particles as described in claim 1 in the detection of small molecule metabolites, characterized in that, The biological small molecules in the urine sample include sugars and amino acids.

3. The application of the cerium oxide micro-nano particles as described in claim 1 in the detection of small molecule metabolites, characterized in that, In Step C, the MALDI mass spectrometry detection adopts the reflection mode, positive ion detection, and the detection range is set to 100 - 400 Da.

4. The application of the cerium oxide micro-nano particles as described in claim 1 in the detection of small molecule metabolites, characterized in that, In Step A, the urine sample needs to be diluted 5 times with the deionized water, and 1 μL is taken and spotted on the target plate.

5. The application of the cerium oxide micro-nano particles as described in claim 1 in the detection of small molecule metabolites, characterized in that, The concentration of the matrix solution in Step B is 1 mg / mL.

6. Use of cerium oxide micro-nano particles in the identification of small molecule metabolites, characterized in that, It includes the following steps: Step a: Mix 1 mg / mL cysteine and hydroquinone in an equimolar ratio. Step b: Disperse the cerium oxide micro-nano particles in deionized water to form a matrix solution with a concentration of 1 mg / mL. Step c: The sample preparation adopts sandwich spotting. After spotting 1 μL of the 1 mg / mL matrix solution, let it dry naturally; cover and spot 1 μL of the mixed sample in Step a and let it dry naturally; finally, cover and spot 1 μL of the 1 mg / mL matrix solution and let it dry naturally to obtain the sample to be identified in-source. Step d: Perform MALDI mass spectrometry detection on the fingerprint spectrum of the sample to be identified in-source. Step e: Analyze the MALDI mass spectrometry detection results to draw a conclusion. The preparation method of cerium oxide micro-nano particles includes the following steps: Step 1, Preparation of the template 3-aminophenol-formaldehyde spheres; The extended method is applicable to the preparation of spherical APF resins with uniform particle sizes: Add 3-aminophenol and formaldehyde to the mixed solution of ammonia water solution, deionized water and ethanol, and stir to obtain a mixture; then transfer the mixture to a high-pressure reaction kettle for hydrothermal reaction; after naturally cooling to room temperature, obtain the spherical APF resin by centrifugation, wash it with water and ethanol respectively, and dry it in an oven for standby. Step 2: Cerium oxide grows on APF to obtain APF-CeO2: Disperse the synthesized spherical APF resin in ethanol and ultrasonically disperse it to obtain a well-dispersed APF solution; dissolve cerium nitrate hexahydrate and hexamethylenetetramine in deionized water, and then mix it with the APF solution; Stir the mixed solution; terminate the reaction by centrifugation and wash it with ethanol and water respectively, and then dry it in an oven to obtain the APF-CeO2 for standby; Step 3, remove the APF template to obtain multi-level structured cerium oxide micro-nano particles: Heat the prepared APF-CeO2 in a muffle furnace to obtain the cerium oxide micro-nano particles.

7. Use of the cerium oxide micro-nano particles according to claim 6 in identification of small molecule metabolites, characterized in that, In step d, the MALDI mass spectrometry detection adopts the reflection mode, positive ion detection, and the detection range is set to 100-400 Da.

8. The application according to any one of claims 1 or 6, characterized in that The cerium oxide micro-nano particles are multi-level structured and have a hollow structure with surface wrinkles, and the diameter is 350 nanometers.

9. The application according to any one of claims 1 or 6, characterized in that In step 2, stir the mixed solution at room temperature for 2 hours, and then stir it at 75 °C for another 4 hours.

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