A method for analyzing aqueous humor based on titanium oxide nanomaterial matrix

Abstract

The present invention proposes a method for analyzing aqueous humor based on a titanium oxide nanomaterial matrix, and the specific steps are as follows: titanium oxide nanomaterials and deionized water are configured into a material dispersion solution, wherein the mass volume ratio of titanium oxide nanomaterials to deionized water is in the range of 1-2 g / L, 1-2 μL of aqueous humor sample is deposited on a target plate, and after the sample is dried, 1-2 μL of material dispersion solution is applied thereon, and then the sample is subjected to MALDI-TOF-MS mass spectrometry analysis to obtain a metabolic fingerprint map, and then the method of machine learning is used to distinguish patients with cataracts related to different diseases. The present invention realizes the collection of metabolic fingerprint maps of aqueous humor samples by preparing a binary laser / desorption ionization matrix with high ionization efficiency, and can quickly distinguish cataracts related to senility, diabetes, and high myopia by combining the means of machine learning, and has broad application prospects for distinguishing cataracts related to different diseases and exploring the molecular mechanism of cataracts.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and specifically relates to an aqueous humor analysis method based on a titanium oxide nano material matrix, and in particular to an aqueous humor analysis method based on a titanium oxide nano material matrix modified by gold nanoparticles. Background Art

[0002] Aqueous humor is the liquid that fills the space between the cornea and the iris lens (anterior chamber) of the human eye. Under normal circumstances, it is colorless and transparent, and its amount and composition are relatively stable. Aqueous humor is a transparent liquid surrounding the anterior and posterior chambers of the lens, and plays an important role in maintaining intraocular pressure, providing nutrients and antioxidants to the lens, and removing metabolic waste from the lens.

[0003] Cataract is a blinding eye disease with multiple symptoms such as lens protein denaturation and metabolic disorders. Its causes include age, high myopia, diabetes, poisoning and radiation. As of 2020, there are more than 15.3 million cases of cataracts worldwide, ranking first among the causes of blindness. When cataracts and other eye diseases occur, the composition, properties, production and discharge of aqueous humor will change significantly. At this time, the analysis of aqueous humor composition is of great significance.

[0004] Metabolomics is an emerging discipline following genomics and proteomics. Unlike DNA and protein, which are limited by epigenetic events and post-translational modifications, metabolites are the final products of biological metabolism and are directly related to phenotypes. Preliminary studies have found that metabolite abnormalities appear in the aqueous humor of cataract patients with diabetes or high myopia, indicating that the aqueous humor may contain useful information that can reflect the lesions related to cataract patients at the metabolic level.

[0005] Mass spectrometry (MS) has been widely used in metabolomics. In recent years, a large number of metabolomics analysis methods based on laser desorption / ionization mass spectrometry (LDI-MS) have been reported. Compared with traditional gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry, laser desorption / ionization mass spectrometry has the advantages of high throughput, high analysis speed and simple sample preparation, and has shown great potential in metabolomics. The most important feature of LDI-MS is the design of the matrix. Currently, many materials are designed for MALDI matrices, such as carbon materials, metal organic frameworks, noble metals, and metal oxides. Compared with traditional matrices (such as CHCA and DHB), they have obvious background elimination capabilities. However, the insufficient ionization ability of these nanomaterials for metabolites leads to the inability to detect metabolites in low-abundance body fluid samples. Secondly, a single material cannot achieve global metabolite coverage. Therefore, it is of great potential to apply composite materials with high ionization efficiency to the analysis of metabolites. Summary of the invention

[0006] The purpose of the present invention is to provide an aqueous humor analysis method based on a titanium oxide nanomaterial matrix.

[0007] The present invention proposes an aqueous humor analysis method based on a titanium oxide nanomaterial matrix, which specifically comprises the following steps: preparing a material dispersion solution with titanium oxide nanomaterial and deionized water, wherein the mass volume ratio of titanium oxide nanomaterial to deionized water is 1-2 g / L, taking 1-2 μL of aqueous humor sample and depositing it on a target plate, and after the sample is dried, spotting 1-2 μL of the material dispersion solution on it, and then entering MALDI-TOF-MS mass spectrometry analysis;

[0008] The titanium oxide nanomaterial is a multilayer titanium oxide binary matrix material modified by gold nanoparticles.

[0009] In the present invention, after the metabolic spectrum obtained by the aqueous humor sample entering the MALDI-TOF-MS mass spectrometry analysis is extracted by software, the same aqueous humor sample is averaged and peak aligned to establish an m / z signal matrix, and then the matrix data is imported into the SIMCA software to establish a partial least squares discriminant analysis model.

[0010] In the present invention, the synthesis method of the multilayer titanium oxide matrix material modified by gold nanoparticles comprises the following specific steps:

[0011] (1) adding the polyoxyethylene polyoxypropylene ether triblock copolymer to a tetrahydrofuran solution, stirring thoroughly to dissolve, adding acetic acid and concentrated hydrochloric acid, stirring thoroughly for about 10 minutes, adding tetrabutyl titanate to the system, reacting for about half an hour, pouring the clear yellow-white solution into two weighing bottles, and drying in a vacuum drying oven at 40° C.;

[0012] (2) The product obtained in step (1) was uniformly dispersed in anhydrous ethanol, and vigorously stirred until completely dissolved. Then, about 15 mL of glycerol was added and stirred for 10 min. The resulting mixed solution was transferred to an autoclave, heated at 100 ° C for 10 h, cooled to room temperature, and the precipitate was collected, washed with deionized water and anhydrous ethanol, and dried in a vacuum drying oven to obtain a white solid;

[0013] (3) calcining the product obtained in step (2) in a muffle furnace at 350° C. for 3 hours;

[0014] (4) dispersing the product obtained in step (3) in a chloroauric acid solution, heating it to a giant boil, reducing it with trisodium citrate, collecting a purple-red solid, washing the obtained product with deionized water and ethanol, and drying it in a vacuum drying oven to obtain the gold nanoparticle-modified titanium oxide binary matrix material.

[0015] The beneficial effects of the present invention are:

[0016] 1. The multilayer titanium oxide matrix modified with gold nanoparticles is a composite matrix that combines the excellent ultraviolet absorption ability of titanium oxide materials and gold nanoparticles, and can efficiently ionize small molecule metabolites;

[0017] 2. In the structure of the multilayer titanium oxide matrix modified with gold nanoparticles, the structure of the titanium oxide material of the sheet is stretchable and can load a large number of gold nanoparticles. Compared with titanium oxide materials with other structures, the loading capacity is greatly increased, which can improve the adsorption and ionization efficiency of the material for small molecule metabolites;

[0018] 3. Multilayer titanium oxide matrix material modified with gold nanoparticles was used for the first time to study the metabolites of aqueous humor samples from cataract patients. Combined with MALDI mass spectrometry analysis of aqueous humor metabolites and machine learning algorithms, it can quickly, efficiently and accurately distinguish cataracts related to different diseases, which is of great significance for the early diagnosis and classification of the disease.

[0019] The present invention proposes a highly efficient ionization MALDI small molecule matrix for the analysis of aqueous humor metabolites and the differentiation of cataracts associated with different diseases. For the first time, a multilayer titanium oxide nanomaterial modified with gold nanoparticles with high ionization efficiency is synthesized and used for the analysis of aqueous humor samples of cataract patients. In the analysis of MALDI mass spectrometry, the material can realize the rapid acquisition of metabolic fingerprints of aqueous humor samples, greatly shortening the time of traditional metabolomics analysis and detection. At the same time, the metabolic fingerprint of the aqueous humor sample obtained is combined with machine learning, which can quickly, efficiently and accurately realize the differentiation of cataracts associated with different diseases, including senile cataracts, diabetic cataracts and high myopia cataracts. The present invention aims to provide a novel method for synthesizing a highly efficient ionization MALDI small molecule matrix and its application in the diagnosis and typing of diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a scanning electron microscope photograph of the multilayer titanium oxide matrix material modified with gold nanoparticles of Example 1;

[0021] Figure 2 This is a transmission electron micrograph of the multilayer titanium oxide matrix material modified with gold nanoparticles of Example 1;

[0022] Figure 3 The X-ray powder diffraction results of the multilayer titanium oxide and gold nanoparticle-modified multilayer titanium oxide matrix material of Example 1;

[0023] Figure 4 is the nitrogen adsorption isotherm of the multilayer titanium oxide matrix material modified with gold nanoparticles in Example 1;

[0024] Figure 5The UV-visible absorption spectrum of the multilayer titanium oxide matrix material modified with the multilayer titanium oxide and gold nanoparticles of Example 1;

[0025] Figure 6 The mass spectrum of the multilayer titanium oxide matrix material modified with gold nanoparticles in Example 1 for analyzing a mixed small molecule metabolite standard sample;

[0026] Figure 7 The metabolic fingerprints of the multilayer titanium oxide matrix material modified with gold nanoparticles in Example 2 were obtained by analyzing aqueous humor samples of cataracts related to three different diseases. Figure A is the metabolic fingerprint of aqueous humor related to senile cataracts, Figure B is the metabolic fingerprint of aqueous humor related to diabetes-related cataracts, and Figure C is the metabolic fingerprint of aqueous humor related to high myopia;

[0027] Figure 8 In order to utilize the aqueous humor metabolic fingerprints of the three different disease-related cataracts in Example 2, a partial least squares discriminant analysis was established to distinguish between age-related cataracts, diabetes-related cataracts and high myopia-related cataracts. DETAILED DESCRIPTION

[0028] The present invention utilizes multilayer titanium oxide nanomaterials modified with gold nanoparticles to analyze aqueous humor samples of age-related cataracts, diabetes-related cataracts, and high myopia-related cataracts, and distinguishes these three groups of cataracts related to different diseases by establishing a machine learning model. The specific implementation method is introduced below.

[0029] Example 1: Synthesis of multilayer titanium oxide nanomaterials modified with gold nanoparticles and analysis of standard products

[0030] (1) adding the polyoxyethylene polyoxypropylene ether triblock copolymer to a tetrahydrofuran solution, stirring thoroughly to dissolve, adding acetic acid and concentrated hydrochloric acid, stirring thoroughly for 10 minutes, adding tetrabutyl titanate to the system, reacting for about 30 minutes, pouring the clear yellow-white solution into two weighing bottles, and drying in a vacuum drying oven at 40°C;

[0031] (2) The product obtained in step (1) was uniformly dispersed in anhydrous ethanol, and vigorously stirred until completely dissolved. Then, 15 mL of glycerol was added and stirred for 10 min. The resulting mixed solution was transferred to an autoclave, heated at 100 ° C for 10 h, cooled to room temperature, and the precipitate was collected, washed with deionized water and anhydrous ethanol, and dried in a vacuum drying oven to obtain a white solid;

[0032] (3) calcining the product obtained in step (2) in a muffle furnace at 350° C. for 3 hours;

[0033] (4) dispersing the product obtained in step (3) in a chloroauric acid solution, heating it to a giant boil, reducing it with trisodium citrate, collecting a purple-red solid, washing the obtained product with deionized water and ethanol, and drying it in a vacuum drying oven to obtain the gold nanoparticle-modified multilayer titanium oxide binary matrix material;

[0034] (5) The prepared gold nanoparticle-modified multilayer titanium oxide binary matrix material was detected by scanning electron microscopy. The detection conditions were: the material was dispersed in anhydrous ethanol, sampled on a copper mesh, and observed under a scanning electron microscope at a scale of 1 micron under a working voltage of 15 kV and vacuum. The detection results were as follows: Figure 1 As shown;

[0035] (6) The prepared gold nanoparticle-modified titanium oxide binary matrix material was tested by transmission electron microscopy. The test conditions were as follows: a small amount of dry material was evenly dispersed in anhydrous ethanol on a copper mesh at a working voltage of 200 kV. After drying, the sample was inserted into the instrument and vacuumed. The transmission electron microscopy image was observed at a scale of 1 micron. The test results are shown in FIG. Figure 2 As shown;

[0036] (7) eight standard amino acids, including arginine, creatine, phenylalanine, aspartic acid, glucose, methionine, histidine and valine, were prepared into a mixed solution (each single amino acid was first prepared at 20 mM, 50 uL of each of the eight amino acids was diluted and mixed together, and then deionized water was added to prepare a mixed solution with a concentration of 1 mM);

[0037] (8) Multilayer titanium oxide and multilayer titanium oxide modified with gold nanoparticles were dispersed in a deionized water solution to prepare 1 mg.mL -1 dispersion of materials;

[0038] (9) Mass spectrometry analysis: Take 1-2 μL of the standard solution mixture in step (7) and apply it to the target plate. After natural drying, cover it with a layer of material dispersion solution. After natural drying, perform mass spectrometry analysis. The mass spectrum is shown in Figure 6.

[0039] Analysis results: Figure 6 It can be seen that multilayer titanium oxide and multilayer titanium oxide modified with gold nanoparticles have excellent ionization efficiency for mixed standard samples, and multilayer titanium oxide modified with gold nanoparticles has enhanced ionization effect for mixed standard samples.

[0040] Example 2 Analysis of actual samples

[0041] (1) preparing a gold nanoparticle-modified multilayer titanium oxide binary matrix material according to steps (1) to (4) of Example 1;

[0042] (2) Preparation of aqueous humor sample: Add 100 μL of deionized water to 100 μL of aqueous humor sample for dilution;

[0043] (3) Mass spectrometry analysis: 1-2 μL of the three diluted aqueous humor samples (senile cataract aqueous humor samples, diabetes aqueous humor samples, and high myopia aqueous humor samples) in step (2) were taken and spotted on the targets respectively. After natural drying, 1-2 μL of the prepared gold nanoparticle-modified multilayer titanium oxide was covered thereon. After natural drying, MALDI analysis was performed (mass spectrometry experimental conditions were: positive ion mode, laser intensity of 70%). The mass spectrum is shown in FIG. Figure 7 As shown;

[0044] Analysis results: Figure 7 It can be seen that the gold nanoparticle-modified titanium oxide matrix material has excellent ionization efficiency for the metabolites of the three aqueous humor samples;

[0045] (4) A total of 183 cataract aqueous humor samples were collected in this experiment, including 67 senile-related, 51 diabetes-related, and 65 high myopia-related. Each aqueous humor sample was subjected to four target tests to obtain a metabolic fingerprint. The raw data of the metabolic spectrum of the aqueous humor samples were extracted using the Flexanalysis 3.4 software. The MALDI quantR language package was used to average and align the peaks of the same aqueous humor sample to establish an m / z signal matrix. The matrix data was then imported into the SIMCA software. The partial least squares discriminant analysis (OPLS-DA) model was established to diagnose the three groups of aqueous humor samples, as shown in Figure 3. Figure 8 shown.

[0046] Analysis results: Figure 8 It can be seen that in the orthogonal partial least squares discriminant analysis (OPLS-DA) model, the three groups of aqueous humor samples can be divided into different areas, and the differences between each group of samples are very small and can achieve good clustering. Secondly, the prediction value (Q 2 ) is 0.923, which proves that the model can quickly and accurately distinguish cataracts related to three different diseases.

Claims

1. A method for analyzing aqueous humor based on a titanium oxide nanomaterial matrix, characterized in that: The specific steps are as follows: Titanium oxide nanomaterials and deionized water are prepared into a material dispersion solution, wherein the mass volume ratio of titanium oxide nanomaterials to deionized water is 1-2 g / L, 1-2 μL of aqueous humor sample is deposited on the target plate, and after the sample is dried, 1-2 μL of the material dispersion solution is applied thereon, and then the sample is subjected to MALDI-TOF-MS mass spectrometry analysis; The titanium oxide nanomaterial is a multilayer titanium oxide binary matrix material modified by gold nanoparticles; the synthesis steps are as follows: (1) adding the polyoxyethylene polyoxypropylene ether triblock copolymer to a tetrahydrofuran solution, stirring thoroughly to dissolve, adding acetic acid and concentrated hydrochloric acid, stirring thoroughly, adding tetrabutyl titanate, reacting for 20-40 minutes, and drying in a vacuum drying oven at 40° C.; (2) the product obtained in step (1) is uniformly dispersed in anhydrous ethanol, stirred until completely dissolved, and then glycerol is added, stirred sufficiently, transferred to an autoclave, heated at 100° C. for 10 hours, cooled to room temperature, and the precipitate is collected, washed sufficiently with deionized water and anhydrous ethanol, and dried in a vacuum drying oven; (3) calcining the product obtained in step (2) in a muffle furnace at 350° C. for 3 hours; (4) dispersing the product obtained in step (3) in a chloroauric acid solution, heating it to a giant boil, reducing it with trisodium citrate, washing it with deionized water and ethanol, and drying it in a vacuum drying oven to obtain the gold nanoparticle-modified multilayer titanium oxide binary matrix material.

2. The aqueous humor analysis method based on titanium oxide nanomaterial matrix according to claim 1, characterized in that: After the metabolic spectrum obtained by MALDI-TOF-MS mass spectrometry analysis of aqueous humor samples was extracted by software, the same aqueous humor samples were averaged and peak aligned to establish the m / z signal matrix, and then the matrix data was imported into SIMCA software to establish the partial least squares discriminant analysis model.

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