阅读说明：本技术 一种多孔合金纳米材料的制备方法及其在检测血浆代谢物中的应用 (Preparation method of porous alloy nano material and application of porous alloy nano material in detection of plasma metabolites ) 是由 钱昆 苏海洋 黄琳 于 2021-07-27 设计创作，主要内容包括：本发明公开了一种多孔合金纳米材料的制备方法及其在检测血浆代谢物中的应用,涉及多孔合金纳米材料合成领域,制备方法包括以下步骤：将Na-(2)PdCl-(4)、H-(2)PtCl-(6)·6H-(2)O、盐酸和F127充分混合,超声溶解；完全溶解后,加入抗坏血酸溶液,并立刻置于水浴超声反应；随后加入HAuCl-(4)·4H-(2)O溶液反应；最后分别用无水乙醇和水离心洗涤后干燥。多孔合金纳米材料作为基质辅助激光解吸电离质谱的基质应用于小分子检测。本发明通过一步法即可合成,步骤简单、成本低；作为激光解吸电离质谱的基质,可以解决传统有机基质的热点效应以及在低分子量区有干扰的问题；血浆样本只需经过简单的预处理,准确率高、成本低、检测通量高。(The invention discloses a preparation method of a porous alloy nano material and application thereof in detecting plasma metabolites, and relates to the field of synthesis of porous alloy nano materials, wherein the preparation method comprises the following steps: mixing Na 2 PdCl 4 、H 2 PtCl 6 ·6H 2 Fully mixing O, hydrochloric acid and F127, and dissolving by ultrasonic; after complete dissolution, adding an ascorbic acid solution, and immediately placing in a water bath for ultrasonic reaction; followed by HAuCl addition 4 ·4H 2 Reacting in an O solution; finally, the mixture is centrifugally washed by absolute ethyl alcohol and water respectively and then dried. The porous alloy nano material is used as a matrix of matrix-assisted laser desorption ionization mass spectrometry for small molecule detection. The invention can be synthesized by a one-step method, and has simple steps and low cost; the organic matrix can be used as a matrix of laser desorption ionization mass spectrometry, and can solve the problems of hot spot effect and interference in a low molecular weight region of the traditional organic matrix; plasma sampleOnly simple pretreatment is needed, the accuracy is high, the cost is low, and the detection flux is high.)

1. A preparation method of a porous alloy nano material is characterized by comprising the following steps:

step 1, adding Na₂PdCl₄、H₂PtCl₆·6H₂Fully mixing O, hydrochloric acid and F127, and dissolving by ultrasonic;

step 2, after complete dissolution, adding an ascorbic acid solution, and immediately placing in a water bath for ultrasonic reaction;

step 3, subsequent addition of HAuCl₄·4H₂Reacting in an O solution;

and 4, finally, respectively carrying out centrifugal washing by using absolute ethyl alcohol and water, and drying to obtain the porous alloy nano material PdPtAu.

2. The method for preparing porous alloy nanomaterial according to claim 1, wherein Na in step 1₂PdCl₄、H₂PtCl₆·6H₂O, hydrochloric acid and F127 respectively using Na with the dosage of 20Mm₂PdCl₄0.6mL of 20Mm H was added₂PtCl₆·6H₂O was added to 3mL, 6.0M hydrochloric acid was added to 60. mu.L, and F127 was added to 60 mg.

3. The method for preparing a porous alloy nanomaterial according to claim 1, wherein the ascorbic acid solution is added in an amount of 3mL in the 0.1M ascorbic acid solution in step 2.

4. The preparation method of the porous alloy nanomaterial as claimed in claim 1, wherein the water bath ultrasonic reaction in the step 2 is a reaction at 45 ℃ for 3 hours.

5. The method for preparing porous alloy nanomaterial according to claim 1, wherein HAuCl is used in step 3₄·4H₂The concentration of the O solution is 10-40 Mm, and the addition amount is 1.2 mL.

6. The method for preparing a porous alloy nanomaterial according to claim 1, wherein the centrifugal washing in step 3 is performed 3 times at 10000rpm at a drying temperature of 50 ℃.

7. Use of a porous alloy nanomaterial according to any of claims 1 to 6 as a matrix for matrix assisted laser desorption ionization mass spectrometry in the detection of plasma metabolites.

8. The use of the porous alloy nanomaterial of claim 7 in the detection of plasma metabolites, comprising the steps of:

step 1, dispersing the porous alloy nano material PdPtAu in deionized water;

step 2, mixing the plasma with a methanol/acetonitrile mixed solution with the same volume ratio of 1:1, shaking the table for 10min, centrifuging for 10min, and taking the supernatant for mass spectrometry;

and 3, preparing a sample on a mass spectrum target plate of the matrix-assisted laser desorption ionization mass spectrum by adopting a positive ion reflection mode, wherein the sample is 1 mu L of plasma extract, and detecting small molecules in the plasma sample.

9. The use of a porous alloy nanomaterial in the detection of plasma metabolites according to claim 8, wherein the small molecules have a molecular weight range of less than 1000 Da.

10. The use of the porous alloy nanomaterial of claim 8 in the detection of plasma metabolites, wherein the small molecule is a carbohydrate or an amino acid.

Technical Field

The invention relates to the field of synthesis of porous alloy nano materials, in particular to a preparation method of a porous alloy and application of the porous alloy in detection of plasma metabolites.

Background

Biomarker (protein, nucleic acid, metabolite, etc.) detection plays an increasingly important role in vitro diagnosis due to its characteristics of being noninvasive, etc. Unlike genes whose function is regulated by epigenetic and proteins that are modified post-translationally, metabolites are direct markers of biochemical activity and are more likely to be associated with phenotypes. Therefore, metabolite analysis, or metabolomics, has become a powerful tool that is increasingly used for in vitro diagnostics. At present, biochemical analysis and a gas chromatography/liquid chromatography mass spectrometry (GC/LC MS) -based combined technology are main means for detecting metabolites, and the methods can realize the detection of the metabolites after pretreatment such as desalting, protein removal, derivatization, concentration and the like are carried out on a sample. Biochemical methods, however, typically detect only one substance at a time and are susceptible to interference from background signals and have low sensitivity and specificity. The GC/LC MS can simultaneously detect various metabolites, and has high sensitivity and strong specificity. However, GC/LC MS has a chromatographic column, needs complex sample pretreatment, and has the problems of long detection time, high price and the like. Low cost, high throughput detection of plasma samples is difficult to achieve and is difficult to use clinically.

In addition, matrix-assisted laser desorption/ionization (MALDI MS) also plays an increasingly important role in metabolite detection, and unlike conventional biochemical analysis and GC/LC MS, MALDI MS requires no or only simple sample pretreatment, and can realize rapid, sensitive and high-throughput detection of metabolites under the premise of a very small amount of samples. The traditional organic matrix has fragmentation peaks in a low molecular weight region, and is not suitable for detecting small molecule metabolites.

The matrix is the core component of MALDI MS. Traditional organic matrices tend to generate strong background signals at the small molecular weight end (m/z <500), which affects the detection of small molecules. In an actual biological sample system, various biological macromolecules exist in a plasma sample, and the detection of the micromolecules is hindered by different pH values and high salinity, so that the traditional organic matrix is difficult to meet the detection requirement of the micromolecules. Although inorganic nano materials (such as carbon-based, silicon-based, noble metal materials, etc.) can be used for detecting small molecule metabolites, there is still a limitation in detecting complex biological samples. Therefore, a new nano material needs to be developed for detecting metabolites of complex biological samples (blood plasma).

Therefore, those skilled in the art have been devoted to developing a MALDI MS matrix with better LDI efficiency for the detection of metabolites in complex biological samples (plasma).

Disclosure of Invention

In view of the above-mentioned drawbacks of the prior art, the technical problem to be solved by the present invention is how to use a MALDI MS matrix with better LDI efficiency for the detection of metabolites in complex biological samples (plasma).

In order to achieve the above object, the present invention provides a method for preparing a porous alloy nanomaterial, the method comprising the steps of:

step 1, adding Na₂PdCl₄、H₂PtCl₆·6H₂Fully mixing O, hydrochloric acid and F127, and dissolving by ultrasonic;

step 2, after complete dissolution, adding an ascorbic acid solution, and immediately placing in a water bath for ultrasonic reaction;

step 3, subsequent addition of HAuCl₄·4H₂Reacting in an O solution;

and 4, finally, respectively carrying out centrifugal washing by using absolute ethyl alcohol and water, and drying to obtain the porous alloy nano material PdPtAu.

Further, step 1 said Na₂PdCl₄、H₂PtCl₆·6H₂O, hydrochloric acid and F127 respectively using Na with the dosage of 20Mm₂PdCl₄Adding into0.6mL of 20Mm H₂PtCl₆·6H₂O was added to 3mL, 6.0M hydrochloric acid was added to 60. mu.L, and F127 was added to 60 mg.

Further, the ascorbic acid solution in step 2 was added in an amount of 3mL of 0.1M ascorbic acid solution.

Further, the water bath ultrasonic reaction in the step 2 is a reaction at 45 ℃ for 3 hours.

Further, the HAuCl in the step 3₄·4H₂The concentration of the O solution is 10-40 Mm, and the addition amount is 1.2 mL.

Further, in step 3, the centrifugal washing times are 3 times, the rotation speed is 10000rpm, and the drying temperature is 50. The application of the porous alloy nano material in detecting plasma metabolites is characterized in that the porous alloy nano material is used as a matrix of matrix-assisted laser desorption ionization mass spectrometry.

Further, the method comprises the following steps:

step 1, dispersing the porous alloy nano material PdPtAu in deionized water;

step 2, mixing the plasma with a methanol/acetonitrile mixed solution with the same volume ratio of 1:1, shaking the table for 10min, centrifuging for 10min, and taking the supernatant for mass spectrometry;

and 3, preparing a sample on a mass spectrum target plate of the matrix-assisted laser desorption ionization mass spectrum by adopting a positive ion reflection mode, wherein the sample is 1 mu L of plasma extract, and detecting small molecules in the plasma sample.

Further, the small molecules have a molecular weight range of less than 1000 Da.

Further, the small molecule is a carbohydrate or an amino acid.

The invention has the following technical effects:

(1) the porous PdPtAu alloy can be synthesized by a one-step method, and has simple synthesis steps and low preparation cost.

(2) The nano material is used as a matrix of laser desorption ionization mass spectrometry, and can solve the problems of hot spot effect of the traditional organic matrix and interference in a low molecular weight region.

(3) In the invention, the plasma sample only needs simple pretreatment, and each sample only needs 1 mu L of plasma extract, so that the micromolecule metabolite in the plasma can be rapidly and sensitively detected. The method has high accuracy, low cost and high detection flux, and meets the requirement of clinical plasma detection.

Drawings

FIG. 1 is a SEM image of a porous PdPtAu nanomaterial according to a preferred embodiment of the invention;

FIG. 2 is a TEM representation of porous PdPtAu nanomaterials according to a preferred embodiment of the invention;

FIG. 3 is a mass spectrum of a standard small molecule detected by matrix-assisted laser desorption ionization mass spectrometry using a porous PdPtAu nanomaterial according to a preferred embodiment of the invention;

FIG. 4 is a mass spectrum of a porous PdPtAu nanomaterial as a matrix-assisted laser desorption ionization mass spectrometry for detecting small molecules containing NaCl and bovine serum albumin according to a preferred embodiment of the invention;

FIG. 5 is a mass spectrum of a porous PdPtAu nanomaterial as a matrix-assisted laser desorption ionization mass spectrometry for detecting small molecule metabolites in a plasma sample according to a preferred embodiment of the invention.

Detailed Description

The technical contents of the preferred embodiments of the present invention will be more clearly and easily understood by referring to the drawings attached to the specification. The present invention may be embodied in many different forms of embodiments and the scope of the invention is not limited to the embodiments set forth herein.

Example 1 preparation of porous alloy nanomaterial

Step 1: preparation of instruments and reagents: matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) in positive ion reflection mode;

step 2: the preparation of the porous PdPtAu alloy comprises the following steps;

step 2.1: 0.6mL of Na₂PdCl₄(20mM)，3mL H₂PtCl₆·6H₂O (20mM), 60. mu.L hydrochloric acid (6.0M) and 60mg F127 were mixed well, and dissolved by sonication until F127 was finishedAfter complete dissolution, 3mL ascorbic acid solution (0.1M) was added, and immediately placed in a water bath at 45 ℃ for ultrasonic reaction for 3h, 1.2mL HAuCl was added₄·4H₂O solution (10 mM, 20mM, 30mM, 40mM, respectively) was reacted for 1 hour. After the reaction, the mixture was washed 3 times with absolute ethanol and water by centrifugation (10000rpm), and dried at 50 ℃ for use.

Step 2.2: dispersing the porous PdPtAu alloy in deionized water to be used as a matrix;

and step 3: dissolving glucose, phenylalanine and lysine in proportion in deionized water, and detecting PdPtAu mass spectrum performances with different apertures and gold contents;

and 4, step 4: mixing plasma with methanol/acetonitrile mixed solution (methanol/acetonitrile, v/v ═ 1:1) of the same volume, shaking the table for 10min, centrifuging for 10min, and taking supernatant for mass spectrum detection;

and 5: preparing a sample on a mass spectrum target plate, wherein the matrix is an optimized PdPtAu material, and drying at room temperature;

step 6: detecting small molecules in the plasma sample;

and 7: and analyzing the mass spectrum detection result to obtain a conclusion.

The results of scanning electron microscopy and energy dispersive X-ray spectroscopy were obtained using Hitachi S-4800, and as shown in fig. 1, the synthesized porous PdPtAu alloy had a particle size of about 170nm, a uniform particle size, and a porous structure.

Transmission electron microscopy results were obtained using JEOL JEM-2100F. As shown in FIG. 2, the nano material has a porous structure, which is consistent with the result of a scanning electron microscope.

Example 2 Standard Small molecule substance detection

(1) Preparation of instruments and reagents: matrix-assisted laser desorption ionization mass spectrometry, and detecting by adopting a cation reflection mode; preparing a porous alloy into a suspension, and preparing a standard small molecule (glucose, phenylalanine and lysine) solution;

(2) preparing a sample on a mass spectrum target plate, and drying at room temperature;

(3) the detection is carried out under a mass spectrometer, and the mass spectrum image is analyzed, the result is shown in fig. 3, and fig. 3a, 3b and 3c are glucose, phenylalanine and lysine standard small molecule solutions respectively.

Example 3 detection of Small molecule substances containing NaCl and bovine serum Albumin

(1) Preparation of instruments and reagents: matrix-assisted laser desorption ionization mass spectrometry, and detecting by adopting a cation reflection mode; preparing a suspension from the porous alloy, and preparing a mixed micromolecule (glucose, phenylalanine and lysine) solution of high-concentration salt or protein;

(2) preparing a sample on a mass spectrum target plate, and drying at room temperature;

(3) the detection is carried out under a mass spectrometer, and the mass spectrum image is analyzed, the result is shown in figure 4, figure 4a is the mass spectrum of detecting glucose, phenylalanine and lysine in the solution containing NaCl, and figure 4b is the mass spectrum of detecting glucose, phenylalanine and lysine in the solution of bovine serum albumin.

Example 4 detection of plasma metabolites

(1) Preparation of instruments and reagents: matrix-assisted laser desorption ionization mass spectrometry, and detecting by adopting a cation reflection mode; preparing the porous alloy into a suspension; simple pretreatment of plasma with organic solvents;

(2) preparing a sample on a mass spectrum target plate, and drying at room temperature;

(3) the detection is carried out under a mass spectrometer, and the mass spectrum images are analyzed, as shown in fig. 5, and fig. 5a and 5b are the mass spectra of the healthy plasma sample and the cancer plasma sample respectively.

The foregoing detailed description of the preferred embodiments of the invention has been presented. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the prior art according to the concept of the present invention should be within the scope of protection defined by the claims.