CN110903444B - Polymer silver-coated micro-nano particle and method for detecting urine micromolecules by using same - Google Patents
Polymer silver-coated micro-nano particle and method for detecting urine micromolecules by using same Download PDFInfo
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- CN110903444B CN110903444B CN201911194988.9A CN201911194988A CN110903444B CN 110903444 B CN110903444 B CN 110903444B CN 201911194988 A CN201911194988 A CN 201911194988A CN 110903444 B CN110903444 B CN 110903444B
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Abstract
The invention discloses a polymer silver-coated micro-nano particle matrix with anti-interference and salt tolerance, and a detection method based on the polymer silver-coated micro-nano particles in urine micromolecule mass spectrometry, wherein the detection steps comprise: the method comprises the following steps: preparation of instruments and reagents: analyzing ionization time-of-flight mass spectrum by matrix-assisted laser, and detecting positive ions in a reflection mode; step two: diluting the urine sample in proportion; step three: sample preparation is carried out on a mass spectrum target plate, and drying is carried out at room temperature; step four: detecting small molecules in the urine sample by using a mass spectrometer based on the polymer silver-coated micro-nano particles; step five: and analyzing the mass spectrum detection result to obtain a conclusion. The detection method can eliminate the interference of background signal noise on detection, improve the detection efficiency and eliminate the influence of a complex urine system on a detection result.
Description
Technical Field
The invention relates to a molecular detection application technology based on matrix-assisted laser desorption ionization mass spectrometry, in particular to application of polymer silver-coated micro-nano particles in urine micromolecular metabolite detection.
Background
The traditional urine detection mainly adopts a biochemical method, carries out qualitative detection aiming at bacteria, white blood cells and red blood cells, and carries out semi-quantitative detection aiming at urine protein, glucose, urine bilirubin and the like. And a great research gap still exists in the accurate detection and analysis of metabolic small molecules in urine. Due to the high complexity of the urine system and the low abundance of metabolic molecules in urine, the raman spectroscopy of the conventional metabolic detection methods, such as electrochemical sensors, is greatly limited.
Compared with the traditional detection technology, the mass spectrometry detection has high flux and high sensitivity, and can carry out molecular identification and structural analysis. Mass spectrometry is a preferred means of detection and analysis due to its superior properties.
The mass spectrum has different types due to different working principles and application ranges, and the most common types include a gas chromatography-mass spectrometer, a liquid chromatography-mass spectrometer and a matrix-assisted laser desorption time-of-flight mass spectrometer. The gas chromatography-mass spectrometer and the liquid chromatography-mass spectrometer have complicated pretreatment steps and long time consumption, so that the urine is difficult to analyze and detect and is applied to clinic. Compared with the two mass spectrum modes, the matrix-assisted laser desorption time-of-flight mass spectrometer has the characteristics of simple sample preparation and high analysis efficiency.
Matrix-assisted laser desorption time-of-flight mass spectrometers have very high requirements on the matrix. The traditional organic matrix is easy to generate strong background signals at the small molecular weight end (m/z is less than 1000), and the noises bring great interference to the detection of the small molecules and influence the detection effect. In an actual urine system, the urine sample has high complexity, various different biomacromolecules, different pH values and high salinity exist, and the detection of the micromolecules is hindered. Therefore, the conventional matrix is difficult to meet the requirement of small molecule detection, and a novel matrix material which can be used for urine detection and has certain interference resistance and certain salt tolerance and an application method thereof are in urgent need of development.
Disclosure of Invention
In view of the above defects of the prior art, the first aspect of the present invention provides a polymer coated silver micro-nano particle matrix, which comprises the following preparation steps:
1) adding ammonia water into a mixed solution of water and ethanol, and reacting for 1 hour;
2) adding resorcinol into the mixed solution, then adding a formaldehyde solution, and reacting for 24 hours;
3) transferring the mixed solution in the step 2) into a Teflon high-pressure reaction kettle, and reacting for 24 hours to form polymer particles;
4) repeatedly washing the polymer particles obtained in the step 3) with ethanol and deionized water, and finally drying at 60 ℃ for later use;
5) resuspending 50 mg of the polymer particles obtained in the step 4) in absolute ethanol, adding 20 ml of freshly prepared silver ammonia solution, and reacting for 1 hour;
6) putting the mixed solution obtained in the step 5) into 50 ml of ethanol solution of polyvinylpyrrolidone, and reacting for 7 hours at 70 ℃ to form polymer silver-coated micro-nano particles;
7) repeatedly washing the polymer silver-coated micro-nano particles obtained in the step 6) with ethanol and deionized water, and finally drying at 60 ℃ for later use;
8) resuspending the polymer-coated silver micro-nano particles obtained in the step 7) in deionized water to be used as a matrix.
Further, the reaction temperature in step 1) and step 2) is 20 ℃ to 30 ℃.
Preferably, the reaction temperature in step 1) and step 2) is 25 deg.C
Further, the reaction temperature of the step 3) is 90 ℃ to 120 ℃.
Preferably, the reaction temperature of step 3) is 100 ℃.
Further, the polymer silver-coated micro-nano particles have ultraviolet absorption.
Further, the silver-coated polymer particles are resorcinol/formaldehyde resin polymer beads.
Furthermore, the polymer silver-coated micro-nano particles have the particle size of 200nm-800nm and uniform size.
Preferably, the particle size of the polymer silver-coated micro-nano particles is 400-450 nm.
Meanwhile, the invention provides an application of the polymer coated silver micro-nano particles in urine micromolecule mass spectrometry detection, which comprises the following specific steps:
the method comprises the following steps: preparation of instruments and reagents: analyzing ionization time-of-flight mass spectrum by matrix-assisted laser, and detecting positive ions in a reflection mode;
step two: diluting the urine sample in proportion;
step three: preparing a sample on a mass spectrum target plate, and drying at room temperature;
step four: detecting small molecules in the urine sample by using a mass spectrometer based on the polymer silver-coated micro-nano particles;
step five: and analyzing the mass spectrum detection result to obtain a conclusion.
Preferably, the detection molecular weight range is less than 1000 Da.
Preferably, the small molecules include saccharides, amino acids.
And the method is applied to the detection of urine micromolecules by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. By adopting the micro-nano particle material as the matrix, the defect of the traditional matrix is overcome, and the urine is detected quickly, with high flux and high sensitivity.
The polymer silver-coated micro-nano particles have low preparation cost and simple synthesis steps, and can be manufactured in large batch. The nano-scale polymer silver-coated micro-nano particles are used as a matrix material in a mass spectrum, so that the problems of the traditional organic matrix, such as background interference and hot spot effect of small molecular segments, can be solved. In the invention, the urine sample does not need any pretreatment steps such as enrichment or separation, and each sample only needs 1 microliter of urine to efficiently and quickly detect and analyze the small molecule metabolites in the urine. The detection method has high accuracy, low cost and high detection flux, meets the requirement of clinical urine detection, and can be applied to clinic.
Compared with the prior art, the invention has the following beneficial effects:
1) the polymer silver-coated micro-nano particles have low preparation cost and simple synthesis steps, and can be manufactured in large batch. The nano-scale polymer silver-coated micro-nano particles are used as a matrix material in a mass spectrum, so that the problems of the traditional organic matrix, such as background interference and hot spot effect of small molecular segments, can be solved.
2) In the invention, the urine sample does not need any pretreatment steps such as enrichment or separation, and each sample only needs 1 microliter of urine to efficiently and quickly detect and analyze the small molecule metabolites in the urine. The detection method has high accuracy, low cost and high detection flux, meets the requirement of clinical urine detection, and can be applied to clinic.
Drawings
FIG. 1 is a SEM representation of silver-coated polymer particles prepared in a preferred embodiment of the present invention;
FIG. 2 is a TEM image of the polymer-coated silver particles prepared in the preferred embodiment of the present invention;
FIG. 3 is a mass spectrum of glucose standard molecule detected by matrix-assisted laser desorption ionization time-of-flight mass spectrometry in example 1;
FIG. 4 is a mass spectrum of lysine standard molecule detected by matrix-assisted laser desorption ionization time-of-flight mass spectrometry in example 2;
FIG. 5 is a mass spectrum of a small molecular weight end of urine detected by matrix-assisted laser desorption ionization time-of-flight mass spectrometry in example 3;
FIG. 6 is the urine micromolecules of different samples detected by matrix assisted laser desorption ionization time-of-flight mass spectrometry in the specific example 4, and the differential diagnosis is carried out in SIMCA software.
Detailed Description
The following description is presented to disclose the invention so as to enable any person skilled in the art to practice the invention. The preferred embodiments in the following description are given by way of example only, and other obvious variations will occur to those skilled in the art.
The specific implementation mode is as follows:
the preparation method comprises the following steps of preparing a polymer silver-coated micro-nano particle matrix, wherein the polymer silver-coated micro-nano particle matrix has ultraviolet absorption, is a resorcinol/formaldehyde resin polymer bead, has a particle size of 200-800 nm and uniform size, and has an optimal particle size of 400-450nm, and the specific preparation steps comprise:
1) adding ammonia water into a mixed solution of water and ethanol, and reacting for 1 hour;
2) adding resorcinol into the mixed solution, then adding formaldehyde solution, and reacting for 24 hours;
3) transferring the mixed solution in the step 2) into a Teflon high-pressure reaction kettle, and reacting for 24 hours to form polymer particles;
4) repeatedly washing the polymer particles obtained in the step 3) with ethanol and deionized water, and finally drying at 60 ℃ for later use;
5) resuspending 50 mg of the polymer particles obtained in the step 4) in absolute ethanol, adding 20 ml of freshly prepared silver ammonia solution, and reacting for 1 hour;
6) putting the mixed solution obtained in the step 5) into 50 ml of ethanol solution of polyvinylpyrrolidone, and reacting for 7 hours at 70 ℃ to form polymer silver-coated micro-nano particles;
7) repeatedly washing the polymer silver-coated micro-nano particles obtained in the step 6) by using ethanol and deionized water, and finally drying at 60 ℃ for later use;
8) resuspending the polymer-coated silver micro-nano particles obtained in the step 7) in deionized water to be used as a matrix.
The reaction temperature in the step 1) and the step 2) is 20-30 ℃, and the effect is better when the reaction temperature is 25 ℃.
The reaction temperature of the step 3) is 90-120 ℃, and the effect is better when the reaction temperature is 100 ℃.
Secondly, detecting small molecules in urine by using a mass spectrometer based on the polymer silver-coated micro-nano particles, and the method specifically comprises the following steps:
the method comprises the following steps: preparation of instruments and reagents: analyzing ionization time-of-flight mass spectrum by matrix-assisted laser, and detecting positive ions in a reflection mode;
step two: diluting the urine sample in proportion;
step three: sample preparation is carried out on a mass spectrum target plate, and drying is carried out at room temperature;
step four: based on the polymer silver-coated micro-nano particles, detecting small molecules in a urine sample by using a mass spectrometer, wherein the detection molecular weight range is less than 1000Da, and the small molecules comprise saccharides and amino acid;
step five: and analyzing the mass spectrum detection result to obtain a conclusion.
In the embodiment, the transmission electron microscope result is obtained by an instrument adopting a NERCN-TC-006 field emission scanning electron microscope, and the transmission electron microscope result is obtained by the NERCN-TC-010-1 field emission transmission electron microscope.
The characterization results are shown in fig. 1 and fig. 2, the prepared polymer silver-coated micro-nano particles are spherical materials with the size of about 400 nanometers, and the scanning electron microscope results (fig. 1) show that the synthesized materials are uniform in size and rough in surface. The transmission electron microscope result (figure 2) shows that the synthesized silver-coated micro-nano particles of the polymer have uniform size, which is consistent with the result in figure 1.
Example one: detection of glucose standards
Preparation of instruments and reagents: matrix-assisted laser desorption ionization time-of-flight mass spectrometer adopts a reflection mode and positive ion detection; the prepared polymer silver-coated micro-nano particles; preparing a prepared glucose standard solution;
preparing a sample on a mass spectrum target plate, and drying at room temperature;
detecting under a mass spectrometer, and analyzing the mass spectrum image, wherein the detection result is shown in figure 3;
example two: detection of lysine standards
Preparation of instruments and reagents: matrix-assisted laser desorption ionization time-of-flight mass spectrometer adopts a reflection mode and positive ion detection; the prepared polymer silver-coated micro-nano particles; preparing a prepared lysine standard solution;
preparing a sample on a mass spectrum target plate, and drying at room temperature;
detecting under a mass spectrometer, and analyzing a mass spectrum image, wherein the detection result is shown in figure 4;
EXAMPLE III: detection of urine sample small molecules
Preparation of instruments and reagents: matrix-assisted laser desorption ionization time-of-flight mass spectrometer adopts a reflection mode and positive ion detection; the prepared polymer silver-coated micro-nano particles;
diluting the urine sample according to a certain proportion;
preparing a sample on a mass spectrum target plate, and drying at room temperature;
the detection is carried out under a mass spectrometer, and the mass spectrum image is analyzed, and the detection result is shown in fig. 5.
Example four: diagnosis methodClinopodium and its subtype
Preparation of instruments and reagents: matrix-assisted laser desorption ionization time-of-flight mass spectrometer adopts a reflection mode and positive ion detection; the prepared polymer silver-coated micro-nano particles; SIMCA analytical software;
diluting the urine sample according to a certain proportion;
preparing a sample on a mass spectrum target plate, and drying at room temperature;
detecting under a mass spectrometer, and collecting mass spectrum data;
the mass spectrum data was preprocessed and analyzed by SIMCA software, and the results are shown in fig. 6.
The foregoing shows and describes the general principles, principal features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are merely illustrative of the principles of the invention, but that various changes and modifications may be made without departing from the spirit and scope of the invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (7)
1. The application of the polymer silver-coated micro-nano particles in a mass spectrum matrix is characterized in that the mass spectrum matrix is the polymer silver-coated micro-nano particles, and the preparation steps of the polymer silver-coated micro-nano particles comprise:
1) adding ammonia water into a mixed solution of water and ethanol, and reacting for 1 hour to obtain a first mixed solution;
2) adding resorcinol into the first mixed solution, then adding a formaldehyde solution, and reacting for 24 hours to obtain a second mixed solution;
3) transferring the second mixed solution into a Teflon high-pressure reaction kettle, and reacting for 24 hours to form polymer particles;
4) repeatedly washing the polymer particles obtained in the step 3) with ethanol and deionized water, and finally drying at 60 ℃ for use;
5) resuspending 50 mg of the polymer particles obtained in the step 4) in absolute ethanol, adding 20 ml of freshly prepared silver ammonia solution, and reacting for 1 hour to obtain a third mixed solution;
6) putting the third mixed solution into 50 ml of ethanol solution of polyvinylpyrrolidone, and reacting at 70 ℃ for 7 hours to form the polymer silver-coated micro-nano particles;
7) repeatedly washing the polymer silver-coated micro-nano particles obtained in the step 6) by using ethanol and deionized water, and finally drying at 60 ℃ for later use;
8) resuspending the polymer silver-coated micro-nano particles obtained in the step 7) in deionized water to be used as a matrix;
the particle size of the polymer silver-coated micro-nano particles is 200nm-800 nm.
2. The application of the silver-coated micro-nano particles in the mass spectrometry matrix according to claim 1, wherein the reaction temperature in the step 1) and the step 2) is 20-30 ℃.
3. The application of the polymer-coated silver micro-nano particles in the mass spectrometry matrix according to claim 1, wherein the reaction temperature in the step 3) is 90-120 ℃.
4. The application of the polymer-coated silver micro-nano particles in the mass spectrometry matrix according to claim 1, wherein the optimal reaction temperature in the step 3) is 100 ℃.
5. The application of the polymer-coated silver micro-nano particles in a mass spectrometry matrix according to claim 1, wherein the polymer-coated silver micro-nano particles have ultraviolet absorption.
6. The application of the polymer-coated silver micro-nano particles in a mass spectrometry matrix according to claim 1, wherein the polymer-coated silver particles are resorcinol/formaldehyde resin polymer beads.
7. The application of the silver-coated micro-nano particles in the mass spectrometry matrix as claimed in claim 1, wherein the particle size of the silver-coated micro-nano particles is 400-450 nm.
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| US4392963A (en) * | 1980-03-26 | 1983-07-12 | Sartorius Gmbh | Resorcinol or phloroglucinol condensation product for aqueous mixture purification |
| CN102901790A (en) * | 2012-09-21 | 2013-01-30 | 中国人民解放军***南京总医院 | Determination method of urine metabolic marker for early diagnosis of diabetic nephropathy. |
| CN106807941A (en) * | 2015-11-30 | 2017-06-09 | 上海交通大学 | A kind of noble metal core-shell structure nanometer particle and its preparation and application |
| CN106841373A (en) * | 2015-12-07 | 2017-06-13 | 中国科学院大连化学物理研究所 | Sub-micron aoxidizes application of the carbon ball as matrix in MALDI-MS |
| CN109293859A (en) * | 2018-10-15 | 2019-02-01 | 西安交通大学 | A kind of hydrophily sulindac molecular engram magnetic Nano resin balls and its preparation method and application |
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| CN101381423B (en) * | 2008-09-10 | 2010-09-08 | 厦门大学 | Method for preparing silver-coating polystyrene nano granule |
| CN104998591B (en) * | 2015-07-28 | 2017-06-16 | 深圳大学 | Carrying nano silver resorcinol formaldehyde resin microballoon and the mesoporous carbosphere of carrying nano silver |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4392963A (en) * | 1980-03-26 | 1983-07-12 | Sartorius Gmbh | Resorcinol or phloroglucinol condensation product for aqueous mixture purification |
| CN102901790A (en) * | 2012-09-21 | 2013-01-30 | 中国人民解放军***南京总医院 | Determination method of urine metabolic marker for early diagnosis of diabetic nephropathy. |
| CN106807941A (en) * | 2015-11-30 | 2017-06-09 | 上海交通大学 | A kind of noble metal core-shell structure nanometer particle and its preparation and application |
| CN106841373A (en) * | 2015-12-07 | 2017-06-13 | 中国科学院大连化学物理研究所 | Sub-micron aoxidizes application of the carbon ball as matrix in MALDI-MS |
| CN109293859A (en) * | 2018-10-15 | 2019-02-01 | 西安交通大学 | A kind of hydrophily sulindac molecular engram magnetic Nano resin balls and its preparation method and application |
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