CN108344793A - A kind of matrix and preparation method thereof, the mass spectral analysis detection method of metabolic molecule - Google Patents
A kind of matrix and preparation method thereof, the mass spectral analysis detection method of metabolic molecule Download PDFInfo
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Abstract
A kind of matrix and preparation method thereof, the mass spectral analysis detection method of metabolic molecule, belong to materials synthesis and Mass Spectrometer Method field.The zinc oxide matrix is synthesized using sodium hydroxide and zinc acetate as raw material by hydro-thermal method, and condition is easily-controllable, time-consuming short, easy to operate.The zinc oxide matrix being prepared is star nano particle, sword shape nanometer rods of the star nano particle by size less than 2 μm, which assemble, to be formed, grain graininess is uniform, has good uv absorption property, therefore with the performance of good help ionized sample molecule parsing.Compared to ball shaped nano zinc oxide matrix, which can remove the background interference and hot spot-effect of traditional matrices, while having the resistance to protein characteristic of superior salt tolerant, can be used in the biological sample system of the complexity such as serum.Meanwhile efficient, the quick detection of metabolic molecule is realized without the sample pretreatments operation such as enrichment, separation in detection process.
Description
Technical field
The present invention relates to materials synthesis and Mass Spectrometer Method fields, in particular to a kind of matrix and preparation method thereof, generation
Thank to the mass spectral analysis detection method of molecule.
Background technology
Matrix-Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF-MS) is one kind developed in recent years
The surface laser resolves mass spectrum of new shape, is mainly realized by the analysis to sample ions mass-to-charge ratio (m/z) and is carried out precisely to sample
Qualitative and quantitative analysis.The molecular weight that large biological molecule can be measured at present is up to 600KDa, due to its detectable molecular weight model
It encloses greatly, sweep speed is fast, resolution ratio, high sensitivity, the simply equal remarkable advantages of apparatus structure, many forward positions in life science
Field, such as protein science, Pharmaceutical Analysis, are widely used.But MALDI-TOF-MS also has limitation：With
MALDI-TOF-MS carries out small molecule (m/z<1000) analysis is generally limited.This is because traditional organic substrate is such as
Alpha-cyano -4- hydroxycinnamic acids (CHCA) and 2,5-dihydroxybenzoic acid (DHB) etc. will appear a large amount of within the scope of low quality
Background matrix mass signal, the measurement of severe jamming small molecule.Meanwhile the crystallization of these organic substrates is uneven, therefore in mass spectrum
It will produce point-to-point effect when detection, lead to the less reproducible of testing result.In addition, for simple standard sample system,
The detection limit of MALDI-TOF-MS still reaches 10-15~10-18, but in the biological samples such as such as serum, a large amount of protein, salt
The presence of the chaff interferents such as class significantly impacts sample to be tested Ionization Efficiency.Therefore traditional matrix is difficult to meet for small point
The demand of son detection, a kind of new shape can be used for biosystem detection, and with certain anti-interference and certain salt tolerance
Host material urgently develop.
Invention content
Based on the deficiencies of the prior art, the present invention provides a kind of matrix and preparation method thereof, metabolic molecule mass spectral analyses
In application, partly or entirely to improve, even solve problem above.
The invention is realized in this way：
In a first aspect, the embodiment of the present invention provides a kind of matrix.
Matrix has ultraviolet absorption characteristic, and matrix includes aggregation, and aggregation includes even-grained multiple nano particles,
Nano particle is star structure, and nano particle is mainly made by zinc oxide, and the size of nano particle is less than 2 μm.
In second aspect, the embodiment of the present invention provides a kind of preparation method of matrix as the aforementioned.
Preparation method includes：
Homogeneous alkaline solution containing zinc ion is provided；
Make alkaline solution that hydro-thermal reaction occur, to obtain particulate matter；
Particulate matter after drying is suspended in the decentralized medium of liquid.
In the third aspect, the embodiment of the present invention provides a kind of Mass Spectrometry detection method of metabolic molecule.
Mass Spectrometry detection method includes：
Step 1：The preparation of instrument and reagent：Matrix-Assisted Laser Desorption Ionization Time of Flight instrument sets signal-to-noise ratio
Mass signal more than 6 is for analyzing；Using the working method of impulse electric field delay extraction and reflection, positive ion mode is examined
It surveys；
Step 2：Prepare the standard solution of the metabolic molecules such as glucose, mannitol, lysine；
Step 3：Compound mass spectral analysis will be carried out such as aforementioned matrix and standard solution.
Advantageous effect：
Above-mentioned Zinc oxide nanoparticle provided in an embodiment of the present invention can parse ion as matrix to metabolism small molecule
Enhance with changing effect selection.Matrix can be used for the Molecular Detection that molecular weight is 100Da~10000Da, including small peptide fragment, nucleosides
Acid monomers, amino acid, carbohydrate, lipid or Medicine small molecule etc..
In addition, matrix provided in an embodiment of the present invention at least has the following advantages that：
It is convieniently synthesized：Using zinc acetate and sodium hydroxide in the preparation method of raw material, to be synthesized by hydro-thermal method, need not move through
The techniques such as calcining, condition is easily-controllable, takes short.
Sensitivity improves：The ambient noise of organic substrate and the interference of hot spot-effect are effectively removed, is realized to small molecule object
The accurate identification of matter.
It is salt tolerance, resistance to Proteinaceous good：Compared to spherical Zinc oxide nanoparticle, biological sample system can be effectively excluded
In Mass Spectrometer Method is generated by salt, protein macromolecule etc. influence.
Description of the drawings
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technology description to be briefly described.
Fig. 1 is the scanning electron microscopic picture for the star Zinc oxide nanoparticle being prepared in the embodiment of the present invention 1；
Fig. 2 a are the background mass spectra figures that gained is measured when carrying out Mass Spectrometer Method using the matrix in the embodiment of the present invention 1；
Fig. 2 b are the background mass spectra figures that gained is measured when carrying out Mass Spectrometer Method using the matrix in comparative example 1；
Fig. 3 a are the mass spectrograms carried out to glucose using the matrix in test example 1 obtained by Mass Spectrometer Method；
Fig. 3 b are the mass spectrograms carried out to mannitol using the matrix in test example 1 obtained by Mass Spectrometer Method；
Fig. 3 c are the mass spectrograms carried out to lysine using the matrix in test example 1 obtained by Mass Spectrometer Method；
Fig. 4 a are to carrying out the mass spectrum obtained by metabolic molecule Mass Spectrometer Method after the matrix progress salt tolerance processing in test example 1
Scheme (signal for the metabolism small molecule that spider lable is in figure)；
Fig. 4 b are to carrying out the mass spectrum obtained by metabolic molecule Mass Spectrometer Method after the matrix progress salt tolerance processing in comparative example 1
Scheme (signal for the metabolism small molecule that spider lable is in figure)；
Fig. 5 a are to carry out carrying out the matter obtained by metabolic molecule Mass Spectrometer Method after resistance to protein processing to the matrix in test example 1
Spectrogram (signal for the metabolism small molecule that spider lable is in figure)；
Fig. 5 b are to carry out carrying out the matter obtained by metabolic molecule Mass Spectrometer Method after resistance to protein processing to the matrix in comparative example 1
Spectrogram (signal for the metabolism small molecule that spider lable is in figure).
Specific implementation mode
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.It is not specified in embodiment specific
Condition person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer is
The conventional products that can be obtained by commercially available purchase.
Matrix below for the embodiment of the present invention and preparation method thereof, the mass spectral analysis detection method of metabolic molecule carry out
It illustrates：
Matrix provided in an embodiment of the present invention has ultraviolet absorption characteristic.In some instances, matrix is in 300~400nm
Ultraviolet range in show absorption characteristic.Alternatively, can also be to be shown in the wave-length coverage of 360~380nm
Absorption characteristic.
Matrix mainly contains aggregation, and aggregation contains even-grained multiple nano particles.Wherein, nano particle is
Star structure, and be mainly made by zinc oxide.The size of nano particle is less than 2000nm.For example, the size of nano particle
It can also be and be less than 1500nm, or be less than 1000nm or 500nm.
More specifically, in some instances, the star-shaped nano particle described in the embodiment of the present invention is by multiple oxygen
Change zinc nanometer rods to be combined into.For example, nanometer can be that zinc oxide is made in rodlike to be grown in a base core upper edge multiple directions
It forms.
Preferably, zinc oxide nano rod is sword shape.
Alternatively, in some instances, a diameter of 100nm~200nm of nanometer rods is preferably 110~
190nm, more preferably 130~160nm, override are 150~170nm.
In addition, the length of nanometer rods can be 1000~2000nm, it is preferably 1050~1800nm, more preferably 1200~
1600nm, override are 1500~1900nm.
Sea of the embodiment of the present invention additionally provides a kind of preparation method of matrix, and preparation method includes：
Homogeneous alkaline solution containing zinc ion is provided；
Make alkaline solution that hydro-thermal reaction occur, to obtain particulate matter；
Particulate matter after drying is suspended in the decentralized medium of liquid.
Alternatively, alkaline solution is the aqueous solution for being dispersed with zinc acetate and sodium hydroxide.For example, aqueous solution pass through it is following
Method is made：Using as the water dissolution zinc acetate of solvent, to obtain zinc acetate solution；Sodium hydroxide is uniformly dispersed
In zinc acetate solution.
Wherein, the condition of hydro-thermal reaction is 180 DEG C, 2 hours.
Preferably, particulate matter is obtained by using ethyl alcohol and the mixed liquor of deionized water washing hydro-thermal reaction product.
It is highly preferred that the method for dry particle object includes：Particulate matter is heated under the conditions of 60 DEG C, most preferably,
Decentralized medium is deionized water.
Based on substrate provided above, the embodiment of the present invention additionally provides a kind of Mass Spectrometry detection method of metabolic molecule.
Mass Spectrometry detection method includes：
Step 1：The preparation of instrument and reagent：Matrix-Assisted Laser Desorption Ionization Time of Flight instrument sets signal-to-noise ratio
Mass signal more than 6 is for analyzing；Using the working method of impulse electric field delay extraction and reflection, positive ion mode is examined
It surveys；
Step 2：Prepare the standard solution of the metabolic molecules such as glucose, mannitol, lysine；
Step 3：The matrix of any one of such as claim 1-4 and standard solution are subjected to compound mass spectral analysis.
A kind of optional example, Matrix-Assisted Laser Desorption Ionization Time of Flight instrument use Nd:YAG laser, wavelength
For 355nm.The analyzing processing that flexAnalysis carries out data may be used in the data that Mass Spectrometer Method obtains.In addition, in order to subtract
The harmful effect of few impurity or pollution to the reliability of data.Clean target plate must be used, the target plate uses formic acid, anhydrous successively
Ethyl alcohol, deionized water are cleaned by ultrasonic 2 hours totally.
Zinc oxide known to inventor is a kind of common semi-conducting material, has larger band gap and exciton bind energy,
With good ultraviolet absorption characteristic, electric charge carrier can be used as.Based on this, zinc oxide is gradually answered as MALDI matrix
With, but preparation method needs the complex operations such as high-temperature calcination at present.Meanwhile when low molecular weight detects, its own background is made an uproar
Sound is still difficult to ignore, and cannot exclude the influence of the chaff interferents such as salt, protein, significantly impacts the effect of its detection.
The Zinc oxide nanoparticle object of matrix provided in an embodiment of the present invention, star structure therein can be to small point of metabolism
The ionization analysis feature of son has preferable raising to act on.In addition, when carrying out Mass Spectrometer Method, ambient noise smaller, effectively
Avoid the background interference and hot spot-effect of traditional matrices.Further, salt tolerant, alkaline-resisting, therefore, is more advantageous to for serum
Deng the Mass Spectrometer Method of complicated biological sample system.It preferably, can be to avoid existing using matrix provided in an embodiment of the present invention
The sample pretreatments operation such as the matrix enrichment, the separation that are widely present when carrying out Mass Spectrometer Method, it is thereby achieved that metabolic molecule
Efficient, quick detection.
With reference to embodiments to the mass spectral analysis detection side of a kind of matrix of the present invention and preparation method thereof, metabolic molecule
Method is described in further detail.
Embodiment 1
The preparation of star Zinc oxide nanoparticle matrix.
Preparation method includes the following steps：
Step 1：Using water as solvent, zinc acetate solution is prepared；
Step 2：Above-mentioned solution is added in sodium hydroxide, and magnetic agitation is disperseed to solution in homogeneous at room temperature；
Step 3：The mixed solution obtained in second step is moved to the stainless steel hydrothermal reaction kettle of teflon lined, and
It keeps reacting 2 hours at 180 DEG C, to form star Zinc oxide nanoparticle；
Step 4：The zinc oxide star nano particle obtained in third step is washed repeatedly with ethyl alcohol and deionized water, in
It is dried at 60 DEG C powdered.
Nanoparticles in matrix are characterized in the following way：
Characterization instrument used：The size and shape of product is characterized in complete in HitachiS-4800 scanning electron microscope (SEM)
At.
Characterization result is：Allusion quotation shape star Zinc oxide nanoparticle is 100nm~200nm, length range 1 by diameter range
~2 μm of sword shape nanometer rods composition, grain graininess are uniform.Refering to fig. 1.
Comparative example 1
Spherical Zinc oxide nanoparticle matrix is bought by Shanghai Chao Wei nanosecond science and technology Co., Ltd, and the production trade mark is cw-
Zno-001, product batch number 20171106001.
Test example 1
The matrix and comparative example that embodiment 1 is provided respectively using Matrix-Assisted Laser Desorption Ionization Time of Flight instrument
1 matrix provided carries out background detection, testing result such as Fig. 2 a and Fig. 2 b.The preparation of instrument and reagent：Ground substance assistant laser solution
Ionization time-of-flight mass spectrometer is analysed, only mass signal of the signal-to-noise ratio more than 6 is for analyzing；Using impulse electric field be delayed extraction and
The working method of reflection, positive ion mode are detected.Before being detected, clean target plate must be used, the target plate according to
Secondary formic acid, absolute ethyl alcohol, deionized water are cleaned by ultrasonic 2 hours totally.The Matrix Assisted Laser Desorption ionizes flight time matter
Spectrometer is Bruker autoflex speed mass spectrographs, using Nd:YAG laser, wavelength 355nm.
Test example 2
Detect dextrose standard sample
Matrix Assisted Laser Desorption is carried out to dextrose standard sample using Zinc oxide nanoparticle matrix and ionizes the flight time
The step of Mass Spectrometer Method, is as follows：
(1) preparation of instrument and reagent：Matrix-Assisted Laser Desorption Ionization Time of Flight instrument, only signal-to-noise ratio are more than
6 mass signal is for analyzing.Using Brukerautoflexspeed mass spectrographs, Nd:YAG laser, wavelength 355nm.It adopts
With the working method of impulse electric field delay extraction and reflection, positive ion mode is detected.Using flexAnalysis observations, place
Reason, analysis data, only mass signal of the signal-to-noise ratio more than 6 is for analyzing.
(2) star Zinc oxide nanoparticle is prepared with reference to embodiment 1.
(3) a series of dextrose standard sample of concentration gradients is configured.
(4) formic acid, absolute ethyl alcohol, deionized water is used to be cleaned by ultrasonic target plate totally 2 hours successively.
(5) sample is put on dry target plate.
(6) ultrasonic vibration disperses Zinc oxide nanoparticle in deionized water, after sample drying, is added dropwise in sample surfaces
Matrix suspension, sample form secondary recrystallization with matrix.
(7) it after dry, is analyzed for Matrix-Assisted Laser Desorption Ionization Time of Flight instrument.Testing result please refers to figure
3a。
Test example 3
Detect mannitol standard items
(1) preparation of instrument and reagent：Matrix-Assisted Laser Desorption Ionization Time of Flight instrument, only signal-to-noise ratio are more than
6 mass signal is for analyzing；
(2) Zinc oxide nanoparticle is prepared with reference to embodiment 1；
(3) a series of mannitol standard items of concentration gradients are configured.
(4) Zinc oxide nanoparticle carries out compound mass spectral analysis with mannitol molecule.
Specific steps are similar to Example 1, and details are not described herein again.Testing result please refers to Fig. 3 b.
Test example 4
Detect lysine standard items
(1) preparation of instrument and reagent：Matrix-Assisted Laser Desorption Ionization Time of Flight instrument, only signal-to-noise ratio are more than
6 mass signal is for analyzing；
(2) Zinc oxide nanoparticle is prepared with reference to embodiment 1；
(3) a series of lysine standard items of concentration gradients are configured.
(4) Zinc oxide nanoparticle carries out compound mass spectral analysis with lysine molecule.
Specific steps are similar to Example 1, and details are not described herein again.Testing result please refers to Fig. 3 c.
Test example 5
Salt resistant character detects
(1) preparation of instrument and reagent：Matrix-Assisted Laser Desorption Ionization Time of Flight instrument, only signal-to-noise ratio are more than
6 mass signal is for analyzing；
(2) Zinc oxide nanoparticle is prepared with reference to embodiment 1；
(3) mixed solution of the standard items such as glucose, mannitol, lysine, arginine is configured, it is molten with high concentration sodium chloride
Liquid mixes, and ultrasound is disperseed to solution in homogeneous.
(4) solution obtained in Zinc oxide nanoparticle and previous step carries out compound mass spectral analysis.
Specific steps are similar to Example 1, and details are not described herein again.Testing result please refers to Fig. 4 a and Fig. 4 b.
Test example 6
The sample of resistance to protein capability
(1) preparation of instrument and reagent：Matrix-Assisted Laser Desorption Ionization Time of Flight instrument, only signal-to-noise ratio are more than
6 mass signal is for analyzing；
(2) Zinc oxide nanoparticle is prepared with reference to embodiment 1；
(3) mixed solution of the standard items such as glucose, mannitol, lysine, arginine is configured, it is molten with bovine serum albumin(BSA)
Liquid mixes, and ultrasound is disperseed to solution in homogeneous.
(4) solution obtained in Zinc oxide nanoparticle and previous step carries out compound mass spectral analysis.
Specific steps are similar to Example 1, and details are not described herein again.Testing result please refers to Fig. 5 a and Fig. 5 b.
The preferred embodiment of the present invention has been described in detail above.It should be appreciated that the ordinary skill of this field is without wound
The property made labour, which according to the present invention can conceive, makes many modifications and variations.Therefore, all technician in the art
Pass through the available technology of logical analysis, reasoning, or a limited experiment on the basis of existing technology under this invention's idea
Scheme, all should be in the protection domain being defined in the patent claims.
Although illustrate and describing the present invention with specific embodiment, it will be appreciated that without departing substantially from the present invention's
Many other change and modification can be made in the case of spirit and scope.It is, therefore, intended that in the following claims
Including belonging to all such changes and modifications in the scope of the invention.
Claims (10)
1. a kind of matrix, which is characterized in that the matrix has ultraviolet absorption characteristic, and the matrix includes aggregation, described poly-
Collective includes even-grained multiple nano particles, and the nano particle is star structure, and the nano particle is mainly by aoxidizing
Zinc is made, and the size of the nano particle is less than 2 μm.
2. matrix according to claim 1, which is characterized in that the star-shaped nano particle is by multiple zinc oxide nano rods
It is combined into, it is preferable that the zinc oxide nano rod is sword shape.
3. matrix according to claim 2, which is characterized in that a diameter of 100nm~200nm of the nanometer rods, preferentially
For 110~190nm, more preferably 130~160nm, override is 150~170nm；
Alternatively, the length of the nanometer rods is 1000~2000nm, it is preferably 1050~1800nm, more preferably 1200~
1600nm, override are 1500~1900nm.
4. matrix according to any one of claim 1-3, which is characterized in that the matrix is in the ultraviolet of 300~400nm
Absorption characteristic is shown in line wave-length coverage.
5. a kind of preparation method of matrix as described according to any one of claim 1-4, which is characterized in that the preparation side
Method includes：
Homogeneous alkaline solution containing zinc ion is provided；
Make the alkaline solution that hydro-thermal reaction occur, to obtain particulate matter；
The particulate matter after drying is suspended in the decentralized medium of liquid.
6. the preparation method of matrix according to claim 5, which is characterized in that the alkaline solution is to be dispersed with zinc acetate
With the aqueous solution of sodium hydroxide.
7. the preparation method of matrix according to claim 6, which is characterized in that the aqueous solution makes by the following method
It forms：
Using as the water dissolution zinc acetate of solvent, to obtain zinc acetate solution；Sodium hydroxide is homogeneously dispersed in the vinegar
In sour zinc solution.
8. the preparation method of matrix according to claim 5, which is characterized in that the condition of the hydro-thermal reaction be 180 DEG C,
2 hours, it is preferable that the particulate matter is obtained by using ethyl alcohol and the mixed liquor of deionized water washing hydro-thermal reaction product
, it is highly preferred that the method for the dry particulate matter includes：The particulate matter is heated under the conditions of 60 DEG C, most preferably
Ground, the decentralized medium are deionized water.
9. a kind of mass spectral analysis detection method of metabolic molecule, which is characterized in that the mass spectral analysis detection method includes：
Step 1：The preparation of instrument and reagent：Matrix-Assisted Laser Desorption Ionization Time of Flight instrument, setting signal-to-noise ratio are more than 6
Mass signal for analyzing；Using the working method of impulse electric field delay extraction and reflection, positive ion mode is detected；
Step 2：Prepare the standard solution of the metabolic molecules such as glucose, mannitol, lysine；
Step 3：Matrix as described in any one of claim 1-4 is subjected to compound mass spectral analysis with the standard solution.
10. the mass spectral analysis detection method of metabolic molecule according to claim 9, which is characterized in that the Matrix-assisted
Laser desorption ionization time of flight mass spectrometry instrument uses Nd:YAG laser, wavelength 355nm.
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| CN112730323A (en) * | 2020-12-18 | 2021-04-30 | 上海交通大学 | Novel hybrid metal nano material, preparation method thereof and application thereof in matrix-assisted laser desorption ionization mass spectrometry |
| CN113484405A (en) * | 2021-07-05 | 2021-10-08 | 上海交通大学 | Preparation method of sub-microreactor and serum metabolite detection method based on sub-microreactor |
| CN115078336A (en) * | 2021-03-10 | 2022-09-20 | 华东师范大学 | Multilayer hollow oxide microsphere material, synthesis method and application in metabolite detection |
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