CN106338542A - Method for detecting serum small molecule metabolites by using mass spectrometry - Google Patents
Method for detecting serum small molecule metabolites by using mass spectrometry Download PDFInfo
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- CN106338542A CN106338542A CN201510395908.1A CN201510395908A CN106338542A CN 106338542 A CN106338542 A CN 106338542A CN 201510395908 A CN201510395908 A CN 201510395908A CN 106338542 A CN106338542 A CN 106338542A
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Abstract
The invention discloses a method for detecting serum small molecule metabolites by using a mass spectrometry. The method employs a micro nano granular material as a matrix, uses an internal standard substance for a quantitative analysis, can reach high sensitivity in a wide detection scope, and only requires to consume little micro biological sample; the method can remove the background interference and hot spot effect of the traditional matrix, and can realize the detection of molecule metabolites in a serum sample. Under prerequisite that enrichment and separating processes are not required, the method can rapidly and efficiently perform quantitative determination and analysis on the metabolite in serum.
Description
Technical field
The present invention relates to a kind of Molecular Detection application technology based on matrix-assisted laser desorption ionization time-of-flight mass spectrometry, specifically relate to
And its application in serum small molecule metabolism analyte detection.
Background technology
Traditional analysis method to serum is directed primarily to albumen, gene and peptide fragment, and the detection of metabolic molecule with
Analysis yet suffers from very big research vacancy.Due to the complexity of system and the low abundance of metabolic molecule, traditional inspection
Survey method is very restricted, thus limiting its application in medical diagnosiss.With traditional detection technique,
Such as electrochemical sensor, Raman spectrum is compared, and Mass Spectrometer Method due to its high flux, sensitivity and can be carried out point
The identification of son and structural analyses, and become a kind of preferred means of detection and analysis.
The most frequently used mass spectrum detection has electron spray ionisation and laser desorption ionization.Compared to electron spray ionisation, swash
Light desorption ionization has the characteristics that sample preparation is simple and analysis efficiency is high.But up to the present, laser desorption electricity
From still suffering from certain defect in the detection and analysis of living things system:
(1) laser desorption ionization typically takes time and effort in the analysis of biological sample, and needs substantial amounts of
Step is reducing the complexity of system.
(2) still needed based on the mass spectrum quantitative technique of laser desorption ionization and improve sensitivity, accuracy further
With detection range such that it is able to the reasonable detection being applied to a large amount of molecules.
(3) traditional mass spectrum organic substrate easily causes high point-to-point effect and low sample repeatability, and
Easily produce strong background signal when detecting small molecule, affect Detection results.
(4) can be good at reducing background signal with inorganic matrix and overcome point-to-point effect, but selecting
Also there is very big restriction in property, sensitivity, accuracy and Clinical detection scope.
Traditional substrate easily produces background noise at small-molecular-weight end (m/z < 1000), for the inspection of small molecule
Measuring tape greatly to disturb.And in the middle of actual living things system, biological sample is generally sufficiently complex.Various biologies
The presence of macromole, and different acid-base value, salinity all can bring obstruction to the detection of small molecule.Therefore pass
The substrate of system is difficult to meet the demand for small molecule detection, and a kind of new living things system that can be used for detects, and
The host material with certain anti-interference and certain salt tolerance is urgently developed.
Content of the invention
In view of the drawbacks described above of prior art, the invention provides a kind of new mass spectrum inspection based on granule auxiliary
Survey technology.It is used as substrate by the granular materialss using micro/nano level, overcome the defect of traditional matrices, quick, high
Special survey molecule in flux, in high sensitivity quantitative serum.
Technical scheme is as follows:
A kind of method of utilization Mass Spectrometer Method serum small molecule metabolite, comprises the following steps:
Step 1: the preparation of instrument and reagent: laser desorption MALDI-MS, using reflective-mode, positive ion detection；
Step 2: prepare metal oxide particle substrate, comprise the following steps；
Step 2.1: slaine and trisodium citrate are dissolved in ethylene glycol solution；
Step 2.2: add sodium acetate in above-mentioned mixed solution, and ultrasonic at room temperature become homogeneous until solution
System；
Step 2.3: reaction is carried out in Teflon autoclave, reacts 10 hours at 200 degrees celsius with shape
Become metal oxide particle；
Step 2.4: the metal oxide particle obtaining in step 2.3 ethanol and deionized water are rinsed repeatedly,
Finally it is dried at 60 c in case using；
Step 2.5: by resuspended for described metal oxide particle in deionized water, use as substrate；
Step 3: dilution proportion is carried out to blood serum sample；
Step 4: carry out sample preparation on mass spectrum target plate, be dried under room temperature；
Step 5: quantitative analyses detection is carried out to the small molecule in blood serum sample using internal standard method；
Step 6: mass spectrum testing result is analyzed it was therefore concluded that.
Preferably, described metal oxide particle is iron oxide particle.
Preferably, the particle diameter of described iron oxide particle is 50nm～1 μm, and size uniformity.
It is highly preferred that the particle diameter of described iron oxide particle is 200nm～300nm.
Preferably, described iron oxide particle has rough surface, described rough surface receiving by below 50nm
Nano-sphere forms.
Preferably, described iron oxide particle is fe2o3、fe3o4Or its mixture.
Preferably, described iron oxide particle has uv absorption.
Preferably, the extension rate of described blood serum sample is less than or equal to 10000 times.
Preferably, detection molecules amount scope is less than 10000da.
Preferably, the material of detection includes saccharide and aminoacid.
The beneficial effects of the present invention is: the metal oxide particle substrate preparation cost of micro/nano level is low, Ke Yi great
Batch making, synthesis step is simple；The metal oxide particle substrate of this micro/nano level can remove traditional matrices
Ambient interferences and hot spot-effect, have high-salt tolerance.The present invention only need to consume trace serum, need not be enriched with, divide
Metabolite that can fast and efficiently in quantitative detecting analysis serum on the premise of operation；Whole detection process step
Suddenly simple, low cost, flux are high；The quantitative result accuracy being obtained is high, can be applicable in clinic.
Below with reference to accompanying drawing, the invention will be further described, to absolutely prove the purpose of the present invention, technical characteristic
And technique effect.
Brief description
Fig. 1 is the sign picture of the iron oxide particle preparing in preferred embodiment of the present invention, and Fig. 1 a is
Sem characterizes picture, and Fig. 1 b characterizes picture for tem；
Fig. 2 is the mass spectrum that in specific embodiment 1, laser desorption ionization technique detects Glucose standards molecule；
Fig. 3 is the mass spectrum that in specific embodiment 2, laser desorption ionization technique detects serum small molecule amount end.
Specific embodiment
Below in conjunction with the accompanying drawings and embodiment the present invention is described further.
The preparation of iron oxide particle substrate comprises the following steps:
Step 1: ferric chloride and trisodium citrate are dissolved in ethylene glycol solution；
Step 2: in above-mentioned mixed solution add sodium acetate, and at room temperature ultrasonic half an hour until molten
Liquid becomes homogeneous system；
Step 3: reaction will be carried out in Teflon autoclave, reacts 10 hours at 200 degrees celsius
To form iron oxide particle；
Step 4: the iron oxide particle obtaining in step 3 ethanol and deionized water are rinsed repeatedly,
It is dried at 60 c afterwards in case using；
Step 5: by resuspended for iron oxide particle in deionized water, use as substrate.
The sign of substrate:
Characterizing instrument has: saturating using jeol jem-2100f instrument acquisition transmission electron microscope picture, high-resolution
Penetrate electron microscopic picture and SEAD style；Scanning electron microscope example is prepared using silicon chip, and passes through hitachi
S-4800 instrument obtains scanning electron microscopic picture；Under room temperature, the ultraviolet light absorption shading value of material adopts aucy uv1900
Photometer obtains；B-H loop adopts vibrating specimen magnetometer vsm, quantum design, physical
Property measurement system obtains；Contact angle adopts easydrop device kruss gmbh,
Germany obtains；N2 adsorption curve negotiating micromeritics asap 2020m records.
Characterization result is:
The granular materialss being obtained are spherical, and size is about 250nm.Can be seen by transmission electron microscope picture
Go out, granular materialss have subunit structure, and obtain high-resolution transmission by choosing granular materialss marginal area
Electron microscopic picture is it can also be seen that regular lattice figure proves that granular materialss are by a lot of nanocrystal sets further
Become.According to the scanning electron microscopic picture recording it can be seen that granular material surface assumes coarse shape, and size uniformity.
From N2 adsorption curve, the specific surface area of functional particles material is more than 100m2/ g, saturation magnetization is more than
50emu/g, surface hydrophilic, in 270～1100nm region, there is to ultraviolet good absorption.Above structure is into
Essential condition for the host material of good biomolecule Mass Spectrometer Method.
To be further elucidated with matrix-assisted laser desorption ionization time-of-flight mass spectrometry below by several typical Application Examples to exist
Application in the detection and analysis of blood serum metabolic molecule.
Embodiment 1: the detection of dextrose standard sample
(1) preparation of instrument and reagent: laser desorption ionization mass spectrometer, using reflective-mode, just from
Son detection；The iron oxide particle of prepared micro/nano level.
(2) configure a series of dextrose standard sample of Concentraton gradient.
(3) carry out sample preparation on mass spectrum target plate, be dried under room temperature.
(4) the mass spectrum picture being obtained is analyzed, result is as shown in Figure 2.
Embodiment 2: the detection of metabolism small molecule in serum sample
(1) preparation of instrument and reagent: laser desorption ionization mass spectrometer, using reflective-mode, just from
Son detection；The iron oxide particle of prepared micro/nano level.
(2) dilute serum sample by a certain percentage.
(3) carry out sample preparation on mass spectrum target plate, be dried under room temperature.
(4) the serum mass spectrum picture being obtained is analyzed, result is as shown in Figure 3.
The preferred embodiment of the present invention described in detail above.It should be appreciated that the ordinary skill of this area need not
Creative work just can make many modifications and variations according to the design of the present invention.Therefore, all in the art
Technical staff passes through logical analysis, reasoning, or a limited experiment under this invention's idea on the basis of existing technology
Available technical scheme, all should be in the protection domain being defined in the patent claims.
Claims (10)
1. a kind of method using Mass Spectrometer Method serum small molecule metabolite is it is characterised in that comprise the following steps:
Step 1: the preparation of instrument and reagent: laser desorption MALDI-MS, using reflective-mode, cation
Detection；
Step 2: prepare metal oxide particle substrate, comprise the following steps；
Step 2.1: slaine and trisodium citrate are dissolved in ethylene glycol solution；
Step 2.2: add sodium acetate in above-mentioned mixed solution, and ultrasonic at room temperature become until solution
Homogeneous system；
Step 2.3: reaction is carried out in Teflon autoclave, reacts 10 hours at 200 degrees celsius
To form metal oxide particle；
Step 2.4: the metal oxide particle obtaining in step 2.3 ethanol and deionized water are rinsed repeatedly,
Finally it is dried at 60 c in case using；
Step 2.5: by resuspended for described metal oxide particle in deionized water, use as substrate；
Step 3: dilution proportion is carried out to blood serum sample；
Step 4: carry out sample preparation on mass spectrum target plate, be dried under room temperature；
Step 5: quantitative analyses detection is carried out to the small molecule in blood serum sample using internal standard method；
Step 6: mass spectrum testing result is analyzed it was therefore concluded that.
2. the method for a kind of utilization Mass Spectrometer Method serum small molecule metabolite according to claim 1, it is special
Levy and be, described metal oxide particle is iron oxide particle.
3. the method for a kind of utilization Mass Spectrometer Method serum small molecule metabolite according to claim 2, it is special
Levy and be, the particle diameter of described iron oxide particle is 50nm～1 μm, and size uniformity.
4. the method for a kind of utilization Mass Spectrometer Method serum small molecule metabolite according to claim 2, it is special
Levy and be, the particle diameter of described iron oxide particle is 200nm～300nm.
5. the method for a kind of utilization Mass Spectrometer Method serum small molecule metabolite according to claim 2, it is special
Levy and be, described iron oxide particle has rough surface, and described rough surface is by the nanometer of below 50nm
Bead forms.
6. the method for a kind of utilization Mass Spectrometer Method serum small molecule metabolite according to claim 2, it is special
Levy and be, described iron oxide particle is fe2o3、fe3o4Or its mixture.
7. the method for a kind of utilization Mass Spectrometer Method serum small molecule metabolite according to claim 2, it is special
Levy and be, described iron oxide particle has uv absorption.
8. the method for a kind of utilization Mass Spectrometer Method serum small molecule metabolite according to claim 1, it is special
Levy and be, the extension rate of described blood serum sample is less than or equal to 10000 times.
9. the method for a kind of utilization Mass Spectrometer Method serum small molecule metabolite according to claim 1, it is special
Levy and be, detection molecules amount scope is less than 10000da.
10. the method for a kind of utilization Mass Spectrometer Method serum small molecule metabolite according to claim 1, it is special
Levy and be, the material of detection includes saccharide and aminoacid.
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Cited By (8)
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| CN107655960A (en) * | 2017-09-18 | 2018-02-02 | 亿纳谱（浙江）生物科技有限公司 | Serum mass spectrometric analysis method, matrix based on gold-palladium and preparation method thereof, application |
| CN111413396A (en) * | 2020-04-21 | 2020-07-14 | 四川大学 | Application of ferric oxide nano material and MA L DI-TOF MS in detection of small molecule metabolites |
| CN111458399A (en) * | 2020-04-14 | 2020-07-28 | 上海交通大学 | Mass spectrum detection method for low-molecular-weight substances based on palladium-gold core-shell micro-nano material |
| CN112834662A (en) * | 2021-02-01 | 2021-05-25 | 上海拜谱生物科技有限公司 | Method for detecting serum small molecule metabolite by using mass spectrum |
| CN113447560A (en) * | 2021-05-24 | 2021-09-28 | 上海交通大学 | Small molecule metabolite fragmentation control method based on metal ion addition and application |
| CN113484404A (en) * | 2021-07-02 | 2021-10-08 | 上海交通大学 | Preparation method of metal organic framework material and small molecule detection application thereof |
| CN113588769A (en) * | 2021-02-20 | 2021-11-02 | 上海交通大学 | Preparation method of porous alloy nano material and application of porous alloy nano material in detection of plasma metabolites |
| CN114414341A (en) * | 2022-01-25 | 2022-04-29 | 厦门元谱生物科技有限公司 | Detection method for blood culture positive reporting |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107655960A (en) * | 2017-09-18 | 2018-02-02 | 亿纳谱（浙江）生物科技有限公司 | Serum mass spectrometric analysis method, matrix based on gold-palladium and preparation method thereof, application |
| CN111458399A (en) * | 2020-04-14 | 2020-07-28 | 上海交通大学 | Mass spectrum detection method for low-molecular-weight substances based on palladium-gold core-shell micro-nano material |
| CN111413396A (en) * | 2020-04-21 | 2020-07-14 | 四川大学 | Application of ferric oxide nano material and MA L DI-TOF MS in detection of small molecule metabolites |
| CN112834662A (en) * | 2021-02-01 | 2021-05-25 | 上海拜谱生物科技有限公司 | Method for detecting serum small molecule metabolite by using mass spectrum |
| CN113588769A (en) * | 2021-02-20 | 2021-11-02 | 上海交通大学 | Preparation method of porous alloy nano material and application of porous alloy nano material in detection of plasma metabolites |
| CN113447560A (en) * | 2021-05-24 | 2021-09-28 | 上海交通大学 | Small molecule metabolite fragmentation control method based on metal ion addition and application |
| CN113484404A (en) * | 2021-07-02 | 2021-10-08 | 上海交通大学 | Preparation method of metal organic framework material and small molecule detection application thereof |
| CN114414341A (en) * | 2022-01-25 | 2022-04-29 | 厦门元谱生物科技有限公司 | Detection method for blood culture positive reporting |
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