CN101172666A - Fe*O* magnetic material of surface finish C8 alkyl chain, preparation method and application thereof - Google Patents
Fe*O* magnetic material of surface finish C8 alkyl chain, preparation method and application thereof Download PDFInfo
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- CN101172666A CN101172666A CNA2007100477010A CN200710047701A CN101172666A CN 101172666 A CN101172666 A CN 101172666A CN A2007100477010 A CNA2007100477010 A CN A2007100477010A CN 200710047701 A CN200710047701 A CN 200710047701A CN 101172666 A CN101172666 A CN 101172666A
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Abstract
The invention belongs to the field of the inorganic material and analysis technology, in particular to a Fe3O4 magnetic microsphere decorated with C8 alkyl chain at the surface as well as a preparing method and the application thereof. The magnetic nanometer material of ferroferric oxide with surface rich in amido is firstly synthesized, and then the surface is chemically decorated by use of C1-C8, finally the magnetic nanometer material is obtained. The magnetic nanometer particle, which fixes the C1-C8 alkyl chain and is larger than the surface, is used as the micro-adsorbent and can enrich the peptide section in organism. The method is simple and effective. The material possesses perfect application prospect in such fields as Proteomics.
Description
Technical field
The invention belongs to inorganic materials and analysis technical field, be specifically related to a kind of Fe of surface finish C 8 alkyl chain
3O
4Magneticsubstance and its production and application.
Background technology
As everyone knows, protein is formed and to be had diversity and mutability, promptly in the different cells of same species or same cell at different times, its protein group Chengdu is constantly changing, it is complicated unusually to cause protein to form.Simultaneously because there is posttranslational modification in protein, so often corresponding a plurality of protein of mRNA, that is to say, proteinic quantity is far away more than the quantity of gene, protein is widely different aspect size, relative abundance, potential of hydrogen and hydrophobicity, only on relative abundance, high-abundance proteins matter and low abundance proteins differ six orders of magnitude even higher; And because protein can't be as DNA quilt " amplification ", these all cause the low protein of many content to be difficult to be detected in large-scale detection.The analysis of low-abundance protein with identify it is one of the emphasis of proteomics research and difficult point content.The albumen of bearing important vital movement in organism often all is low-abundance protein, yet its extremely low content brings difficulty for follow-up analysis and detection, has limited research and the understanding of people to them.Therefore will be from molecular level, the rule of further investigation vital process, explore the secret of biological phenomena, must analyze monitoring to some low abundance proteinses with important physiological function, this can't but be a stern challenge to present analytical technology and means.
Effectively concentrating of low-abundance protein is to realize one of its accurate essential condition of analyzing and identifying.In fact all relate to effective enrichment of sample in the proteomics research process aspect many, be example with the analysis of enzymolysis sample in the glue: the volume of peptide hydrolysis extracting solution is too big, must concentrate before mass spectroscopy.The most frequently used sample concentration method has solvent evaporated method and chromatogram method of enrichment at present.Solvent evaporated method is wasted time and energy, and the absorption of vessel surface can cause a large amount of peptide section losses in drying process, and impurity such as while inorganic salt also can be concentrated, and influence the sensitivity that mass spectrum is identified.The chromatogram method of enrichment is to utilize the chromatogram interaction partners sample between sample and the sorbent material to concentrate, and sorbent material generally is the silica gel that adopts alkyl chain to modify.This method can be implemented in carries out effective spissated while to sample, removes salinity and other impurity in the sample.Especially when liquid chromatography and mass spectrometry, enter mass spectrum and influence signal detection in order to prevent salt in the sample impurity that grades, all be to have adopted before separator column, to connect a short and small anti-phase pre-column, to realize effect to sample concentration and desalination.Carried out spissated commercial prod Zip-tip of sample desalination and Zip-plate by what people extensively adopted, also all be based on chromatogram and concentrate ratio juris.Be respectively to fill a little reverse phase filler, operate loaded down with trivial details relatively at the rifle head tip or the bottom of 96 orifice bores; And because filler seldom, therefore the spissated sample size of energy enrichment is very limited.In recent years, nano material with its development fast and application potential and being applied in proteome analysis more and more.The former professor of Yang Peng group etc. successfully is applied to the zeolite nanoparticle enrichment of trace peptide section, but this method needs high speed centrifugation sample separation and zeolite mixture, operates loaded down with trivial details relatively.Therefore the method that develops a kind of simple and convenient and effective separation and concentration albumen and peptide section becomes an importance of proteomics research.
Magnetic polymer microsphere is easy to finishing, solution good dispersity and sensitive magnetic field induction with it and provides possibility for its separation and concentration aspect that is applied to trace peptide section in the proteome analysis.The Fe 3 O 4 magnetic polymer microballoon has been synthesized in this research, and then has synthesized the magnetic polymer material of novel surface finish C 8 alkyl chain.Utilize this magnetic polymer material as sorbent material, carry out the enrichment of separation and concentration and MALDI-TOF/MS direct analysis and Preliminary Applications polypeptide in actual blood sample of trace polypeptide.Thereby simplified the assay determination program of lower concentration polypeptide, solved the analysis difficulty of trace samplings; Also opened up new approach for the application of magnetic polymer microsphere.
Summary of the invention
The object of the present invention is to provide a kind of simple to operate, efficient is high, effective, can carry out the Fe of the finishing alkyl chain of enrichment to polypeptide
3O
4Amino-magnetic microballoon and its production and application.
The Fe of finishing alkyl chain provided by the invention
3O
4Magneticsubstance is to adopt hydrothermal method to synthesize with amido modified ferroferric oxide magnetic nano-material earlier, carries out chemically modified with its surface of dimethyl-octa base silane reaction pair then, generates the magnetic microsphere of surface finish C 8 alkyl chain, and its structure is shown below:
Promptly connect the C8 alkyl chain by similar Silanization reaction to magnetic ball surface amino groups.
The preparation method of the magnetic nano-particle of above-mentioned surface finish C 8 alkyl chain is as follows:
(1) has the superparamagnetic nanoparticle of amino Z 250 with the hydrothermal method synthetic surface: adopt 3.0 gram FeCl
36H
2O is a raw material, is dispersion system with 80-90mL ethylene glycol, adds 6-12 gram anhydrous sodium acetate, 10-20 gram 1, the 6-hexanediamine, temperature of reaction is 195-200 ℃, reaction times is 4-6 hour, generates the surface and has amino magnetic nano-particle, and its particle diameter is 20-100nm;
(2) at first, will be by step (1) synthetic Fe
3O
4The amino-magnetic microballoon is used deionized water wash repeatedly, to remove water-soluble impurity, then product is obtained black powder with 45-50 ℃ of oven dry.Secondly, to Fe
3O
4The amino-magnetic microballoon carries out finishing, gets exsiccant Fe
3O
4Amino-magnetic microballoon 10mg is dispersed in the anhydrous pyridine of 1-2mL, adds 0.1-0.2g dimethyl-octa base silane again, and after room temperature mechanical stirred 12-24h, with second alcohol and water repetitive scrubbing, products therefrom is dispersed in the 1mL deionized water, and was standby.
The magnetic microsphere of synthetic surface finish C 8 alkyl chain of the present invention, synthetic method is simple effectively and have a good magnetic field induction, can directly put into the sample that contains polypeptide, need not special processing, after the peptide section is attached to magnetic Nano material, need not centrifugation, adopt the simple magnetic field action can realize the enrichment of peptide section.
The magnetic microsphere of surface finish C 8 alkyl chain provided by the invention provides new method for the enrichment of low-abundance protein, peptide section in the organism, and expanded the practical application of magnetic Nano material, in fields such as bioanalysis researchs good practical value and application prospect are arranged.
Description of drawings
Fig. 1 is the synthetic method of the magnetic nano-particle of surface finish C 8 group.
Fig. 2 is the Fe of hydrothermal method synthetic finishing amino
3O
4Transmission electron microscope picture of magnetic nano-particle (a) and sem photograph (b).It has good homogeneity and dispersiveness.
Fig. 3 is amino-magnetic sodium rice corpuscles (a) and the infrared figure contrast of the amino-magnetic sodium rice corpuscles of modifying with C8 (b), marks peak (2900cm-among the figure
1) Strength Changes represent the increase of nanoparticle surface carbochain, as seen, we have successfully prepared the magnetic sodium rice corpuscles of surface finish C 8 alkyl chain through after the finishing.
Embodiment
Embodiment is further specifying of the separation and concentration analytic process of hanging down abundance peptide section of the magnetic Nano material to the surface finish C 8 alkyl chain of superparamagnetism provided by the present invention.
Synthesizing of the magnetic silicon ball material of embodiment 1 surface finish C 8 alkyl chain
Synthetic being divided into of the magnetic silicon ball material of surface finish C 8 alkyl chain was three steps.
At first, the superparamagnetic nanoparticle that has amino Z 250 with the hydrothermal method synthetic surface; Adopt 3.0 gram FeCl
36H
2O is a raw material, is dispersion system with 90mL ethylene glycol, adds 6-12 gram anhydrous sodium acetate, 10-20 gram 1, and the 6-hexanediamine, temperature of reaction is 198 ℃, and the reaction times is 6 hours, generates the surface and has amino magnetic nano-particle, and its particle diameter is 20-100nm;
Secondly, will synthesize Fe
3O
4The amino-magnetic microballoon is used deionized water wash repeatedly, to remove water-soluble impurity, at last product is obtained black powder with 50 ℃ of oven dry.Again to Fe
3O
4The amino-magnetic microballoon carries out finishing, gets exsiccant Fe
3O
4Amino-magnetic microballoon 10mg is dispersed in the anhydrous pyridine of 1mL, adds 0.1g dimethyl-octa base silane again, and after room temperature mechanical stirred 12-24h, with second alcohol and water repetitive scrubbing, products therefrom was dispersed in the 1mL deionized water standby.
The magnetic nano-particle of embodiment 2 surface finish C 8 alkyl chains is used for the enrichment of peptide section
(1) protein solution enzymolysis:
Get the 1.0mg protein sample, comprise bovine serum albumin (BSA), cytochrome C (Cytochrome C), horse myocardium protein (Myoglobin), cattle beta-casein (β-casein) be dissolved in respectively in the 1.0mL water, after the heat denatured, add ammonium bicarbonate soln regulation system PH in the solution and be about 8, add 25 μ g trypsinase with 1: 50 (mass ratio between enzyme and the albumen).Under 37 ℃ temperature, enzymolysis stopped enzymolysis after 12 hours, and enzymolysis solution places-80 ℃ of refrigerators freezing stand-by.
(2) separation and concentration of peptide section:
With 20 μ L concentration is 2mg mL
-1The magneticsubstance dispersion liquid to join 1mL concentration respectively be 5fmol μ L
-1In the trypsin digestion peptide section mixture of standard peptide section or standard protein, 37 ℃, vibration 90min.Add under the action of a magnetic field remove supernatant after, clean repeatedly three times with pure water; Be dispersed in 5 μ L CHCA (5mg/mL then; Be dissolved among 50% (v/v) acetonitrile and 0.1% (v/v) TFA) 1 μ L is contained by the dispersion liquid point of enrichment peptide section to target plate, carry out MALDI-TOF MS after waiting to do and analyze.
(3) enrichment of peptide section in the salts solution:
With 20 μ L concentration is 10mg mL
-1The magneticsubstance dispersion liquid to join 1mL concentration respectively be 5fmol μ L
-1In the proteic trypsin digestion peptide of the Myoglobin section mixture, 37 ℃, vibration 60min.Add under the action of a magnetic field remove supernatant after, with 20 μ L pure water cleaning 2 times; Be dispersed in 5 μ L CHCA (5mg/mL then; Be dissolved among 50% (v/v) acetonitrile and 0.1% (v/v) TFA) 1 μ L is contained by the dispersion liquid point of enrichment peptide section to target plate, carry out MALDI-TOF MS after waiting to do and analyze.
(4) detection of MALDI-TOF-MS
Get magnetic microsphere dispersion liquid point that 1 μ L is enriched with the peptide section on the MALDI target plate, and then the last 0.5 μ LCHCA of point.After treating the sampling liquid drying, crystallization on the target plate, put target plate into mass spectrograph, carry out the MALDI-TOF mass spectroscopy.The experiment of MALDI-TOF MS mass spectrum is finished on 4700 Proteomics Analyzer (Applied Biosystems); Laser apparatus is a Nd-YAG laser, wavelength 355nm, laser pulse frequency 200Hz; Acceleration voltage 20kV, positive ion mode detects under the reflective TOF condition.
The nano magnetic material of embodiment 3 surface finish C 8 alkyl chains is used for the enrichment of human serum peptide section
Get 10 μ L HASs, add 20 μ L PBS buffered soln dilutions after, adding 20 μ L concentration again is the magneticsubstance of the surface finish C 8 alkyl chain of 10mg/mL; With liquid-transfering gun piping and druming 5 times, behind the 30s, magnetic separates removes supernatant.With enrichment the material PH of peptide section be that 7.0 PBS buffered soln cleans 2 times, supernatant is removed; The material that cleaned is with 5 μ LCHCA (5mg/mL; Be dissolved among 50% (v/v) acetonitrile and 0.1% (v/v) TFA) disperse; 1 μ L is contained by the dispersion liquid point of enrichment peptide section to target plate, put 0.5 μ L CHCA again after doing.Carrying out MALDI-TOF MS after waiting to do analyzes.
Claims (3)
1. the Fe of a surface finish C 8 alkyl chain
3O
4Magnetic microsphere is characterized in that:
Be the ferroferric oxide magnetic nano-material that adopts the hydrothermal method synthesizing amino to modify earlier, carry out chemically modified with its surface of dimethyl-octa base silane reaction pair then, generate the magnetic microsphere of surface finish C 8 alkyl chain, its structure is shown below:
2. the preparation method of the magnetic microsphere of a surface finish C 8 alkyl chain as claimed in claim 1 is characterized in that concrete steps are as follows:
(1) has the superparamagnetic nanoparticle of amino Z 250 with the hydrothermal method synthetic surface: adopt 3.0 gram FeCl
36H
2O is a raw material, is dispersion system with 80-90mL ethylene glycol, adds 6-12 gram anhydrous sodium acetate, 10-20 gram 1, the 6-hexanediamine, temperature of reaction is 195-200 ℃, reaction times is 4-6 hour, generates the surface and has amino magnetic nano-particle, and its particle diameter is 20-100nm;
(2) at first, will be by step (1) synthetic Fe
3O
4Magnetic microsphere is used deionized water wash repeatedly, to remove water-soluble impurity, then product is obtained black powder with 45-50 ℃ of oven dry; Secondly, to Fe
3O
4Magnetic microsphere carries out finishing, gets exsiccant Fe
3O
4Amino-magnetic microballoon 10mg is dispersed in the anhydrous pyridine of 1-2mL, adds 0.1-0.2g dimethyl-octa base silane again, and after room temperature mechanical stirred 12-24h, with second alcohol and water repetitive scrubbing, products therefrom is dispersed in the 1mL deionized water, and was standby.
3. the application in albumen of the rich end and the peptide section is hanged down in the enrichment in organism of the magnetic microsphere of the described surface finish C 8 alkyl chain of claim 1.
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| CNA2007100477010A CN101172666A (en) | 2007-11-01 | 2007-11-01 | Fe*O* magnetic material of surface finish C8 alkyl chain, preparation method and application thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101805026A (en) * | 2010-03-12 | 2010-08-18 | 南京大学 | Method for preparing spherical super-paramagnetic ferroferric oxide nano-clusters |
| CN106990250A (en) * | 2017-05-23 | 2017-07-28 | 华中科技大学 | A kind of preparation method and application for reducing self-assembled protein coated magnetic microballoon |
| CN108970631A (en) * | 2018-07-28 | 2018-12-11 | 张剑 | A kind of nano-copper base catalyst and preparation method thereof |
-
2007
- 2007-11-01 CN CNA2007100477010A patent/CN101172666A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101805026A (en) * | 2010-03-12 | 2010-08-18 | 南京大学 | Method for preparing spherical super-paramagnetic ferroferric oxide nano-clusters |
| CN106990250A (en) * | 2017-05-23 | 2017-07-28 | 华中科技大学 | A kind of preparation method and application for reducing self-assembled protein coated magnetic microballoon |
| CN106990250B (en) * | 2017-05-23 | 2017-12-08 | 华中科技大学 | A kind of preparation method and application for reducing self-assembled protein coated magnetic microballoon |
| CN108970631A (en) * | 2018-07-28 | 2018-12-11 | 张剑 | A kind of nano-copper base catalyst and preparation method thereof |
| CN108970631B (en) * | 2018-07-28 | 2021-06-29 | 茂名市科达化工有限公司 | Nano copper-based catalyst and preparation method thereof |
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Open date: 20080507 |