CN1975429B - Nano-magnetic microsphere with amino having fixed protease at surface, preparing method and application thereof - Google Patents
Nano-magnetic microsphere with amino having fixed protease at surface, preparing method and application thereof Download PDFInfo
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- CN1975429B CN1975429B CN2006101472503A CN200610147250A CN1975429B CN 1975429 B CN1975429 B CN 1975429B CN 2006101472503 A CN2006101472503 A CN 2006101472503A CN 200610147250 A CN200610147250 A CN 200610147250A CN 1975429 B CN1975429 B CN 1975429B
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Abstract
The invention relates to an nm magnetic micro ball with the amino which the surface is fixed with the protease and the preparation method, also it is applied in the protein enzymolysis. The micro ball is composed by the one-step hydro-thermal method and with the particle size of 15-200nm. The glutaraldehyde is as the cross linker and the protease is fixed on the surface of the nm magnetic micro ball by which the protein is solution, slug, target decomposed. The method of the invention is simple, so it is proper for separation and identification of the protein in complex biological sample and it has the good future in the protein study.
Description
Technical field
The invention belongs to the biochemical analysis technical field, be specifically related on a kind of nano-magnetic microsphere of the band amino that has fixed protease at surface and preparation method thereof and the application in proteolysis.
Technical background
In proteinic identification and analysis, proteinic enzymolysis is a crucial step.Loaded down with trivial details, the consuming time length of traditional in-solution digestion method steps is unfavorable for realizing automated operation.Along with of the extensive expansion of the present whole world, press for more effective, the simpler proteolysis technology of development to proteomics research in the organism.
Immobilized enzyme replaces traditional in-solution digestion to have many advantages: (1) improves enzyme stability, and is reusable; (2) improve the enzyme concn of unit volume, thereby increase the enzyme service efficiency, reduce enzymolysis time; (3) be easy to immobilized enzyme is separated with substrate, product, do not occur in the peptide spectrum enzyme from the hydrolysis peak; (4) can be used for constructing enzyme reactor, batch reactions and successive reaction help realizing flux and automatization repeatedly.Magnetic microsphere has attracted to pay close attention to more and more in recent years, with its carrier as immobilized enzyme, has the advantage of handled easily and good reproducibility.Nano-magnetic microsphere is to be born on the basis of former magnetic microsphere.Magnetic microsphere refers to a kind of microballoon of shell/nuclear structure, and nuclear is made up of metal oxide (as the oxide compound of iron, cobalt, nickel), and shell is made up of macromolecular material, examines the different macromolecular material of exoperidium according to application need at magnetic Nano.The nano-magnetic microsphere that utilizes traditional method to make has monodispersity and degree of crystallinity preferably, yet, before the process finishing, these nano-magnetic microspheres that adopt pyrolysis techniques to make only have good dispersiveness in non-polar solvent, this has hindered their application at biological technical field greatly.Recently, bibliographical information has been arranged a kind of method of nano-magnetic microsphere of one-step synthesis band amino, prepared magnetic microsphere particle diameter distributes wide; Has superparamagnetism; And in water, has a good monodispersity; Have broad application prospects at biological technical field.
Summary of the invention
The purpose of this invention is to provide a kind of amino nano-magnetic microsphere of band and preparation method thereof and in proteolysis, using of having fixed protease at surface.
The method of ankyrin enzyme on the amino nano-magnetic microsphere of band that the present invention proposes, concrete steps are as follows:
(1) the band amino-magnetic microballoon of exsiccant particle diameter between the 15-200 nanometer is scattered in 50mM Ammoniom-Acetate damping fluid and (is designated as CB, its composition: NH
4OAc is 50mM, CaCl
21mM, MnCl
21mM, pH8.0-8.5) in, the content of magnetic microsphere in solution is 1-10mg/mL;
(2) hold nano-magnetic microsphere with magnet, remove CB, it is scattered in again (in this solution, the weight content of GA is 5%-10%, pH6.5-7.0), and at room temperature activates 1-3 hour in the CB solution of glutaraldehyde (GA);
(3) will be scattered in the CB solution of proteolytic enzyme by GA activatory nano-magnetic microsphere and at room temperature soak 3-5 hour with immobilized enzyme, wherein the content of proteolytic enzyme be 2-5mg/mL in the CB solution, NaCNBH
3Weight content be 0.8-1.2%;
(4) nano-magnetic microsphere that will fix proteolytic enzyme is scattered in the CB solution of glycine and at room temperature soaks 1-2 hour with the aldehyde radical of immobilized enzyme not on the sealing magnetic microsphere, and wherein the content of glycine is 0.5%-1.0% in the CB solution, NaCNBH
3Weight content be 0.8-1.2%.
Nano-magnetic microsphere by the immobilized enzyme of method for preparing has superparamagnetism, and promptly the magnetic grain has only and just shows strong magnetic under the outside magnetic field effect, and after foreign field removes, then no longer includes magnetic, its saturation magnetization height, and remanent magnetism and coercive force are little.General almost spherical, globule size can not coexist as required, and the 15-200nm scope is interior to be selected, and the nano-magnetic microsphere particle diameter ratio that makes is more even, and particle size distribution is narrow, within ± 10%.Nano-magnetic microsphere can be scattered in the aqueous solution preferably, is difficult for precipitation and coagulates wadding mutually.The fixed amount of proteolytic enzyme is 50-100 μ g/mg.
Among the present invention, described nano-magnetic microsphere is the Nano microsphere of shell/nuclear structure of being often referred to, and its nuclear is made up of metal oxide (as the oxide compound of iron, cobalt, nickel etc.), and shell is made up of macromolecular material.
The nano-magnetic microsphere of the present invention preparation can be applicable to proteolysis, comprises on protein soln enzymolysis, protein chip enzymolysis and the protein target enzymolysis etc.
The application of nano-magnetic microsphere in the protein soln enzymolysis that is fixed with proteolytic enzyme of the present invention specifically comprises the steps:
(1) treats that the protein soln of enzymolysis was at 90-100 ℃ of following thermally denature 15-30 minute;
(2) nano-magnetic microsphere that has fixed protease at surface is scattered among the CB, adds the protein of sex change, at 30-40 ℃ of following enzymolysis 0.5-2 hour, wherein the content of magnetic microsphere in CB solution was 1-3mg/mL;
(3) hold magnetic microsphere with magnet after, supernatant liquor is put on the target plate;
(4) the acetonitrile solution point that will contain alpha-cyano-4-hydroxyl meat silicic acid (CHCA) is again sent into mass spectrograph analysis after the drying on the sample target.
The application of nano-magnetic microsphere in the protein chip enzymolysis that is fixed with proteolytic enzyme of the present invention specifically comprises the steps:
(1) magnet is placed the below of chip microchannel;
(2) the magnetic microsphere dispersion liquid of surperficial immobilized enzyme flows through chip microchannel under the promotion of pump;
(3) magnetic microsphere is fixed in the chip microchannel under the action of a magnetic field, forms the packed bed of 1-3mm.
(4) treat that the protein soln of enzymolysis was at 90-100 ℃ of following thermally denature 15-30 minute;
(5) protein solution is pushed in the chip enzyme reactor based on functionalized magnetic microsphere with pump, sealed at both ends, at 20-60 ℃ of following enzymolysis 2-10 minute;
(6) will release with pump based on the enzymolysis product in the chip enzyme reactor of functionalized magnetic microsphere, collect the back point on target plate;
(7) the acetonitrile solution point that will contain alpha-cyano-4-hydroxyl meat silicic acid (CHCA) is again sent into mass spectrograph analysis after the drying on the sample target.
The nano-magnetic microsphere application in the enzymolysis on the protein target that is fixed with proteolytic enzyme of the present invention specifically comprises the steps:
(1) treats that the protein soln of enzymolysis was at 90-100 ℃ of following thermally denature 15-30 minute;
(2) the magnetic microsphere dispersion liquid point that 1 μ L is had fixed protease at surface is to the MALDI target plate;
(3) 1 μ L is treated the protein solution point of enzymolysis is to same target spot;
(4) at 20-60 ℃ of following enzymolysis 2-10 minute;
(5) siphon away nano-magnetic microsphere with magnet;
(6) the acetonitrile solution point that will contain alpha-cyano-4-hydroxyl meat silicic acid (CHCA) is again sent into mass spectrograph analysis after the drying on the sample target.
Embodiment
Below by embodiment the present invention is further described in detail.
Embodiment 1. is being with amino nano-magnetic microsphere surface ankyrin enzyme:
(1) be that the nano-magnetic microsphere of the band amino of 500nm is scattered in 200 μ L 50mM Ammoniom-Acetate damping fluid (CB, CaCl with 1mg exsiccant particle diameter
21mM, MnCl
21mM, pH8.3) in;
(2) hold nano-magnetic microsphere with magnet, remove CB, it is scattered in again (GA 5%-10% pH6.8), and at room temperature activates 1.5 hours in the CB solution of 200 μ L glutaraldehyde (GA);
(3) will be scattered in the CB solution of 200 μ L proteolytic enzyme by GA activatory nano-magnetic microsphere and at room temperature soak 3h with immobilized enzyme, wherein the content of proteolytic enzyme be 5mg/mL in the CB solution, NaCNBH
3Content be 1%;
(4) nano-magnetic microsphere that will fix proteolytic enzyme is scattered in the CB solution of 200 μ L glycine and at room temperature soaks 1 hour with the aldehyde radical of immobilized enzyme not on the sealing magnetic microsphere, and wherein the content of glycine is 0.5%-1.0% in the CB solution, NaCNBH
3Content be 1%.
After tested, the amount of fixed proteolytic enzyme is the 50-100 microgram on the nano magnetic material of every milligram of band amino.
The nano-magnetic microsphere that embodiment 2. will fix proteolytic enzyme is applied to the protein soln enzymolysis:
(1) the CB solution of the cytochrome C of 0.2mg/mL was 95 ℃ of following thermally denatures 15 minutes;
(2) the 1mg nano-magnetic microsphere of having fixed proteolytic enzyme is scattered among the 1mLCB, goes 10 μ L magnetic microsphere dispersion liquids to add the 10 μ L cytochrome c solution of sex change, and 37 ℃ of following enzymolysis 1 hour, wherein the content of magnetic microsphere in CB solution was 1-3mg/mL;
(3) hold magnetic microsphere with magnet after, supernatant liquor is put on the target plate;
(4) the acetonitrile solution point that will contain alpha-cyano-4-hydroxyl meat silicic acid (CHCA) is again sent into mass spectrograph analysis after the drying on the sample target.
The result of mass spectroscopy is similar to the result that traditional in-solution digestion obtains, and the peptide section number on the cytochrome C coupling is 12, and the amino acid number is 81, and peptide section fraction of coverage is 77%.
The nano-magnetic microsphere that embodiment 3. will fix proteolytic enzyme is applied to the protein chip enzymolysis:
(1) magnet is placed the below of chip microchannel;
(2) the magnetic microsphere dispersion liquid of immobilized enzyme flows through chip microchannel under the promotion of pump;
(3) magnetic microsphere is fixed in the chip microchannel under the action of a magnetic field, forms the packed bed of 1-3mm.
(4) the CB solution of the cytochrome C of 0.2mg/mL was 95 ℃ of following thermally denatures 15 minutes;
(5) cytochrome c solution of 0.5 μ L sex change is pushed in the chip enzyme reactor based on functionalized magnetic microsphere with pump, sealed at both ends, 50 ℃ of following enzymolysis 5 minutes;
(6) will release with pump based on the enzymolysis product in the chip enzyme reactor of functionalized magnetic microsphere, collect the back point on target plate;
(7) the acetonitrile solution point that will contain alpha-cyano-4-hydroxyl meat silicic acid (CHCA) is again sent into mass spectrograph analysis after the drying on the sample target.
The result of mass spectroscopy shows that the peptide section number on the cytochrome C coupling is 9, and the amino acid number is 81, and peptide section fraction of coverage is 77%.
The nano-magnetic microsphere that embodiment 4. will fix proteolytic enzyme is applied to enzymolysis on the protein target:
(1) 1 μ L has been fixed the magnetic microsphere dispersion liquid point of proteolytic enzyme to the MALDI target plate;
(2) the CB solution of the cytochrome C of 0.2mg/mL was 95 ℃ of following thermally denatures 15 minutes;
(3) cytochrome c solution of 1 μ L sex change is put on the same target spot;
(4) 50 ℃ of following enzymolysis 5 minutes;
(5) siphon away nano-magnetic microsphere with magnet;
(6) the acetonitrile solution point that will contain alpha-cyano-4-hydroxyl meat silicic acid (CHCA) is again sent into mass spectrograph analysis after the drying on the sample target.
The result of mass spectroscopy shows that the peptide section number on the cytochrome C coupling is 13, and the amino acid number is 80, and peptide section fraction of coverage is 76%.
Claims (6)
1. the preparation method of the nano-magnetic microsphere of a band amino that has fixed protease at surface is characterized in that described nano-magnetic microsphere has amino and is fixed with proteolytic enzyme, and the fixed amount of proteolytic enzyme is 50-100 μ g/mg, and particle diameter is 15-200nm; The concrete steps of preparation are as follows:
(1) be that the band amino-magnetic microballoon of 15-200 nanometer is scattered in the ammonium acetate buffer with particle diameter, remember that this damping fluid is CB, the content of magnetic microsphere in CB is 1-10mg/mL;
(2) hold nano-magnetic microsphere with magnet, remove CB, it is scattered in the CB solution of glutaraldehyde again, and at room temperature activates 1-3 hour, the content of glutaraldehyde is 5%-10% in the CB solution of described glutaraldehyde, and the pH value is 6.5-7.0;
(3) will be scattered in the CB solution that contains proteolytic enzyme by glutaraldehyde activatory nano-magnetic microsphere and at room temperature soak 3-5 hour with immobilized enzyme, wherein, in the CB solution of described proteolytic enzyme, the content of proteolytic enzyme is 2-5mg/mL, NaCNBH
3Content be 0.8-1.2%;
(4) nano-magnetic microsphere that will fix proteolytic enzyme is scattered in the CB solution of glycine, and at room temperature soaks 1-2 hour with the aldehyde radical of immobilized enzyme not on the sealing magnetic microsphere; In the CB solution of described glycine, the content of glycine is 0.5%-1.0%, NaCNBH
3Content be 0.8-1.2%.
2. method according to claim 1 is characterized in that, the consisting of of step (1) described ammonium acetate buffer: NH
4OAc is 50mM, CaCl
2Be 1mM, MnCl
2Be 1mM, the pH value is 8.0-8.5.
3. nano-magnetic microsphere application in the enzymolysis on protein soln enzymolysis, protein chip enzymolysis or protein target of the band amino that has fixed protease at surface of the method for claim 1 preparation.
4. application according to claim 3, the concrete steps that it is characterized in that being applied to the protein soln enzymolysis are as follows:
(1) treats that the protein soln of enzymolysis was at 90-100 ℃ of following thermally denature 15-30 minute;
(2) nano-magnetic microsphere that is fixed with proteolytic enzyme is scattered among the CB, adds the protein of thermally denature, and at 30-40 ℃ of following enzymolysis 0.5-2 hour, wherein, the content of magnetic microsphere in CB solution was 1-3mg/ml;
(3) hold magnetic microsphere with magnet after, supernatant liquor is put on the target plate;
(4) the acetonitrile solution point that will contain alpha-cyano-4-hydroxyl meat silicic acid is again sent into mass spectrograph analysis after the drying on the sample target.
5. application according to claim 3, the concrete steps that it is characterized in that being applied to the protein chip enzymolysis are as follows:
(1) magnet is placed the below of chip microchannel;
(2) the magnetic microsphere dispersion liquid of surperficial immobilized enzyme flows through chip microchannel under the promotion of pump;
(3) magnetic microsphere is fixed in the chip microchannel under the action of a magnetic field, forms the packed bed of 1-3mm;
(4) treat that the protein soln of enzymolysis was at 90-100 ℃ of following thermally denature 15-30 minute;
(5) protein solution is pushed in the chip enzyme reactor based on functionalized magnetic microsphere with pump, sealed at both ends, at 20-60 ℃ of following enzymolysis 2-10 minute;
(6) will release with pump based on the enzymolysis product in the chip enzyme reactor of functionalized magnetic microsphere, collect the back point on target plate;
(7) the acetonitrile solution point that will contain alpha-cyano-4-hydroxyl meat silicic acid is again sent into mass spectrograph analysis after the drying on the sample target.
6. application according to claim 3, the concrete steps that it is characterized in that being applied to enzymolysis on the protein target are as follows:
(1) treats that the protein soln of enzymolysis was at 90-100 ℃ of following thermally denature 15-30 minute;
(2) the magnetic microsphere dispersion liquid point that 1 μ L is had fixed protease at surface is to the MALDI target plate;
(3) 1 μ L is treated the protein solution point of enzymolysis is to same target spot;
(4) under 20-60 ℃ of temperature enzymolysis 2-10 minute;
(5) siphon away nano-magnetic microsphere with magnet;
(6) the acetonitrile solution point that will contain alpha-cyano-4-hydroxyl meat silicic acid is again sent into mass spectrograph analysis after the drying on the sample target.
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| CN2006101472503A CN1975429B (en) | 2006-12-14 | 2006-12-14 | Nano-magnetic microsphere with amino having fixed protease at surface, preparing method and application thereof |
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| CN1975429B true CN1975429B (en) | 2011-11-02 |
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| CN106732437A (en) * | 2017-03-10 | 2017-05-31 | 深圳市瑞赛生物技术有限公司 | A kind of affine magnetic microsphere and its preparation and application for purifying and detecting carbamate and organophosphorus pesticide |
| CN107899552B (en) * | 2017-10-31 | 2020-06-30 | 苏州博进生物技术有限公司 | Metal chelating affinity chromatography medium using magnetic polymer microsphere as matrix |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1296032A (en) * | 2000-11-21 | 2001-05-23 | 上海博纳科技发展有限公司 | Polyacrylonitrile magnetic pearl and preparation process and application thereof |
| CN1376792A (en) * | 2001-03-27 | 2002-10-30 | 内蒙古师范大学 | Process for preparing magnetic microsphere immobilized cellulase |
| CN1688001A (en) * | 2005-03-30 | 2005-10-26 | 深圳市人民医院 | Nano-magnetic microsphere and nona-magnetic immuno-microsphere, and preparing process and application thereof |
| CN101177678A (en) * | 2006-11-09 | 2008-05-14 | 中国科学院大连化学物理研究所 | Magnetic nano particle enzyme immobilization as well as preparation method and uses thereof |
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2006
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1296032A (en) * | 2000-11-21 | 2001-05-23 | 上海博纳科技发展有限公司 | Polyacrylonitrile magnetic pearl and preparation process and application thereof |
| CN1376792A (en) * | 2001-03-27 | 2002-10-30 | 内蒙古师范大学 | Process for preparing magnetic microsphere immobilized cellulase |
| CN1688001A (en) * | 2005-03-30 | 2005-10-26 | 深圳市人民医院 | Nano-magnetic microsphere and nona-magnetic immuno-microsphere, and preparing process and application thereof |
| CN101177678A (en) * | 2006-11-09 | 2008-05-14 | 中国科学院大连化学物理研究所 | Magnetic nano particle enzyme immobilization as well as preparation method and uses thereof |
Non-Patent Citations (3)
| Title |
|---|
| JP平2-128690A 1990.05.17 |
| JP特开2004-256445A 2004.09.16 |
| 丁丽俐，等.氨基末端磁性载体固定化中性蛋白酶的研究.《生物化学与生物物理进展》.2001,第28卷(第5期),691-694. * |
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