CN1281961C - Method for fixing protein on surface of protein chip - Google Patents
Method for fixing protein on surface of protein chip Download PDFInfo
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- CN1281961C CN1281961C CN 03157199 CN03157199A CN1281961C CN 1281961 C CN1281961 C CN 1281961C CN 03157199 CN03157199 CN 03157199 CN 03157199 A CN03157199 A CN 03157199A CN 1281961 C CN1281961 C CN 1281961C
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Abstract
The present invention relates to a method for fixing a protein on the surface of a protein chip. A protein chip is soaked in an anhydrous alcohol solution of a component A and a component B, and the component A is an organic compound capable of causing the surface of the protein chip to be hydrophobic; the component B is an organic compound, wherein one end of the component B contains active groups which can be covalently connected with a protein molecule needing fixing, and one end contains polar groups; then, a common method is used for activating the polar groups of the component B; finally, the protein chip is placed into a protein solution needing fixing to be soaked. The protein fixed by the method is stable, the interaction between the surface of the protein chip and the protein molecule can be effectively reduced, and the influence of the surface of the protein chip on a space image of the protein molecule is reduced. Compared with the biological activity of the protein fixed by physical adsorption or simple the component B, the biological activity of the protein fixed by the method is enhanced by more than one time.
Description
Technical field
The present invention relates to a kind of at protein-chip surface method for immobilizing protein.
Background technology
Protein is a kind of composition, baroque organic macromolecule, almost can take place to interact and adsorb from the teeth outwards with all solids surface.The fixed target protein molecule has been widely used in biomedical sectors such as protein purification, solid-phase immunity detection and biomaterial preparation on the protein-chip surface.
At present the main method of fixing protein is by physisorption on the protein-chip surface, and just the static by between the two, hydrophobic and hydrogen bond etc. interact protein is fixing to solid surface.As Jonsson, U.; Ivarsson, B.; Lundstrom, I.; Berghem, L J.Colloid.Interface Sci.1982 described in 90,148., at protein-chip surface fixing protein, has been to use dichlorodimethylsilane modification silicon face by physisorption.But, use the method for physisorption, the biologically active that is fixed on protein-chip proteins on surfaces molecule all is lower than the protein active under the solution state usually, its main cause is that protein molecule is when being adsorbed onto solid surface, its space structure picture also can change thereupon, and the biologically active of protein molecule is depend on its space structure picture stable.In addition, Gu Ding protein is very unstable by this method, on the one hand, is having under the environment of working fluid, is fixed on the protein-chip proteins on surfaces and comes off easily; On the other hand, because competitive Adsorption, other protein also can substitute original proteins on surfaces that is fixed on, and causes its loss.
Another kind of method for immobilizing protein on the protein-chip surface comparatively commonly used is by covalently bound mode.As Haodan Yuan, Wayne M.Mullett and Janusz Pawliszyn, Biological sample analysiswith immunoaffinity solid-phase microextraction, The Analyst, 2001,126, pass through covalent approach at protein-chip surface fixing protein described in the 1456-1461., be to use aminopropyl triethoxysilane modification silicon face.The covalent bond that the method forms is more firm than physisorption, makes protein relatively stable, can partly solve the protein instability problem that the physisorption method exists.But, the group that is used for Covalent Immobilization protein on the protein-chip surface mostly is the group of polarity, this can further strengthen the electrostatic interaction between the protein-protein chip surface, thereby makes the space structure picture of protein that bigger change take place, and causes bioactive reduction.
Summary of the invention
The objective of the invention is to overcome existing method for immobilizing protein and make protein space structure picture that low, the fixing unsettled defective of protein of bigger change, protein biologically active take place, thus provide a kind of protein stabilization of fix, space structure picture influenced little, biologically active is high, at the surperficial method for immobilizing protein of protein-chip.
The objective of the invention is to realize by the following technical solutions:
The invention provides a kind of at protein-chip surface method for immobilizing protein, comprise following step: the 1) ethanol solution of formulation components A and B component, wherein the concentration of component A is 1 mM～1 mole, and the mol ratio of component A and B component is 20～100: 1; Described component A is for can make the protein-chip surface be hydrophobic organic compound; Described B component is that an end contains the organic compound that can contain polar group with required fixing protein molecule with covalently bound reactive group, an end;
2) substrate of protein-chip is positioned over soak in the solution of step 1) and carried out modification in 30 minutes～5 hours; Clean solid substrate 3 times with absolute ethyl alcohol; Re-use the polar group of conventional method activating component B;
3) will add 0.05～1wt% non-ionics in the required fixing protein solution;
4) with step 2) substrate of the protein-chip of the modification protein solution of putting into step 3) soaked 30 minutes.
Described component A is methyl triethoxysilane, undecane mercaptan or methyltrimethoxy silane.
Described B component is aminopropyl triethoxysilane, carboxyl undecane mercaptan or sulfydryl trimethoxy silane.
The substrate of described protein-chip is glass, silicon chip, metal or plastics.
Described protein comprises immunoglobulin G while, human albumin.
Described non-ionics is Tween 20 (tween).
The principle of this method is by two kinds of organic compounds---component A and B component are used simultaneously, at protein-chip surface fixing protein.Wherein, component A is used for closed protein matter chip surface, makes the protein-chip surface be strong-hydrophobicity, electric neutrality, does not form hydrogen bond with protein molecule, as methyl triethoxysilane, alkanethiol etc.; Contain and to contain the organic compound of polar group with required fixing protein molecule with covalently bound reactive group, an end and B component is an end, as amino triethoxysilane, carboxyl alkanethiol etc.
Provided by the invention at protein-chip surface method for immobilizing protein compared with the prior art, its advantage is:
1, on the one hand, this method is used the reactive group of B component, forms covalent bond between protein molecule and solid surface, and fixing protein is more stable;
2, simultaneously, because the covalent bond quantity that forms between protein molecule and the solid surface is few more, just helping protein molecule more keeps its space structure picture, so amount by control component A and B component, make on the area that a protein molecule covers, only connect with a covalent bond, just be enough to make it to be fixed on the protein-chip surface, and can effectively reduce interaction between protein-chip surface and protein molecule, reduce of the influence of protein-chip surface to the space structure picture of protein molecule;
3, can select and the molecular weight of protein molecule and volume differences organic molecule apart from big surface modification, the organic molecule that is used for Covalent Immobilization protein so only need sparsely intersperse among the protein-chip surface, just be enough to form saturated protein molecule rete, can effectively reduce the interaction between protein-chip surface and protein molecule, reduce of the influence of protein-chip surface the space structure picture of protein molecule;
4, by adding a spot of non-ionics, effectively Profilin matter molecule keeps the space structure picture of protein molecule in the lip-deep physisorption of protein-chip;
5, by the ratio of regulation and control two organic molecular species, can control protein quantitatively in the lip-deep fixed amount of protein-chip
6, the biological activity ratio of fixing protein improves more than 1 times by physisorption or the fixing protein biologically active of simple B component by this method.
Embodiment
Embodiment 1
The mixed ethanol solution of preparation methyl triethoxysilane and aminopropyl triethoxysilane (mol ratio of the two is 20: 1), the concentration of methyl triethoxysilane is 1 mM;
To be that the protein-chip of substrate is positioned over to soak in the above-mentioned solution and carried out modification in 5 hours with the silicon chip; Clean solid substrate 3 times with absolute ethyl alcohol; Re-use the amino on the glutaraldehyde activating ammonia propyl-triethoxysilicane;
Preparation 1mg/ml immunoglobulin G while PBS (phosphate buffer) solution wherein contains 0.05wt%Tween 20; The protein-chip of modification is put into this immunoglobulin G while solution to be soaked 30 minutes.
Embodiment 2
The mixed ethanol solution of preparation undecane mercaptan and carboxyl undecane mercaptan (mol ratio of the two is 100: 1), the concentration of undecane mercaptan is 1 mole;
To be that the protein-chip of substrate is positioned over to soak in the above-mentioned solution and carried out modification in 30 minutes with the gold; Clean solid substrate 3 times with absolute ethyl alcohol; Re-use the carboxyl of ethyl carbodiimide (EDC) activated carboxyl undecane mercaptan;
Preparation 1mg/ml human albumin PBS solution wherein contains 1wt%Tween 20;
The protein-chip of modification is put into this human albumin's solution to be soaked 30 minutes.
Claims (4)
1, a kind of at protein-chip surface method for immobilizing protein, comprise following step:
1) ethanol solution of formulation components A and B component, wherein the concentration of component A is 1 mM～1 mole, the mol ratio of component A and B component is 20～100: 1; Described component A is methyl triethoxysilane, undecane mercaptan or methyltrimethoxy silane; Described B component is aminopropyl triethoxysilane, carboxyl undecane mercaptan or sulfydryl trimethoxy silane;
2) substrate of protein-chip is positioned over soak in the solution of step 1) and carried out modification in 30 minutes～5 hours; Clean solid substrate 3 times with absolute ethyl alcohol; Re-use the polar group of conventional method activating component B;
3) will add 0.05～1wt% non-ionics in the required fixing protein solution;
4) with step 2) substrate of the protein-chip of the modification protein solution of putting into step 3) soaked 30 minutes.
2, as claimed in claim 1 at protein-chip surface method for immobilizing protein, it is characterized in that: the substrate of described protein-chip is glass, silicon chip, metal or plastics.
3, as claimed in claim 1 at protein-chip surface method for immobilizing protein, it is characterized in that: described protein comprises immunoglobulin G while, human albumin.
4, as claimed in claim 1 at protein-chip surface method for immobilizing protein, it is characterized in that: described non-ionics is Tween 20.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 03157199 CN1281961C (en) | 2003-09-18 | 2003-09-18 | Method for fixing protein on surface of protein chip |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 03157199 CN1281961C (en) | 2003-09-18 | 2003-09-18 | Method for fixing protein on surface of protein chip |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1598587A CN1598587A (en) | 2005-03-23 |
| CN1281961C true CN1281961C (en) | 2006-10-25 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 03157199 Expired - Lifetime CN1281961C (en) | 2003-09-18 | 2003-09-18 | Method for fixing protein on surface of protein chip |
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Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101745495B (en) * | 2008-12-12 | 2012-04-25 | 中国科学院理化技术研究所 | Method for inhibiting non-specific adsorption on solid material surface |
| CN101776546B (en) * | 2010-01-08 | 2011-07-27 | 广西大学 | Method for fixing protein molecules on organic glass |
| CN101833001B (en) * | 2010-04-21 | 2013-08-28 | 广州瑞博奥生物科技有限公司 | Protein chip kit for detecting inflammatory factors and preparation method thereof |
| CN102175668B (en) * | 2011-01-28 | 2012-10-17 | 上海铭源数康生物芯片有限公司 | Method for inhibiting trailing of point of solid-phase protein chip by changing chemiluminescence mode |
| CN104502311B (en) * | 2014-12-15 | 2017-09-05 | 国家纳米科学中心 | A kind of protein fixing means based on surface plasma resonance image-forming biologic sensor chip |
| CN109610006A (en) * | 2018-10-12 | 2019-04-12 | 深圳市瀚海基因生物科技有限公司 | The fixing means and chip of a kind of preparation method of chip, DNA or protein |
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2003
- 2003-09-18 CN CN 03157199 patent/CN1281961C/en not_active Expired - Lifetime
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| Publication number | Publication date |
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| CN1598587A (en) | 2005-03-23 |
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Application publication date: 20050323 Assignee: GUIZHOU ZHONGKE JINJIU BIOTECHNOLOGY CO.,LTD. Assignor: INSTITUTE OF MECHANICS, CHINESE ACADEMY OF SCIENCES Contract record no.: 2015990000793 Denomination of invention: Method for fixing protein on surface of protein chip Granted publication date: 20061025 License type: Common License Record date: 20150906 |
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