CN1381723A - Protein chip and its preparing process - Google Patents
Protein chip and its preparing process Download PDFInfo
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- CN1381723A CN1381723A CN 02111698 CN02111698A CN1381723A CN 1381723 A CN1381723 A CN 1381723A CN 02111698 CN02111698 CN 02111698 CN 02111698 A CN02111698 A CN 02111698A CN 1381723 A CN1381723 A CN 1381723A
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- protein
- chip
- metal ion
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Abstract
A protein chip for biochemical reactions is prepared through cross-linking the metal ion chelate onto the surface of solid substrate by covalent binding, chelating the metal ion chelate with metal ion under the alkaline condition, and chelating protein molecule with the said metal ion via its his-tag at its terminal. Its solid substrate can be used cyclically. The protein fixed on this protein chip can perform different biochemical reactions and be applied to the research of life science.
Description
Technical field
The invention belongs to technical field of molecular biology, relate to a kind of protein-chip and preparation method thereof.Specifically, relate to a kind of directed protein-chip of histidine tagged protein matter and preparation method thereof that fixedly has of chelate of metal ion (nitrilotriacetic acid (NTA)) that utilizes, this chip can carry out various biochemical reactions, can be used for life science, medical diagnosis on disease and drug screening etc.
Background technology
In recent years, the biotechnology of develop rapidly has driven the innovation of traditional medical diagnosis on disease mode and drug screening mode.Because advantages such as chip technology has fast, micro-, accurate, convenient and high flux, thereby paid much attention to.Biochip has genetic chip, protein-chip and chip lab three classes.Genetic chip can be measured expression conditions or gene mutation situation, needs to use nucleic acid amplification in vitro technology usually.Protein-chip is to detect interactional biochip between the protein, at present the protein biochip technology in the research is mostly based on the principle of receptors ligand specific bond, multiple proteins is combined on the solid-phase matrix the corresponding protein of the combination of selectivity with it that is contained in the detection of biological.Aspect drug screening, protein-chip has its advantage of not replacing.
The key that protein-chip is made is how reactive protein to be fixed in stromal surface with orientation, solid support commonly used now has materials such as slide, plastics, porous silicon, polystyrene, nitrocellulose membrane, nylon membrane, common set mode has absorption, methods such as stromal surface modification.Wherein, plastic material utilizes the fixing big molecule of the character of molecular adsorption more, all be beneficial to more macromolecular absorption because of having electrostatic attraction as polystyrene and polychloroethylene film, but the anchor effect of this absorption is not good, easily causes macromolecular peeling off.Nitrocellulose membrane and nylon membrane mainly are the characteristics with micropore, help the absorption of protein molecule, but nitrocellulose membrane and nylon membrane are not had a light transmission, be difficult for being applied to chip, and protein is adsorbed on the film with any direction, make its fixed-direction various, be unfavorable for the exposure of ligand receptor action site.
Summary of the invention
One object of the present invention is to provide a kind of protein-chip, utilizes the directed easily fixed target protein of chelate of metal ion and histidine mark (His-tag).
Another object of the present invention is to provide a kind of method for making of effective cost-effective protein-chip, overcome shortcomings such as the protein positioning direction is different in the protein-chip that existing method makes, protein bound intensity is less, used easier method to be fixed in the solid matrix surface to the protein orientation that has histidine mark.
The protein-chip that the present invention proposes is made up of solid matrix and protein bound, wherein, chelate of metal ion is arranged in that solid matrix is surface-crosslinked, and protein has histidine mark, and mediates by metallic ion and to be positioned on the solid matrix.Here chelate of metal ion is by three carboxyls and the metal ion-chelant of its C end.The histidine mark of protein (His-tag) can be at the N of protein end or C end, the histidine mark of protein N terminal or C end can with above-mentioned metal ion-chelant, thereby the protein orientation is fixed on the slide.
In the protein-chip of the present invention, chelate of metal ion can be that class N end is-NH
2, the C end is-CH (COOH) N (CH
2COOH)
2, the centre is the compound of any organic group (comprising phenyl); Solid matrix can be selected from slide, polystyrene, nitrocellulose membrane, nylon membrane, plastics, porous silicon etc.; Metallic ion can be Ni
2+, Cu
2+, Fe
3+Deng a kind of.
Protein-chip of the present invention, the histidine mark of protein surface combines with chelate of metal ion by the metallic ion mediation, thus orientation is fixed in solid matrix (as slide) surface.The solid matrix surface is connected to the organic compound chelate of metal ion, and making has certain space length between protein and the slide, and this chelate is hydrophilic compounds, is beneficial to protein and exists with correct three-dimensional conformation; The mode of this kind conjugated protein has certain selectivity, has reduced the situation of non-specific bond; Bigger by metallic ion with the bond strength of the protein chelating that has 6 histidines, the protein difficult drop-off; This kind combination is reversible, and the high concentration imidazoles can elute the protein that is incorporated on the slide, and the protein-chip that makes the present invention make has another advantage, and the chip of promptly making can reuse, and effectively saves cost.The present invention also proposes the method for making of above-mentioned protein-chip.Before chelate of metal ion is fixed in the solid matrix surface, can carry out pre-service to matrix earlier, pretreated method can be with matrix aldehyde radicalization or carboxylated, then the amino of chelate of metal ion N end and pretreated matrix (as slide) thus react and be covalently bonded in surface of glass slide.Concrete step is as follows: (1) carries out aldehyde radicalization or hydroxylation pre-service with the solid matrix through cleaning, specifically can be earlier after silanizing agent, reductive agent and oxidizer treatment, make its surface form reactive group (Nathalie Zammatteo, Laurent Jeanmart, Sandrine Hamels, Stephane Courtois, Pierre Louette, Laszlo Hevesi, and Jose Remacle (2000) Comparison betweenDifferent Strategies of Covalent Attachment of DNA to Glass Sufaces to Build DNA Microarrays.Analytical Biochemistry.280,143-150); (2) activated matrix is dipped in the described chelate of metal ion solution of 5mM-50mM incubated at room 0.5-2 hour; (3) be dipped in the described metal ion solution of 5mM-0.1M 5-30 minute again; (4) use the above damping fluid balance of pH7.5 10-30 minute; (5) can place 20 ℃ of water bath incubations 30-60 minute the protein spots that has histidine mark thereon then, protein can be incorporated on the solid matrix.In this process,, in sample, can add the glycerine of its 40-60% weight for guaranteeing the activity of protein sample.Incubation can utilize chip to carry out next step detection after finishing.
In the said method, the above damping fluid of pH7.5 can adopt damping fluids such as PBS, TBS or Hepes.
Embodiment
Embodiment 1 aldehyde radical fix N TA makes protein-chip () slide cleaning
1, slide is dipped in solution (1/3 30%H
2O
2, 2/3 18M H
2SO
4) in 30 minutes.
2, use rinsed with deionized water 1 time.
3, in boiling water, boil 10 minutes.
4, argon gas drying.(2) aldehyde radicalization
1, slide is dipped in 1mM TETU (CL
3Si (CH
2)
10COOCH
2CF
3, be dissolved in toluene) in 1 hour, in fresh
Continuous ultrasound ripple give a baby a bath on the third day after its birth time (10 minutes/time) in the toluene.
2, argon gas drying.
3, in 200mM LiALH
4Soaked in (being dissolved in absolute ether) 2 hours.
4, in 10%HCL, soak 1 hour.
5, rinsing 2 times in the deionized water, rinsing once in the acetone.
6,120 ℃ were dried by the fire 10 minutes.
7, in 100mM PCC (pyridinium chlorochromate is dissolved in methylene chloride), soaked 2 hours.
8, continuous ultrasound ripple give a baby a bath on the third day after its birth time (10 minutes/time) in fresh methylene chloride.
9, washed 5 minutes in the deionized water for ultrasonic ripple.
10, argon gas drying.(3) NTA's is fixing
1, slide is dipped among the 5mM NTA (being dissolved in the MES damping fluid of 0.1M pH6.5), and incubation is 1 hour in 20 ℃ of water baths.
2, a large amount of rinsed with deionized water.(4) NTAization back protein is fixing
The slide of NTAization is dipped in 5mM NiSO
410 minutes, after a large amount of deionized waters embathe, PBS balance 10 minutes, the protein spots that has histidine mark placed 20 ℃ of water bath incubations 30-60 minute in its surface, and protein can be incorporated on the slide.
Embodiment 2 carboxylated fix N TA make protein-chip
1. slide is dipped in solution (1/3 30%H
2O
2, 2/3 18M H
2SO
4) in 30 minutes.
2. use rinsed with deionized water 1 time.
3. in boiling water, boiled 10 minutes.
4. argon gas drying.
5. slide is dipped in 10mM TETU (CL
3Si (CH
2)
10COOCH
2CF
3) in 1 hour, continuous ultrasound ripple give a baby a bath on the third day after its birth time (10 minutes/time) in fresh toluene.
6. argon gas drying.
7. be dipped among the 8M HCL 95 ℃ of reactions 2 hours.
8. in deionized water for ultrasonic ripple give a baby a bath on the third day after its birth time (10 minutes/time).
9. argon gas drying.
10. slide is dipped among the 5mM NTA (NTA is dissolved in the MES damping fluid of 0.1M pH6.5, comprises 5mM EDC in the MES damping fluid, 0.33mM NHSS), and incubation is 2 hours in 20 ℃ of water baths, fix N TA.
11. a large amount of rinsed with deionized water.
The fixed form of protein is with embodiment 1.
Claims (8)
1, a kind of protein-chip is made up of solid matrix and protein bound, it is characterized in that the solid matrix surface is submitted to be associated with chelate of metal ion, and protein has histidine mark, and is positioned on the solid matrix by the metallic ion mediation.
2, protein-chip according to claim 1 is characterized in that chelate of metal ion is that the N end is-NH
2, the C end is-CH (COOH) N (CH
2COOH)
2, the centre is the compound of any organic group.
3, protein-chip according to claim 1 is characterized in that solid bodies matrix is selected from a kind of of slide, plastics, porous silicon, nylon membrane, polystyrene, nitrocellulose membrane.
4, protein-chip according to claim 1 is characterized in that metallic ion is Ni
2+, Cu
2+, Fe
3+A kind of.
5, protein-chip according to claim 1, the histidine mark that it is characterized in that protein can be at the N of protein end or C end.
6, a kind of method for making of protein-chip as claimed in claim 1, it is characterized in that the protein orientation that will have histidine mark in easy mode is fixed in chip surface, concrete steps are as follows: (1) carries out aldehyde radicalization or carboxylated pre-service with the solid matrix through cleaning, and makes its surface form reactive group; (2) activated matrix is dipped in the chelate of metal ion solution of 5mM-50mM 5-30 minute, incubated at room 0.5-2 hour; (3) this matrix is dipped in the 5mM-0.1M metal ion solution 5-30 minute; (4) use the above damping fluid balance of pH7.5 10-30 minute; (5) then thereon, placed 20 ℃ of water bath incubations 30-60 minute the protein spots that has histidine mark.
7, the method for making of protein-chip according to claim 6 is characterized in that being added with in the protein example glycerine of its 40-60% weight.
8, the method for making of protein-chip according to claim 6 is characterized in that the above damping fluid of pH7.5 can be PBS, TBS or Hepes damping fluid.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 02111698 CN1226628C (en) | 2002-05-16 | 2002-05-16 | Protein chip and its preparing process |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 02111698 CN1226628C (en) | 2002-05-16 | 2002-05-16 | Protein chip and its preparing process |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1381723A true CN1381723A (en) | 2002-11-27 |
| CN1226628C CN1226628C (en) | 2005-11-09 |
Family
ID=4741696
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 02111698 Expired - Fee Related CN1226628C (en) | 2002-05-16 | 2002-05-16 | Protein chip and its preparing process |
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| Country | Link |
|---|---|
| CN (1) | CN1226628C (en) |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1304843C (en) * | 2003-07-02 | 2007-03-14 | 许洋 | Protein fingerprint method for biological sample analysis |
| CN1320361C (en) * | 2003-09-18 | 2007-06-06 | 中国科学院力学研究所 | Method for modifing protein chip surface |
| CN100408697C (en) * | 2004-02-16 | 2008-08-06 | 三星电子株式会社 | Method for noncovalently immobilizing a biomolecule on a solid substrate and microarray |
| CN100467610C (en) * | 2005-05-11 | 2009-03-11 | 中国科学院上海生命科学研究院 | Preparation of N-aceto-D-neuraminic acid by N-aceto-D-neuraminic acid aldonase immobilizing method |
| CN103197081A (en) * | 2013-04-17 | 2013-07-10 | 无锡优创生物科技有限公司 | Protein chip and preparation and detection method thereof |
| WO2014007628A1 (en) * | 2012-07-04 | 2014-01-09 | Nederlandse Organisatie Voor Toegepast-Natuurwetenschappelijk Onderzoek Tno | Fluid analysis with libs |
| CN105738451A (en) * | 2016-02-01 | 2016-07-06 | 大连理工大学 | Direct electron transfer type glucose biosensor and preparation method and application |
| CN115193422A (en) * | 2022-07-21 | 2022-10-18 | 西安交通大学 | His-tag bonded cell membrane chromatographic column based on natural nanodisk SMALP and preparation method and application thereof |
-
2002
- 2002-05-16 CN CN 02111698 patent/CN1226628C/en not_active Expired - Fee Related
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1304843C (en) * | 2003-07-02 | 2007-03-14 | 许洋 | Protein fingerprint method for biological sample analysis |
| CN1320361C (en) * | 2003-09-18 | 2007-06-06 | 中国科学院力学研究所 | Method for modifing protein chip surface |
| CN100408697C (en) * | 2004-02-16 | 2008-08-06 | 三星电子株式会社 | Method for noncovalently immobilizing a biomolecule on a solid substrate and microarray |
| CN100467610C (en) * | 2005-05-11 | 2009-03-11 | 中国科学院上海生命科学研究院 | Preparation of N-aceto-D-neuraminic acid by N-aceto-D-neuraminic acid aldonase immobilizing method |
| WO2014007628A1 (en) * | 2012-07-04 | 2014-01-09 | Nederlandse Organisatie Voor Toegepast-Natuurwetenschappelijk Onderzoek Tno | Fluid analysis with libs |
| CN103197081A (en) * | 2013-04-17 | 2013-07-10 | 无锡优创生物科技有限公司 | Protein chip and preparation and detection method thereof |
| CN103197081B (en) * | 2013-04-17 | 2016-08-10 | 无锡优创生物科技有限公司 | A kind of protein chip and preparation thereof and detection method |
| CN105738451A (en) * | 2016-02-01 | 2016-07-06 | 大连理工大学 | Direct electron transfer type glucose biosensor and preparation method and application |
| CN115193422A (en) * | 2022-07-21 | 2022-10-18 | 西安交通大学 | His-tag bonded cell membrane chromatographic column based on natural nanodisk SMALP and preparation method and application thereof |
| CN115193422B (en) * | 2022-07-21 | 2023-09-29 | 西安交通大学 | His-tag bonding type cell membrane chromatographic column based on natural nano-disc SMALP, and preparation method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1226628C (en) | 2005-11-09 |
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