CN1952658B - Process of sugar bio-chip - Google Patents
Process of sugar bio-chip Download PDFInfo
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- CN1952658B CN1952658B CN2006100713574A CN200610071357A CN1952658B CN 1952658 B CN1952658 B CN 1952658B CN 2006100713574 A CN2006100713574 A CN 2006100713574A CN 200610071357 A CN200610071357 A CN 200610071357A CN 1952658 B CN1952658 B CN 1952658B
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Abstract
A Method for preparation of sugar biological chip is disclosed that the epoxyethane radical derived solid carrier is covalence coupled with the amidol of coupling agents by the epoxyethane radical contained on the surface, the aldolisation condensation reaction occurred between the hydroxyl which is in the other side of the coupling agents and the deacidizing end of the deacidizing sugar to form the glycosidic bond, the deacidizing sugar is coupled by covalent formula to the solid carrier to form the sugar chip. Or the amidol radical derived solid carrier is covalence coupled with the acid group which is activated by the N-hydroxy succinimide and the dicyclo hexylcar bodiimide of the coupling agents by the amidol radical contained on the surface, the aldolisation condensation reaction occurred between the hydroxy which is in the other side of the coupling agents and the deacidizing end of the deacidizing sugar to form the glycosidic bond, the deacidizing sugar is coupled by covalent formula to the solid carrier to form the sugar chip. The invention resolves the technical matters of that the deacidizing sugar is friable, the Method for preparation is complex, and the cost is high. The invention is simple and quick; the bioactivity of the sugar chip is stable, and easy to apply dimensionally.
Description
Technical field
The present invention relates to a kind of preparation method of sugar bio-chip.Be specifically related to a kind of reduction end of utilizing reducing sugar, with the preparation method of the sugar bio-chip of glycosidic bond form covalent coupling to the solid phase carrier of hydroxyl derivatization.
Background technology
Carbohydrate-binding protein has important biological function, and it shows intercellular communication and identification, the interaction of cell and pathogenic microorganism, the transportation of intracellular protein, interaction between protein, albumen and aspects such as the specificity of acceptor combines.Three probing directions of biochip technology promptly are genetic chip, protein-biochips and sugar bio-chip.The superiority of sugar bio-chip is: one, biologically active is stable.The long preservation biologically active is constant at ambient temperature for sugar bio-chip.Its two, have wide range of applications.The sugar bio-chip technology is applied on the clinical medicine, can carries out quick diagnosis simultaneously multiple infectiousness and Non Communicable Diseases (NCD).Because all cell surfaces all have the sugar substance of containing, these glycosylated molecules play an important role in the identifying of cellular elements.For example, the mensuration of blood group, the human immunocyte is to the identification of microbial pathogens and some tumour etc.
At present, the research to the carbohydrate chip that is used for carbohydrate-binding protein research still is in the exploratory stage in the world.Major obstacle is to lack high-throughout technology and means, particularly lack the research tool that is similar to DNA chip and protein chip, need to solve the technical barrier that actual chips is made, comprise how containing that sugar substance is fixed on the chip, the biologically active of chip monitoring upper sugar body how.
The preparation of existing carbohydrate chip mainly is based on physisorption, or to the further chemical modification of reducing sugar, and then the reducing sugar covalent coupling to such as on the solid phase carriers such as nitrocellulose filter or slide.Physisorphtion prepares carbohydrate chip, and the reducing sugar that is fixed on the solid phase carrier easily comes off in experimentation, influences result's accuracy.Further chemical modification prepares carbohydrate chip to reducing sugar, be the reducing sugar covalent coupling to solid phase carriers such as nitrocellulose filter or slide, its method complexity, the cost height be difficult for to be implemented, is promoted.
Summary of the invention
The object of the present invention is to provide a kind of preparation method of sugar bio-chip, it has solved, and reducing sugar easily comes off in the background technology, result's poor accuracy, preparation method's complexity, the technical matters that cost is high.
Technical solution of the present invention is:
A kind of preparation method of sugar bio-chip, its special character is: the performing step of this method is as follows
1) gets coupling agent and be dissolved in the organic solvent, be mixed with the solution of 10～50mol/L;
2) solid phase carrier with the ethylene oxide group derivatization immerses wherein, hatches under the room temperature 3 hours;
3) organic solvent and the absolute ethyl alcohol with the dissolving coupling agent cleans secondary with solid phase carrier respectively, each 2 minutes;
4) with solid phase carrier in the room temperature centrifugal drying, be stored in the drying receptacle standby;
5) with the dissolving of reducing sugar water, transfer to required concentration;
6) the acid point sample damping fluid of adding equal volume, pH3～6 carries out point sample to solid phase carrier;
7) solid phase carrier behind the point sample got final product in 25～60 ℃ of incubations in 12 hours.
Above-mentioned coupling agent is the coupling agent that contains an amino and a hydroxyl.
Above-mentioned coupling agent specifically can adopt 4-hydroxybenzamide or 4 hydroxybutyric acid hydrazine etc.
Above-mentioned organic solvent can adopt ethyl acetate, methyl acetate, acetonitrile or dimethyl formamide etc.; Reducing sugar can adopt monose, oligosaccharides or polysaccharide etc.; Solid phase carrier can adopt slide, nitrocellulose filter or ceramics etc.
The water of above-mentioned dissolving and reducing sugar can adopt ultrapure water, distilled water, deionized water or pure water etc.; Point sample is meant manual point sample and machine point sample; The heated culture temperature of solid phase carrier is good with 37 ℃ behind the point sample.
A kind of preparation method of sugar bio-chip, its special character is: the performing step of this method is as follows
1) gets coupling agent and be dissolved in the organic solvent, be mixed with the solution of 10～50mol/L;
2) in the solute ratio:
Coupling agent: N-hydroxy-succinamide: dicyclohexylcarbodiimide=1: 2: 2～4,
Add N-hydroxy-succinamide and dicyclohexylcarbodiimide, the acidic group that coupling agent is activated generates active ester derivant;
3) solid phase carrier with the amino group derivatization soaks wherein, hatches under the room temperature 3 hours;
4) organic solvent and the absolute ethyl alcohol with the dissolving coupling agent cleans secondary with solid phase carrier respectively, each 2 minutes;
5) with solid phase carrier in the room temperature centrifugal drying, be stored in the drying receptacle standby;
6) with the dissolving of reducing sugar water, transfer to required concentration;
7) the acid point sample damping fluid of adding equal volume, pH3～6 carries out point sample to solid phase carrier;
8) solid phase carrier behind the point sample got final product in 25～60 ℃ of incubations in 12 hours.
Above-mentioned coupling agent can adopt the coupling agent that contains a carboxyl and a hydroxyl.
Above-mentioned coupling agent specifically can adopt 4-hydroxy phenyl valeric acid or 1 6-hydroxyl, ten hexacarboxylic acids etc.
Above-mentioned organic solvent can adopt ethyl acetate, methyl acetate, acetonitrile or dimethyl formamide etc.; Reducing sugar can adopt monose, oligosaccharides or polysaccharide etc.; Solid phase carrier can adopt slide, nitrocellulose filter or ceramics etc.
The water of above-mentioned dissolving and reducing sugar can adopt ultrapure water, distilled water, deionized water or pure water etc.; Point sample is meant manual point sample and machine point sample; The heated culture temperature of solid phase carrier is good with 37 ℃ behind the point sample.
The present invention has the following advantages:
1. fixedly the connected mode of reducing sugar is simple, quick, and cost is lower.
2. the biologically active of carbohydrate chip is stable, at ambient temperature long preservation and biologically active is constant.
3. usage range is wide, is easy to large-scale promotion, use.
Description of drawings
Fig. 1 is the principle schematic of the invention process method one.Fig. 2 is the principle schematic of the invention process method two.
Fig. 3 a, b are the present invention checks sugar coupling effect on solid phase carrier as chromogenic reagent with the sulfuric acid solution of orcin synoptic diagram.
Fig. 4 a, b are the effect synoptic diagram that the present invention is used for the agglutinin canavalin of the direct mark of CyDye fluorescent dye carbohydrate chip.
Embodiment
The present invention mainly is to be model with the reducing sugar, and to be hemiacetal (ROH) (OH) prepare carbohydrate chip on the solid phase carrier of derivatization with the form covalent coupling of glycosidic bond to hydroxyl to the reduction end that makes reducing sugar.
The principle of implementation method one of the present invention: the solid phase carrier of ethylene oxide group derivatization, because of containing one deck ethylene oxide group in its surface, can with the amino covalence coupling of coupling agent.(OH) reduction end with reducing sugar is that 1 aldehyde radical generation aldol reaction forms glycosidic bond to the hydroxyl of the coupling agent other end, and reducing sugar is coupled on the solid phase carrier in the mode of covalency, prepares carbohydrate chip.
In this method, reducing sugar can adopt monose, oligosaccharides or polysaccharide etc.Solid phase carrier can adopt slide, nitrocellulose filter or ceramics etc.Coupling agent can adopt the coupling agent that contains an amino and a hydroxyl, as 4-hydroxybenzamide or 4 hydroxybutyric acid hydrazine etc.
The performing step of the embodiment of the invention one:
1. get coupling agent 4-hydroxybenzamide or 4 hydroxybutyric acid hydrazine and be dissolved in the organic solvent dimethyl formamide (DMF), be mixed with the solution of 50mL, 20mol/L.
2. the slide with the ethylene oxide group derivatization immerses wherein, hatches about 3 hours under the room temperature.
3. clean the slide secondary respectively with dimethyl formamide (DMF) and absolute ethyl alcohol, each about 2 minutes.
With slide in the room temperature centrifugal drying, be stored in 4 ℃ of drying receptacles standby.
5. reducing sugar is dissolved with ultrapure water, transfer to required concentration.
6. add the acid point sample damping fluid of equal volume, pH3～6, slide is carried out manual point sample and machine point sample.
7. get final product about 12 hours in 37 ℃ of incubations.
The principle of implementation method two of the present invention: the solid phase carrier of amino group derivatization, because of containing one deck amino group in its surface, can with the acidic group covalent coupling through N-hydroxy-succinamide (NHS) and dicyclohexylcarbodiimide (DCC) activation of coupling agent.The hydroxyl of the other end of coupling agent (can be that 1 aldehyde radical generation aldol reaction forms glycosidic bond with the reduction end of reducing sugar OH), reducing sugar is coupled on the solid phase carrier in the mode of covalency, prepare carbohydrate chip.In this method, reducing sugar can adopt monose, oligosaccharides, polysaccharide etc.Solid phase carrier can adopt slide, nitrocellulose filter, ceramics etc.Coupling agent can adopt and contain a carboxyl and a hydroxyl, as 4-hydroxy phenyl valeric acid, 16-hydroxyl ten hexacarboxylic acids etc.
The performing step of the embodiment of the invention two:
1. get coupling agent 4-hydroxy phenyl valeric acid or 16-hydroxyl ten hexacarboxylic acids are dissolved in the organic solvent ethyl acetate, be mixed with the solution of 50mL, 20mol/L.
2. add N-hydroxy-succinamide (NHS) and dicyclohexylcarbodiimide (DCC), the acidic group that coupling agent is activated generates active ester derivant.
3. the slide with the amino group derivatization soaks wherein, hatches about 3 hours under the room temperature.
4. clean the slide secondary respectively with dimethyl formamide (DMF) and absolute ethyl alcohol, each about 2 minutes.
With slide in the room temperature centrifugal drying, be stored in 4 ℃ of drying receptacles standby.
6. reducing sugar is dissolved with ultrapure water, transfer to required concentration.
7. add acid point sample damping fluids equal volume, pH3～6, slide is carried out manual point sample and machine point sample.
8. get final product about 12 hours in 37 ℃ of incubations.
Select the slide of reducing sugar and handle through the sealing damping fluid, ultrapure water cleans, the room temperature centrifugal drying.Drip the sulfuric acid solution of orcin in the point sample district, then to this zone heating.As seen the sulfuric acid solution of orcin is by colourless yellowing gradually.Fig. 3 a, 3b are respectively the design sketchs of preparing carbohydrate chip with the inventive method one, two.
With the D-mannose is model, prepares carbohydrate chip with the inventive method one, two, and the agglutinin concanavalin A (Concanavalin A, Con A) of the direct mark of CyDye fluorescent dye is used for this carbohydrate chip, the results are shown in Figure 4a, 4b.
Claims (4)
1. the preparation method of a sugar bio-chip is characterized in that, the performing step of this method is as follows:
1) gets coupling agent and be dissolved in the organic solvent, be mixed with the solution of 10～50mol/L; Described coupling agent is the coupling agent that contains an amino and a hydroxyl;
2) solid phase carrier with the ethylene oxide group derivatization immerses wherein, hatches under the room temperature 3 hours;
3) organic solvent and the absolute ethyl alcohol with the dissolving coupling agent cleans secondary with solid phase carrier respectively, each 2 minutes;
4) with solid phase carrier in the room temperature centrifugal drying, be stored in the drying receptacle standby;
5) with the dissolving of reducing sugar water, transfer to required concentration;
6) the acid point sample damping fluid of adding equal volume, pH3～6 carries out point sample to solid phase carrier;
7) solid phase carrier behind the point sample got final product in 25～60 ℃ of incubations in 12 hours.
2. the preparation method of sugar bio-chip according to claim 1, it is characterized in that: described coupling agent is 4-hydroxybenzamide or 4 hydroxybutyric acid hydrazine.
3. the preparation method of sugar bio-chip according to claim 1 and 2, it is characterized in that: described organic solvent is ethyl acetate, methyl acetate, acetonitrile or dimethyl formamide; Described reducing sugar is monose, oligosaccharides or polysaccharide; Described solid phase carrier is slide, nitrocellulose filter or ceramics.
4. the preparation method of sugar bio-chip according to claim 3, it is characterized in that: the water of described dissolving and reducing sugar is ultrapure water, distilled water, deionized water or pure water; Described point sample is meant manual point sample and machine point sample; The heated culture temperature of solid phase carrier is 37 ℃ behind the described point sample.
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| CN2006100713574A CN1952658B (en) | 2005-10-19 | 2006-03-21 | Process of sugar bio-chip |
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| CN200510096211.0 | 2005-10-19 | ||
| CN200510096211 | 2005-10-19 | ||
| CN2006100713574A CN1952658B (en) | 2005-10-19 | 2006-03-21 | Process of sugar bio-chip |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101308141B (en) * | 2007-05-16 | 2012-08-29 | 陕西北美基因股份有限公司 | Method for analyzing glucoprotein |
| US10408717B2 (en) | 2009-06-29 | 2019-09-10 | Nicolai Vladimirovich Bovin | Printing of FSL constructs |
| CA2766110C (en) * | 2009-06-29 | 2018-07-17 | Nicolai Vladimirovich Bovin | Printing of fsl constructs |
| CN101643321B (en) * | 2009-09-01 | 2012-07-18 | 博奥生物有限公司 | High-polymer three-dimensional amino-group substrate as well as preparation method and application thereof |
| CN103901212B (en) * | 2014-03-28 | 2015-07-22 | 西北大学 | Glycan microarray for identifying serial liver diseases based on saliva glycan-binding proteins, and application of glycan microarray |
| CN109107620A (en) * | 2017-06-23 | 2019-01-01 | 天津科技大学 | A method of quickly preparing carbohydrate chip |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1375507A (en) * | 2001-03-20 | 2002-10-23 | 清华大学 | Surface cladding and radical functino modification method of magnetic microsphere, thus obtained microsphere and its application |
| EP1265071A2 (en) * | 2001-06-05 | 2002-12-11 | Fuji Photo Film Co., Ltd. | Reactive solid support and DNA fragment detection tool |
| CN1627069A (en) * | 2003-12-11 | 2005-06-15 | 中国科学院大连化学物理研究所 | Micro flow control chip of silicon rubber and method for face finish |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1375507A (en) * | 2001-03-20 | 2002-10-23 | 清华大学 | Surface cladding and radical functino modification method of magnetic microsphere, thus obtained microsphere and its application |
| EP1265071A2 (en) * | 2001-06-05 | 2002-12-11 | Fuji Photo Film Co., Ltd. | Reactive solid support and DNA fragment detection tool |
| CN1627069A (en) * | 2003-12-11 | 2005-06-15 | 中国科学院大连化学物理研究所 | Micro flow control chip of silicon rubber and method for face finish |
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Owner name: SHAANXI BAIMEI GENE CO., LTD. Free format text: FORMER NAME: SHAANXI LIFEGEN CO., LTD. |
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Address after: 386 box 710069, Northwestern University, 229 Taibai North Road, Shaanxi, Xi'an Patentee after: Shaanxi Baimei Gene Co., Ltd. Address before: 386 box 710069, Northwestern University, 229 Taibai North Road, Shaanxi, Xi'an Patentee before: Shaanxi Beimei Gene Co., Ltd. |