CN101832996B - Substrate of traditional Chinese medicine biochip as well as preparation method and use thereof - Google Patents
Substrate of traditional Chinese medicine biochip as well as preparation method and use thereof Download PDFInfo
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- CN101832996B CN101832996B CN 201010151913 CN201010151913A CN101832996B CN 101832996 B CN101832996 B CN 101832996B CN 201010151913 CN201010151913 CN 201010151913 CN 201010151913 A CN201010151913 A CN 201010151913A CN 101832996 B CN101832996 B CN 101832996B
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Abstract
The invention provides a substrate of a traditional Chinese medicine biochip formed by a silanizated slide and an organic active group connected through -O-. The substrate of the traditional Chinese medicine biochip is characterized in that the organic active group is -(CH2)2SO2CHCH2, an active ethenyl is at the end of the group and can be in cross-linking reaction with micromolecular organic matters such as carbonyls, glycosides and alkaloids and the like so that the organic micromolecule can be fixed on the substrate to prepare the traditional Chinese medicine biochip using the micromolecular organic matters as a probe, for example, a high throughput biochip which screens active ingredients of Chinese medicinal herb.
Description
Technical field
The present invention relates to biology and chemical field.
Background technology
Biochip refers to that high density is fixed on the microarray of the biological information molecule on the fixing phase supporting dielectric, and each molecule and position in the array are known.Biochip technology originates from making nucleic acid molecular hybridization, and namely so-called genetic chip can detect in real time to genome and the expression thereof of biology.Through the development of more than ten years, biochip develops in the direction of diversification, has realized the detection to accurate, the quick and large information capacity of the biological components such as compound, protein, polypeptide, DNA, biological tissue.
Biochip is comprised of substrate and probe two parts.Substrate generally is through materials such as the glass sheet of organic active base group modification, silicon chip or nylon membranes.Probe be can with the material of biological components specific binding, such as nucleic acid, antibody etc., select different type probes can realize being fixed on detection to different biological components on the substrate by being combined with the organic active group of modifying on the substrate surface.At present, can be divided into two kinds by it with the classification of probe combination on the bio-chip substrate of selling on the market: a kind of to be the organic active group be combined (such as the substrate of polylysine modification) by the Electrostatic Absorption mode with probe, since electrostatic interaction a little less than, this class substrate and probe in conjunction with insecure, probe in hybridization, elution process easily comes off; Another kind is that the organic active group is combined with probe (such as amido modified substrate and aldehyde group modified substrate) by covalent manner, and this class substrate is combined comparatively firm with probe, is applicable to contain enrich hydroxyl or amino material, such as DNA, protein.
The research and development of biochip technology provides a good platform for the Large-scale Screening of Chinese herbal medicine effective ingredients.The traditional die drug screening method be first with drug effect in tumour cell, change the effect of observation medicine from the gene expression profile of its cell.This screening be on the mechanism to medicine screen, compatibility, be not the system of a targeting screening medicament effective component, exist the cycle of screening long, expend very large, the shortcoming such as efficient is not high.The medium-height grass the effective elements of the medicine mostly is the small organic molecules such as aldoketones, glycoside and alkaloids, but the exploitation of at present commercially available bio-chip substrate mainly is for being used for fixing biomacromolecules such as connecting protein, nucleic acid, not reporting for the sheet base of special fixedly small molecule organic compound.How to utilize new method, prepare a kind of can be efficiently fixing small molecule organic compound, and develop the screening that Chinese medicine biochip take these small molecule organic compounds as probe is used for targeted drug, important society and realistic price are arranged.
Summary of the invention
The technical problem to be solved in the present invention provides a kind of organic micromolecular Chinese medicine biochip substrate of fixing.
The technical scheme that the present invention addresses the above problem is:
A kind of substrate of Chinese medicine biochip, this substrate is made of silanization slide and its surperficial organic active group, it is characterized in that described organic active group is-(CH
2)
2SO
2CH=CH
2, this group is covalently bound by the silane group on-O-and the described silanization surface of glass slide.
Substrate of the present invention is actually and utilizes surperficial synthetic technology, and the derivative active vinyl of the Si-O-key on the silanization slide obtains (its structure such as Figure of description shown in Figure 1), and its preparation method is comprised of following steps:
(1) clean: slide is dipped in the strong base solution that concentration is 0.5～2M 2～5 hours, and clean with sterile water wash, then placing volume percent content is 1～10% hydrochloric acid or sulfuric acid soaked overnight, uses at last sterile water wash;
(2) silanization: the slide after will cleaning is put into silanization solution and was soaked 0.5～2 hour, and sulfuric acid or the hydrochloric acid of then putting into 0.01～0.5M are hydrolyzed 1～2 hour, cleans 3～5 times with aqua sterilisa again, dries, and obtains the silanization slide;
(3) activation: will be the slide of silanization in containing the activating solution of divinylsulfone, soaked 1～2 hour, make divinylsulfone in the activating solution and the silane group reaction of silanization surface of glass slide, with organic active group-(CH
2)
2SO
2CH=CH
2Be attached to the surface of the slide of silanization, then get final product with the unreacted divinylsulfone of acetone flush away surface of glass slide;
In the above-mentioned steps,
Described strong base solution is potassium hydroxide solution or sodium hydroxide solution;
Described silanization solution is the ethanolic solution of the diglycidyl-propoxyl group trimethoxy silane (GOPS) of 1～15% (v/v);
Described activating solution is that volume ratio is divinylsulfone (DVS): the mixed solution of aqueous sodium carbonate=1: 10, the concentration of wherein said aqueous sodium carbonate is 0.5mol/L.
In the reactivation process of above-mentioned steps (3), the carbon-carbon double bond that the divinylsulfone molecule in the activating solution is ruptures afterwards and the Si-O-covalent bond of silanization surface of glass slide, thereby introduces organic active group-(CH in the silanization surface of glass slide
2)
2SO
2CH=CH
2Because organic active group-(CH
2)
2SO
2CHCH
2In vinyl very active, can with the small organic molecules such as aldoketones, glycoside and alkaloids in alcoholic extract hydroxyl group, phenolic hydroxyl group, carbonyl, amino or unsaturated double-bond etc. addition reaction occurs forms covalent bond, therefore substrate of the present invention can be used for preparing the biochip of the screening effective components of Chinese medicinal take small organic molecule as probe.Substrate of the present invention is the process of hydroxyl small molecule organic compound such as Figure of description shown in Figure 2 fixedly.
Because middle the effective elements of the medicine mostly is the small organic molecules such as aldoketones, glycoside and alkaloids, therefore available middle the effective elements of the medicine is as the probe of chip substrate of the present invention, obtain a kind of biochip that can carry out to effective ingredient in the Chinese herbal medicine targeting screening, the present invention is called Chinese medicine biochip with this chip, and its preparation method can be with reference to following steps:
With biochip point sample instrument Chinese medical extract is printed (point sample) on the substrate of Chinese medicine biochip of the present invention, then rapidly 120～130 ℃ of lower dryings, wash 3～5 times to seal unnecessary active ethylene group with confining liquid at last; Described confining liquid is the mixed aqueous solution by sodium carbonate and 2 mercapto ethanol, and wherein the concentration of sodium carbonate is 0.5mol/L, and the volume percent content of 2 mercapto ethanol is 1～5%.
Chinese medicine biochip of the present invention is actually numerous Chinese medicinal compound molecules is integrated on the solid phase carrier (being substrate of the present invention), can realize effectively treating in the Chinese herbal medicine targeting and the high flux screening of composition, its method is as follows: with Chinese medicine from component be fixed on the glass chip of chemical treatment, make Chinese medicine biochip, hybridize with target protein and chip, and then use the antibody hybridization check, find out and the interactional component of target protein.Can realize medicative component in quick and the high flux screening Chinese herbal medicine with Chinese medicine biochip of the present invention, help the fast development of traditional Chinese medicine and pharmacy research.
Description of drawings
Fig. 1 is the structural representation of Chinese medicine biochip substrate of the present invention, and wherein rectangle frame choosing part is the silanization slide, and linker represents diglycidyl-propoxyl group trimethoxy silane.
Fig. 2 is the fixedly process schematic diagram of hydroxyl small molecule organic compound of Chinese medicine biochip substrate of the present invention, and wherein rectangle frame choosing part is the silanization slide, and linker represents diglycidyl-propoxyl group trimethoxy silane.
Fig. 3 is the results of hybridization figure of Chinese medicine biochip of the present invention (being Chinese medicine biochip) and protein interaction.
Embodiment
In order to understand better the present invention, the below will introduce the technique effect that the present invention has by concrete example.
Example 1
One, the preparation of Chinese medicine biochip substrate of the present invention
1, getting slide, is to soak 2 hours in the sodium hydroxide solution of 1M in concentration, clean with sterile water wash, and then placing volume percent content is 5% watery hydrochloric acid soaked overnight, uses at last sterile water wash;
2, to put into volume percent content be that 95% ethanolic solution of 10% diglycidyl-propoxyl group trimethoxy silane soaked 1 hour to the slide after will cleaning, and then puts into the hydrochloric acid hydrolysis 1.5 hours of 0.3M, cleans 3 times airing with aqua sterilisa again;
3, the slide of silanization reacted in activating solution 1 hour, was placed in the fuming cupboard during reaction and constantly rocked.After finishing, reaction cleans 3 times with acetone.
Activating solution in the step 3 is prepared as follows: get the 10ml divinylsulfone, be dissolved in the sodium carbonate liquor of 100ml 0.5mol/L.
Two, the preparation of Chinese medicine biochip
1, the preparation of probe
(1) material: choose the certified Chinese medicine that has antitumor action or contain antitumor component of research and test.The Chinese herb kind mainly contains: ginseng, the Radix Astragali, kuh-seng, subprostrate sophora, sun plant, Chinese yew, Radix Angelicae Sinensis, rhizoma alismatis, cassia seed, the root of Chinese wild ginger, prepared rhizome of rehmannia, deer horn, the seed of Chinese dodder, the fruit of Chinese wolfberry, rhizoma Gastrodiae, semen armeniacae amarae, dark plum, Semen Lablab Album etc. adopt the methods such as extraction, precipitation or crystallization to separate its active princlple as material.Studies confirm that at present these Chinese herbal medicines have antitumor action or contain antitumor component.
(2) extraction of effective constituent: Chinese herbal medicine powder is broken to 40 order fine powders drops in the extraction kettle, set the extraction route as follows: CO
2Steel cylinder-refrigeration system-high-pressure pump-extraction kettle-separation reactor I-separation reactor I I-clarifier-CO
2Basin (circulation is used).Extract by above-mentioned route: at first respectively extraction kettle, separation reactor I, separation reactor I I are heated, when temperature reaches respectively 45 ℃, 40 ℃, 33 ℃, by high-pressure pump extraction kettle, two separating stills are pressurizeed, when pressure reaches respectively 30MPa, 16MPa, 8MPa (temperature and pressure are for proposing definite value), the beginning cycling extraction, and the maintenance constant temperature and pressure, CO
2Flow velocity 3.3L/h (proposing definite value).(acted in 30-60 minute) at regular intervals during extract and separate and emit extraction product.
Extraction product utilizes ABI BIOCAD 700E HPLC quantitatively with 96 orifice plates of different compound separation to 2ml, then divides 96 orifice plates that are filled to 0.5ml, and vacuum is drained, and is for subsequent use.The chromatographic condition of HPLC: R120 post, mobile phase are methyl alcohol: and water (V: V=75: 25), ultraviolet detects wavelength 260nm, flow velocity 1.0ml/min, and temperature is room temperature.
Behind the above-mentioned respectively separating-purifying, chosen respectively first 10 dna purities preferably the Chinese medicine component carry out fixed test of sheet primary surface.
2, the preparation of chip
To handle Chinese medical extract well and be splined on 96 orifice plates, and remove Chinese drug-treated group especially, other establishes two positive controls (antibody of P53, MDM2 antibody), a negative control (50% DMSO solution).With Pixsys5500 gene chip sample applying instrument these probes are distinguished point sample with probe on substrate of the present invention by 13 * 10 array, each component point sample 4 times, point and dot spacing 300 μ m; Chip after printing is dry in 120 ℃ hot glass disc rapidly, then wash 3 times to seal unnecessary active ethylene group (notes: because positive control is as protein with the 0.5mol/L sodium carbonate liquor that contains 3% (v/v) 2 mercapto ethanol, be rich in free amino in the molecule, can occur covalently bound with surface of glass slide and be fixed).
Three, the hybridization of Chinese medicine biochip and receptor protein and detection
1, material: test albumen P53 and MDM2, available from Sigma company; P53 antibody and MDM2 antibody (mouse-anti human monoclonal antibodies) are available from SANTACRUZ company; TBST:50mMTrisHCl, pH7.5,0.15MNacl, 0.05%Tween20.
2, hybridization:
(1) get P53 albumen and MDM2 albumen, with after the TBST dissolving 37 ℃ of lower and biochip hybridizations 2 hours;
(2) remove unconjugated albumen with the TBST washing, then drying added P53 antibody and MDM2 antibody, 37 ℃ of lower hybridization 1 hour;
(3) remove unconjugated antibody with the TBST washing, then drying added two of CY3 mark and resists (sheep anti mouses), 37 ℃ of lower hybridization 1 hour;
(4) use TBST cyclic washing, ddH
2The O rinsing; Place Scanarray Lite scanner with laser intensity 90% chip after the drying, PMT70%, resolution 5 μ m scan detection.
3, result: scanning result as shown in Figure 3.By the fluorescence signal judgement of scanning background, the background of this chip is low, and the hybridization point is mellow and full, favourable identity.Show from the laser scanning result data: the average background signal intensity is 861.5, average signal strength is 1029.3 in the blank, average signal strength is 1117.4 in the negative control, its average signal strength of sample of obvious hybridization signal is arranged between 3350.1-5768.6, its average signal strength of the sample that can not obviously judge is between 1350.1-2768.6, determine that thus whether stationary probe with the effective criterion of active Chinese drug component molecule is: analyzing spot is high-visible, and more than 3 times of the negative contrast of fluorescence signal when probe then are judged to be the positive.Obviously as seen there are 5 samples to demonstrate positive findings from Fig. 3.In addition, this presentation of results the effective constituent in the Chinese medicine can be fixed on the sheet base.The above results is also pointed out, and can further remove to carry out the cytology confirmatory experiment to the traditional Chinese medicine ingredients that is filtered out by target.
Example 2
The preparation of Chinese medicine biochip substrate of the present invention
1, getting slide, is to soak 3 hours in the sodium hydroxide solution of 1M in concentration, clean with sterile water wash, and then placing volume percent content is rare H of 3%
2SO
4Soaked overnight is used sterile water wash at last;
2, to put into volume percent content be that 95% ethanolic solution of 15% diglycidyl-propoxyl group trimethoxy silane soaked 1.5 hours to the slide after will cleaning, and then puts into the sulfuric acid hydrolysis 1 hour of 0.3M, cleans 3 times airing with aqua sterilisa again;
3, the slide of silanization reacted in activating solution 2 hours, was placed in the fuming cupboard during reaction and constantly rocked.After finishing, reaction cleans 3 times with acetone.
Activating solution in the step 3 is prepared as follows: get the 10ml divinylsulfone, be dissolved in the sodium carbonate liquor of 100ml 0.5mol/L.
Example 3
The preparation of Chinese medicine biochip substrate of the present invention
1, getting slide, is to soak 3 hours in the potassium hydroxide solution of 2M in concentration, clean with sterile water wash, and then placing volume percent content is rare HCl soaked overnight of 10%, uses at last sterile water wash;
2, to put into volume percent content be that 95% ethanolic solution of 10% diglycidyl-propoxyl group trimethoxy silane soaked 2 hours to the slide after will cleaning, and then puts into the hydrochloric acid hydrolysis 1 hour of 0.4M, cleans 3 times airing with aqua sterilisa again;
3, the slide of silanization reacted in activating solution 1.5 hours, was placed in the fuming cupboard during reaction and constantly rocked.After finishing, reaction cleans 3 times with acetone.
Activating solution in the step 3 is prepared as follows: get the 10ml divinylsulfone, be dissolved in the sodium carbonate liquor of 100ml 0.5mol/L.
Claims (5)
1. the substrate of a Chinese medicine biochip, this substrate is made of silanization slide and its surperficial organic active group, it is characterized in that described organic active group is-(CH
2)
2SO
2CH=CH
2, this group is covalently bound by the silane group on-O-and the described silanization surface of glass slide.
2. the preparation method of substrate claimed in claim 1, the method is comprised of following steps:
(1) clean: slide is dipped in the strong base solution that concentration is 0.5～2M 2～5 hours, and clean with sterile water wash, then placing volume percent content is 1～10% hydrochloric acid or sulfuric acid soaked overnight, uses at last sterile water wash;
(2) silanization: the slide after will cleaning is put into silanization solution and was soaked 0.5～2 hour, and sulfuric acid or the hydrochloric acid of then putting into 0.01～0.5M are hydrolyzed 1～2 hour, cleans 3～5 times with aqua sterilisa again, dries, and obtains the silanization slide;
(3) activation: will be the slide of silanization in containing the activating solution of divinylsulfone, soaked 1～2 hour, make divinylsulfone in the activating solution and the silane group reaction of silanization surface of glass slide, with organic active group-(CH
2)
2SO
2CH=CH
2Be attached to the surface of the slide of silanization, then get final product with the unreacted divinylsulfone of acetone flush away surface of glass slide;
In the above-mentioned steps,
Described strong base solution is potassium hydroxide solution or sodium hydroxide solution;
Described silanization solution is the ethanolic solution of the diglycidyl-propoxyl group trimethoxy silane (GOPS) of 1～15% (v/v);
Described activating solution is that volume ratio is divinylsulfone (DVS): the mixed solution of aqueous sodium carbonate=1: 10, the concentration of wherein said aqueous sodium carbonate is 0.5mol/L.
3. the application of the described substrate of claim 1 in the high flux screening effective component of Chinese herbal medicine.
4. Chinese medicine biochip that is used for the screening effective components of Chinese medicinal, this chip be take the described substrate of claim 1 as solid phase carrier, and the small organic molecule of hydroxyl, carbonyl, amino or unsaturated double-bond in the Effective Component of Chinese Medicine is as probe.
5. the preparation method of the described Chinese medicine biochip of claim 4, the method is comprised of following steps:
Select respectively on the substrate of the described Chinese medicine biochip of claim 1 point and dot spacing 300 μ m with a plurality of components that biochip point sample instrument will be separated from Chinese medicine; Then rapidly 120～130 ℃ of lower dryings, wash 3～5 times to seal unnecessary active ethylene group with confining liquid at last; Described confining liquid is the mixed aqueous solution of sodium carbonate and 2 mercapto ethanol, and wherein the concentration of sodium carbonate is 0.5mol/L, and the volume percent content of 2 mercapto ethanol is 1～5%.
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| CN102242190B (en) * | 2011-04-25 | 2013-04-24 | 辽宁中医药大学 | Method for screening effective medicinal flavors of Chinese herbal compounds |
| WO2015006934A1 (en) * | 2013-07-17 | 2015-01-22 | 国家纳米科学中心 | Small-molecule drug screening chip, construction method therefor and application thereof |
| CN106546580B (en) * | 2015-09-16 | 2019-06-28 | 北京博肽未名生物技术有限公司 | It is a kind of for identifying the polypeptide microarrays chip in the ginseng place of production |
| CN105295447A (en) * | 2015-09-17 | 2016-02-03 | 大连理工大学 | Method for biological functionalization of silicon-based material surface |
| CN108503565B (en) * | 2018-04-04 | 2019-08-09 | 大连理工大学 | A kind of bio-chip substrate, preparation method and application |
| CN112972771A (en) * | 2021-04-07 | 2021-06-18 | 大连理工大学 | Preparation method of bioactive surface coating based on polymerization of bis (vinylsulfonyl) methane |
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| CN1462885A (en) * | 2002-05-29 | 2003-12-24 | 中国科学院力学研究所 | Protein chip for covalent fixing biomolecular and its preparation method |
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| CN1462885A (en) * | 2002-05-29 | 2003-12-24 | 中国科学院力学研究所 | Protein chip for covalent fixing biomolecular and its preparation method |
Non-Patent Citations (2)
| Title |
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| 司士辉等.抗体固载于电聚合物膜的压电免疫型细菌传感器.《分析测试学报》.2002,第21卷(第5期),33-36. |
| 抗体固载于电聚合物膜的压电免疫型细菌传感器;司士辉等;《分析测试学报》;20020930;第21卷(第5期);33-36 * |
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