CN1584052A - Gene chip surface processing method - Google Patents
Gene chip surface processing method Download PDFInfo
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- CN1584052A CN1584052A CN 200410027316 CN200410027316A CN1584052A CN 1584052 A CN1584052 A CN 1584052A CN 200410027316 CN200410027316 CN 200410027316 CN 200410027316 A CN200410027316 A CN 200410027316A CN 1584052 A CN1584052 A CN 1584052A
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Abstract
A gene chip surface processing method includes: pretreating slide by siliconizing reagent, acrylic acid and acrylic acid monomer polymerizing on slide surface by polymer polyreacting, forming covalent connecting, inducing active molecule when polyreacting, chemical reacting with amide group of polymer, and forming amino-sensitive chemical surface. It achieves low cost, simple process, and good stability.
Description
Affiliated technical field
The present invention relates to the treatment process on a kind of gene chip surface.
Background technology
The problem that primarily needs solution in the gene chip preparation is dna fragmentation and oligonucleotide probe deposition and be combined in surface of solid phase carriers, the i.e. immobilization of nucleic acid.This fixing or in conjunction with not only want accurately, reproducibility is good, will guarantee nucleic acid or part stable existence in ligation and analysis subsequently simultaneously.Therefore, can fix various nucleic acid probes special at carrier surface, fast and stably and will directly influence the efficient of microarray.
At present, the chip carrier material is mainly based on slide.But because slide is a two dimensional structure, applied sample amount is little, makes that the sensitivity that detects is low.In order to overcome this shortcoming, the researchist is devoted to surface of glass slide is carried out modification (or modification) both at home and abroad, improves itself and nucleic acid bonded characteristic.The physics and the chemical treatment method of DNA chip carrier are a lot, but it is omnipotent up to the present not having a kind of vehicle treated method, and deficiency is always arranged, and mainly has the problem of following several respects:
1) activation of carrier.Carry out chemical conversion treatment in surface of glass slide, applied activator is connected dual mode with non-specific adsorption with covalent linkage and is coated on carrier surface.Wherein the non-specific adsorption bag is simple, cheap by surperficial carrier surface process, but this surface stability is poor, the surface properties inequality, and storage period is short.And excited the Si-OH group in the slide, the advantage of this treatment process in the covalent linkage method of attachment is based on the Si-OH group, can further cause next step surface chemical reaction, thereby the surface stability that this method makes is good, the character homogeneous.
2) fixed efficiency.Present fixing means for nucleic acid molecule mainly contains multiple, but mainly contains two kinds of methods on the slide carrier, and a kind of is physical adsorption; Another kind is covalently bound (being also referred to as chemical coupling).Preceding a kind of method is simple, easy to operate, mainly utilize the principle of physical adsorption, at the surface of glass slide bag by the material of positive charges such as Poly-L-Lysine or tensio-active agent, by electrostatic attraction and ionic bond, with the electronegative phosphate group effect adsorption of DNA in the nucleic acid backbone, be a kind of combination of non covalent bond.There is the point sample heterogeneity in this method, and joint efficiency is low, in conjunction with unstable, shortcoming such as easily peels off.Another kind is that surface of glass slide is carried out modification, introduce various functional groups (as amino, aldehyde radical, carboxyl, epoxy group(ing), sulfydryl etc.) on the surface by various chemical reactions, make DNA and surface functional group that covalently bound reaction take place, stably solidify in support surface, increase applied sample amount, strengthen the print density of dot matrix.But in the second approach, different treating processes is on nucleic acid fixed kind and length, concentration selective (or specific aim).The slide of modifying as amination is mainly used to the Oligo of fixing cDNA or long-chain; Aldehyde radical modification slide is fit to be used for fixing amido modified oligonucleotide molecules or dna fragmentation.
3) fluorescence background.The material of surface attachment can bring some background backgrounds in slide is handled; The chip hybridization process also can increase some non-specially fluorescence backgrounds of property.In chip manufacturing, if not sealing (or deactivation) of the unconjugated free active group of DNA fixing back chip surface, meeting DNA in the bonding mark sample in crossover process, and the non-specially hybridization signal of property that generation can't be differentiated, cause rolling up of background, also reduced hybridization efficiency simultaneously.
4) operating process and cost.The general method surface treatment process complexity that adopts covalent linkage to connect, particularly in the slide preparation process of polyamino modification, this complicated operating process, the time-consuming reagent that takes, cost is higher.At present, the main dependence on import of chip carrier of domestic gene chip experiment chamber, the slide of an import is about 160 yuans.Therefore, in the chip surface preparation, improving treating processes, make surface treatment to low toxicity, the conversion of low-cost direction, is the main developing direction of gene chip carrier treatment process.
Summary of the invention
The object of the present invention is to provide a kind of treatment process of novel gene chip surface, the form that the chip surface after handling with this method can covalent linkage is the fixing dna molecular of all kinds and length stably.
The treatment process of a kind of novel gene chip surface provided by the invention comprises the following steps:
(1) slide is dipped in the alkali lye that concentration is 0.5～2M 2～5 hours, is placed on the shaking table in the immersion process and rocks, put into concentration after cleaning up with aqua sterilisa and be 1～10% diluted acid soaked overnight, clean with aqua sterilisa again;
(2) slide after will cleaning was put into the silication of silanization solution after 0.5～2 hour, cleaned with ethanolic soln, cleaned 3～5 times oven dry again with aqua sterilisa;
(3) be that 1～8% vinylformic acid and methene acrylamide are dissolved in the distilled water with concentration, and 50～100 ℃ of heating 10～50 minutes, add concentration then and be 0.5～4% Ammonium Persulfate 98.5 solution, mixing, 4～20 ℃ of preservations of solution are standby;
(4) make the activation mixed solution in the adding activation solution set of dispense of vinylformic acid for preparing and the medium volume of methene acrylamide derivative monomer, the activation solution component by a kind of homotype molecule of functional group and a kind of energy easily and the primary amino activated molecule that form amido linkage are dissolved in that PH is 5.0～9.0, concentration is 10～30% K
2HPO
4/ KH
2PO
4Prepare in the damping fluid;
(5) slide behind the silanization is immersed in the activation mixed solution prepare, normal-temperature reaction 2～4 hours is placed on the oscillator during reaction and shakes, and takes out slide with cleaning in the deionized water 3～5 times, 40～60 ℃ of oven dry, and room temperature is transferred the usefulness of purchasing.
Alkali lye described in the step of the present invention (1) is NaOH or KOH, and described diluted acid is rare HCl or rare H
2SO
4Silanization solution described in the step (2) is that 1～15% aminopropyl trimethoxysilane (APS), 3-Racemic glycidol propoxy-Trimethoxy silane (GOPS) or γ-An Bingjisanyiyangjiguiwan (APTES) are dissolved in the methyl alcohol and prepare by volume; A kind of homotype molecule of functional group described in the step (4) and a kind of activated molecule easy and primary amino formation amido linkage are respectively carbodiimide (EDC) and N-hydroxy-succinamide (NHS).
The present invention has following technique effect:
1, at first adopt silylating reagent that slide is carried out pre-treatment, utilize macromolecular polymerization reaction to make vinylformic acid and acrylamide monomer then in the surface of glass slide polymerization, the formation covalent linkage connects, introduce activated molecule in when polyreaction takes place, make the amide group generation chemical reaction in itself and the polymkeric substance, formation is beneficial to the fixing of next step DNA to amino responsive chemical surface.
2, polymer molecule synthesizes after surface of glass slide, utilize the swelling character of polymer molecule, overcome the shortcoming of slide two dimensional structure, increase the probe applied sample amount, simultaneously the amino in the nucleic acid molecule can be further and activated molecule generation chemical reaction, be covalently bound on the polymer molecule, improved fixed efficiency and bonded stability, and for required fixedly goal gene (as the oligonucleotide of short chain) need not to its carry out next step 5 ' ' or 3 ' end modified.
3, after chip prepares, can to chip surface utilize homemade confining liquid to chip surface not print area carry out inactivation treatment, to reduce background, improve the sensitivity that detects.
4, in the preparation process the crosslinked connection and the surface active process of polymer molecule are carried out simultaneously, make operating process simple.Owing to do not have the use of organic solvents such as benzene, acetone, ethylene dichloride in the production process, make that production process toxicity is low.Simultaneously, selected for use inexpensive vinylformic acid and acrylamide compound as polymerization single polymerization monomer, reduced production cost, the generation cost of a slide only needs 3～6 yuan, and the import slide is 130～160 a yuan/sheet.
The present invention is further illustrated below in conjunction with accompanying drawing, embodiment and testing data.
Description of drawings
Fig. 1 is the hybrid experiment figure of different probe concentration;
Fig. 2 is the thermostability lab diagram;
Fig. 3 is the first hybridization figure of chip;
Fig. 4 is the 4th hybridization figure of chip;
Fig. 5 is phage cDNA chip figure;
Fig. 6 is SARS oligonucleotide chip figure.
Embodiment
1) cleaning of slide and silanization (pre-treatment of slide):
The cleaning of slide: slide is dipped in 2h among the NaOH of 1M, is placed on the shaking table in the immersion process and rocks, put into rare HCL soaked overnight of 5% after cleaning up with aqua sterilisa, clean with aqua sterilisa again.
The silanization of slide: after slide was put into silanization solution (silanization solution is dissolved in methyl alcohol by 9% aminopropyl trimethoxysilane (APS) and prepares) silication 30min, the ethanolic soln with 95% cleaned, and cleans 3 times oven dry again with aqua sterilisa.
2) surface chemical reaction (polyacrylamide derivative polymeric coating and surface active):
1.2% vinylformic acid and 0.8% methene acrylamide are dissolved in the distilled water, and, add 0.6% Ammonium Persulfate 98.5 solution then at 70 ℃ of heating 30mins, mixing, 4 ℃ of preservations of solution are standby.
Adding activation solution set of dispense at vinylformic acid for preparing and the medium volume of methene acrylamide derivative monomer (1: 1) is made the activation mixed solution.The main ingredient of activation solution by the N-hydroxy-succinamide (NHS) of the carbodiimide (EDC) of 0.1M and 20mM be dissolved in PH6.0, concentration is the K of 0.1M
2HPO
4/ KH
2PO
4Prepare in the damping fluid.
The slide that pre-treatment is good immerses in the activation mixed solution for preparing, and normal-temperature reaction 3h is placed on the oscillator during reaction and shakes, and the taking-up slide is used in the deionized water and cleaned 3 times, 45 ℃ of oven dry, and room temperature is transferred the usefulness of purchasing.The stationary phase of this slide is minimum to be half a year.
3) preparation of chip and DNA's is fixing:
Required fixed probe with gene chip sample applying instrument point sample on the above chip surface of handling well, is carried out the fixing of DNA simultaneously.Fixation procedure: print the back slide and place the wet box of 42 ℃ of 3 * SSC salt solution top, rehydratedization of face down.Rapidly dry in 120～140 ℃ hot glass disc, put into UV-crosslinked instrument then, crosslinked with the total energy irradiation of 65mJ, then slide is put with saturated NaCl temperature box in 42 ℃ spend the night.Make the probe combination and be solidificated in surface of glass slide.
4) fixedly aftertreatment: this process mainly comprises sealing and cleans, sealing be to not with zone that DNA is connected in the free activated molecule carry out inactivation treatment, cleaning mainly is the nucleic acid probe of flush away in conjunction with instability or non-specific binding.Seal and cleaning process: the chip that fixes is placed on rocks reaction 20～30mins in the confining liquid (mixed solution of 2% diacetyl oxide, 80% pyridine and 18% tetrahydrofuran (THF)), meanwhile water that will about 800mL in the beaker of 2L is heated to 95 ℃ (slide will immerse in the water fully), boils behind the 2mins and slide to be transferred in 95% the ethanol.Again slide is placed on the microtitre tray rack with the centrifugal 5mins of the rotating speed of 500g (placing paper handkerchief under the carriage in advance) with imbitition.Use chip immediately or it is kept in the slide box.
Process of the test and result
The patent applicant has carried out quality control to handled chip surface, has investigated surperficial fixed efficiency, thermostability and chemical stability, but and reuse and apply it to oligonucleotide chip and the making of cDNA chip in.
By hybrid experiment, utilize fluorescence intensity behind the chip scanning to determine the fixed efficiency of probe indirectly, obtain best applied sample amount.The Oligo1 probe (1～160 μ M) of different concns is printed as 12 * 8 array (two 6 * 8 little array, 8 points of every row, dot spacing is 300 μ m), array use with concentration be hybridizing of 2 μ M with Oligo1 probe sequence part complementary fluorescent mark oligonucleotide Oligo2.Fig. 1 has shown the relation between the concentration and probe concentration and hybridization signal intensity behind the slide point sample of acrylamide polymer bag quilt of preparation.The result shows, last sample concentration is between 1～10 μ mol/L the time, and crossbreeding effect increases with the increase of concentration and probe concentration, illustrates that fixed efficiency increases with concentration.When last sample concentration was 10～160 μ mol/L, that strength of signal increases and not obvious, promptly fixed efficiency did not have obvious variation, illustrated that (10 μ mol/L) chip had higher fixed efficiency when concentration and probe concentration was low.Detect fluorescence signal intensity value analysis revealed, the hybridization efficiency of chip is good, the favourable identity of hybridization back point, and the probe of lower concentration can reduce the chip manufacturing cost in chip manufacturing.
The thermostability of covalent linkage is an important parameters between chip surface and the oligonucleotide.Finish in the process of thermotolerance in following thermal cycling on printing chip surface, (50 ℃ of 1min change 72 ℃ of 2min again over to then, totally 25 circulations for 10mMTris-HCl PH8.3,50mMKCl) 94 ℃ of 1min in the PCR damping fluid.Array is hybridized then as stated above with sterilization washing, drying subsequently, scanning.From the results of hybridization of Fig. 2 as can be known, the chip surface Heat stability is good, stand the cleaning of certain temperature, potential of hydrogen and ionic strength after, carry out hybrid experiment, the fluorescence signal intensity value height that is detected.Utilize the ratio of QuantArray software analysis mean fluorecence strength of signal and background signal intensity, the ratio of chip is 39.4, and fluorescence background is low.
Oligonucleotide probe combines a stable important feature with surface of glass slide be can utilize after the hybridization again.For the stability on further detection chip surface, whether the check oligonucleotide is stable, firm with combining of surface of glass slide, and the patent applicant carries out repeatedly hybrid experiment to chip, but investigates the reuse of chip.After the each hybridization of chip, (the 2.5mM Sodium phosphate dibasic soaks 15min among 0.1% (v v) SDS) to chip at 95 ℃ the liquid that strips off.Water cleans then, drying.This step repeats, and removes fully until complementary hybridization sample, so that hybridization next time.Determine the removal and the background of the probe of mark with the scanner scanning fluorescence intensity.Our chip hybridize stripping process, quadruplication, result such as Fig. 3 and Fig. 4.Chip is after the 4th hybridization as can be seen from the results, and fluorescence signal intensity is still better, can reach examination criteria.
At last the slide of being handled well has been applied in the making of SARS oligonucleotide chip and phage cDNA chip, the result shows this surface of glass slide fixedly cDNA fragment, the PCR product of long-chain, oligonucleotide probe also fixedly short chain, that need not to modify, result such as Fig. 5 are shown in Figure 6.
Claims (4)
1, a kind of treatment process of novel gene chip surface comprises the following steps:
(1) slide is dipped in the alkali lye that concentration is 0.5～2M 2～5 hours, is placed on the shaking table in the immersion process and rocks, put into concentration after cleaning up with aqua sterilisa and be 1～10% diluted acid soaked overnight, clean with aqua sterilisa again;
(2) slide after will cleaning was put into the silication of silanization solution after 0.5～2 hour, cleaned with ethanolic soln, cleaned 3～5 times oven dry again with aqua sterilisa;
(3) be that 1～8% vinylformic acid and methene acrylamide are dissolved in the distilled water with concentration, and 50～100 ℃ of heating 10～50 minutes, add concentration then and be 0.5～4% Ammonium Persulfate 98.5 solution, mixing, 4～20 ℃ of preservations of solution are standby;
(4) make the activation mixed solution in the adding activation solution set of dispense of vinylformic acid for preparing and the medium volume of methene acrylamide derivative monomer, the activation solution component by a kind of homotype molecule of functional group and a kind of energy easily and the primary amino activated molecule that form amido linkage are dissolved in that PH is 5.0～9.0, concentration is 10～30% K
2HPO
4/ KH
2PO
4Prepare in the damping fluid;
(5) slide behind the silanization is immersed in the activation mixed solution prepare, normal-temperature reaction 2～4 hours is placed on the oscillator during reaction and shakes, and takes out slide with cleaning in the deionized water 3～5 times, 40～60 ℃ of oven dry, and room temperature is transferred the usefulness of purchasing.
2, according to the treatment process described in the claim 1, it is characterized in that the alkali lye described in the step (1) is NaOH or KOH, described diluted acid is rare HCl or rare H
2SO
4
3,, it is characterized in that the silanization solution described in the step (2) is is that 1～15% aminopropyl trimethoxysilane, 3-Racemic glycidol propoxy-Trimethoxy silane or γ-An Bingjisanyiyangjiguiwan are dissolved in the methyl alcohol and prepare by volume according to the treatment process described in the claim 1.
4,, it is characterized in that a kind of homotype molecule of functional group described in the step (4) and a kind of activated molecule easy and primary amino formation amido linkage are respectively carbodiimide and N-hydroxy-succinamide according to the treatment process described in the claim 1.
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| CN 200410027316 CN1271216C (en) | 2004-05-26 | 2004-05-26 | Gene chip surface processing method |
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| CN 200410027316 CN1271216C (en) | 2004-05-26 | 2004-05-26 | Gene chip surface processing method |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100334229C (en) * | 2005-06-17 | 2007-08-29 | 东南大学 | Preparation method of DNA microarray chip based on gel fixed nucleic acid |
| CN111100785A (en) * | 2018-10-25 | 2020-05-05 | 深圳市真迈生物科技有限公司 | Solid phase substrate, method for treating same and method for determining treatment conditions |
| CN111100786A (en) * | 2018-10-25 | 2020-05-05 | 深圳市真迈生物科技有限公司 | Solid phase substrate, method for treating same and use thereof |
| CN114324291A (en) * | 2021-12-27 | 2022-04-12 | 浙江工业大学 | Photo-crosslinking molecular probe fixed on special glass slide and preparation method and application thereof |
-
2004
- 2004-05-26 CN CN 200410027316 patent/CN1271216C/en not_active Expired - Fee Related
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100334229C (en) * | 2005-06-17 | 2007-08-29 | 东南大学 | Preparation method of DNA microarray chip based on gel fixed nucleic acid |
| CN111100785A (en) * | 2018-10-25 | 2020-05-05 | 深圳市真迈生物科技有限公司 | Solid phase substrate, method for treating same and method for determining treatment conditions |
| CN111100786A (en) * | 2018-10-25 | 2020-05-05 | 深圳市真迈生物科技有限公司 | Solid phase substrate, method for treating same and use thereof |
| CN111100786B (en) * | 2018-10-25 | 2022-03-25 | 深圳市真迈生物科技有限公司 | Solid phase substrate, method for treating same and use thereof |
| CN111100785B (en) * | 2018-10-25 | 2023-08-11 | 深圳市真迈生物科技有限公司 | Solid phase substrate, method of treating the same, and method of determining treatment conditions |
| CN114324291A (en) * | 2021-12-27 | 2022-04-12 | 浙江工业大学 | Photo-crosslinking molecular probe fixed on special glass slide and preparation method and application thereof |
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| Publication number | Publication date |
|---|---|
| CN1271216C (en) | 2006-08-23 |
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