CN1490414A - Plastic carriers for producing biochips - Google Patents
Plastic carriers for producing biochips Download PDFInfo
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- CN1490414A CN1490414A CNA021473366A CN02147336A CN1490414A CN 1490414 A CN1490414 A CN 1490414A CN A021473366 A CNA021473366 A CN A021473366A CN 02147336 A CN02147336 A CN 02147336A CN 1490414 A CN1490414 A CN 1490414A
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- plastic slide
- slide
- plastic
- groove
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Abstract
A plastic carrier for preparing biochip is prepared from polystyrene and has at least one recess with 0.01-0.5 mm in depth. Its surface is chemically treated for immobilizing the protein, peptide or small-molecular compound, that is, oligonucleotide or polynucleotide, on its surface to prepare the DNA biochip.
Description
Technical field
The present invention relates to can be used for making the plastic slide of biochip.Specifically, the present invention relates to have the molded plastics slide glass of special tectonic, it can be used for preparation surface through chemically treated plastic slide, and the fixing biological sample of microarray and form spot on the described treated surface is made biochip thus.
Background of invention
Oneself is widely used as making the base material of DNA chip and protein chip glass slide.Yet, use glass slide to show some shortcomings at present as the base material of biochip.At first, glass slide itself is frangible, and needs handled in operation.The surface of glass slide is difficult for accepting chemical treatment, thereby can't successfully biological micromolecule compound (such as the metabolite of plant or herbal medicine) be fixed on the surface after the processing to reach direct bonded purpose.Glass slide also can produce disadvantageous high background signal, and this can be to after the analysis of the biochip that marker is handled causes interference.Therefore, this field need develop novel material or develop to be easy to modify or the material of modification, to replace the base material of glass material as biochip.
Recently, be that the biochip of base material has been made some effort to the exploitation plastic slide, as the plastic slide of biochip substrate not only preparation cost be lower than glass slide, and can utilize jet molding commonly used to be molded as required shape easily.
WO00/55627 has disclosed a kind of biochip that is used to detect the target analyte, and it comprises is fixed in the biological binding partner of the row of one on the non-fluorescent acrylic substrate.
WO00/36145 has disclosed a kind of method of making biochip, and it comprises and grafts to abiotic probe on the electric conductive polymer.
EP1026259 has disclosed a kind of DNA chip, and it comprises solid carrier and be fixed in oligonucleotide or polynucleotide on this solid carrier in the presence of hydrophilic polymer.
Though plastic material has been used to make the DNA chip, yet at present used plastic slide all is smooth, promptly on upper surface without any groove or projection, they can produce disadvantageous high background signal, cause interference when biochip is analyzed.Still nobody successfully uses the treated plastic slide in surface protein, peptide or micromolecular compound to be fixed (being advisable with the microarray form) on treated surface so far, particularly under the not modified situation of described protein, peptide or micromolecular compound.
In addition, prior art does not disclose or points out the surface of polystyrene plastic slide glass to make oligonucleotide or polynucleotide can fix (being advisable with the microarray form) on treated surface by a simple step agent treated.
Summary of the invention
The present invention relates to a kind of plastic slide, its surface is used in fixing protein, peptide or micromolecular compound on this surface through chemical treatment.Therefore plastic slide of the present invention can be used for making biochip.This plastic slide has at least one groove, and the degree of depth of each groove is identical or different, is about 0.01-0.5mm.
The invention still further relates to the plastic slide of being made by polystyrene, its surface is used in immobilized oligonucleotide or polynucleotide on the treated surface through chemical treatment, thereby can be used for making the DNA chip.This plastic slide has at least one groove, and the degree of depth of each groove is identical or different, is about 0.01-0.5mm.
Description of drawings
Fig. 1 shows the skeleton view of plastic slide in the present invention's one preferred implementation.
Fig. 2 A shows the present invention through surface treatment and through the fluorescent image of the plastic slide of Cy3-SA/Cy5-SA mark, and wherein green fluorescent point is the point through the Cy3-SA mark, and red phosphor dot is the point through the Cy5-SA mark.
Fig. 2 B shows the fluorescent image of prior art plastic slide.
Detailed Description Of The Invention
The present invention relates to a kind of plastic slide, its surface is used in ankyrin on this surface through chemical treatment Zhi, Tai or micromolecular compound are with Zao biochip processed, and described plastic slide has the few groove of Zhi, and each is recessed The degree of depth of groove is identical or different, Yue is 0.01-0.5mm. Utilize the biochip of plastic slide of the present invention Zao processed can The platform of Yong Zuo shotgun screening (shotgun screening), described shotgun screening can be with subject matter guidance quality side Formula filters out bioactivator, thereby realizes high efficiency.
The material of plastic slide of the present invention can be homopolymers or the copolymer that one or more monomer Zhi of You become, institute State monomer and be selected from ethene, halogen ethene, propylene, the halogen propylene, acrylate, methacrylate, butadiene, Acrylonitrile, norbornene are Yu styrene, and wherein You selects cinnamic polymer. The material of plastic slide of the present invention Also Merlon. The Zai sheet that the size conforms microarray device of described plastic slide or laser scanner are habitually practised Size.
The advantage of using the present invention to have the plastic slide of special tectonic is that compare with smooth plastic slide of the prior art, the background signal that slide glass of the present invention produced (can cause interference to analyzing biochips) is much lower.
Use another advantage of plastic slide of the present invention to be, can use various chemical agent to handle or modify the surface of plastic slide, feasible not only macromolecular cpd (such as protein and DNA), and micromolecular compound (such as the metabolite of plant or herbal medicine) also can be fixed on the surface of this plastic slide.Compare the fact that conventional glass slide only can be used for fixing macromolecular cpd (such as protein and DNA), this advantage seems and is even more important.Moreover plastic slide of the present invention can be molded as required shape easily, and has the low advantage of cost.In preferred implementation of the present invention, plastic slide like this, can carry out at least 2 different reactions simultaneously through molded and have at least 2 grooves in each groove of same plastic slide.The degree of depth of each groove is identical or different, is about between the 0.01-0.5mm.In addition, can be used for the support glass lid at the molded respectively railing in the relative both sides of each groove, described glass cover is used to prevent to be loaded on this groove interior solution evaporation or loss.
Multiple functional radical aldehyde is used earlier in the preparation surface when chemically treated plastic slide, being immersed in then provides NH
2In the precursor solution of group,, make and contain active amine on the pretreated plastic slide surface with this pre-treatment plastic slide.The described NH that provides
2The precursor of group can be organism or inorganics, can be selected from NH
4OH, primary amine, in secondary amine and the tertiary amine, described primary amine, the aliphatics of secondary amine and tertiary amine and/or aromatic series partly can be used for useing as extra spacer.With regard to the described NH that provides
2The precursor of group preferably directly provides free NH
2The NH of group
4OH and primary amine.
Then, on pretreated plastic slide, applying the multiple functional radical molecule (for example multiple functional radical epoxide) that one deck serves as spacer, make surface of the present invention through chemically treated plastic slide.On function, the effect of this multiple functional radical epoxide is to be connected and fixed on required composition in the specimen (being advisable with the microarray form) of this surface on chemically treated plastic slide.The active epoxy base of this multiple functional radical epoxide one end with through the reaction of the lip-deep amido of pretreated plastic slide, the active epoxy base of this multiple functional radical epoxide the other end then adsorbs composition required in the specimen or reaction with it.Specifically, contain free hydroxyl in the specimen, the composition of sulfydryl and/or amido can form at least one covalent linkage with the active epoxy radical reaction of this multiple functional radical epoxide the other end, thereby is connected to this surface on chemically treated plastic slide.This multiple functional radical epoxide is good with the long-chain that contains 6 to 24 carbon atoms, can make required composition be unlikely direct combination like this or is adsorbed on pretreated plastic slide.Required composition is firm with the surface through combining of chemically treated plastic slide, even can tolerate high strength disengaging flushing.In the present invention, not only macromolecular cpd (such as protein and polypeptide), and micromolecular compound (such as the metabolite of plant or herbal medicine) can homogeneous phase or heterogeneous mode be fixed to plastic slide on chemically treated surface.
Therefore, the preparation surface comprises the following steps: to form the plastic slide with groove through chemically treated plastic slide, utilizes multiple functional radical aldehyde to handle, and being immersed in then provides NH
2The precursor solution of group is (with NH
4OH solution is good) in this plastic slide of pre-treatment, apply surface with multiple functional radical molecule (is good with the multiple functional radical epoxide) through pretreated plastic slide.
Described surface can be used for preparing biochip through chemically treated plastic slide, wherein contains to be fixed on plastic slide through chemically treated lip-deep homogeneous phase or heterogeneous microarray sample point.The biochip of gained can be used for required composition in the subject matter guidance quality mode filler test sample point.Above-mentioned screening method can comprise the following steps: that the solution that will contain label probe is added in the above-mentioned biochip to carry out chemical reaction (wherein, can cover on each groove with sheet glass to prevent to contain the solution evaporation of label probe), and with appropriate device (for example laser scanner) develop and identify can with reaction of this label probe or bonded sample point.The marker that is used for label probe can be dyestuff or radioactive substance.In addition, the probe that is used for hybridization can be based on known molecular mechanism and be homogeneous phase or heterogeneous known mark thing, for example, micromolecular compound, competitive part or antibody, their energy antagonisms are such as cell, acceptor, enzyme or protein through selecting.
On the other hand, the invention still further relates to the plastic slide of making by polystyrene, its surface through chemical treatment and can be on this treated surface immobilized oligonucleotide or polynucleotide, be advisable with micro-array, thereby can be used for making the DNA chip.This plastic slide has at least one groove, and the degree of depth of each groove is identical or different, and generally is about 0.01-0.5mm.The DNA chip of being made by the treated polystyrene plastic slide glass in the present invention surface can be used as the platform that detects DNA, and whether this platform serviceable indicia probe contains required DNA in the test sample spot under hybridization conditions.
The size of polystyrene plastic slide glass used in the present invention also meets the slide glass size that microarray device or laser scanner are habitually practised.As previously mentioned, this polystyrene plastic slide glass also should have at least 2 grooves by molded, thereby can carry out at least 2 different hybridizations in the groove of same plastic slide simultaneously.The degree of depth of each groove is identical or different, is about 0.01-0.5mm.In addition, can be at the relative molded railing of both sides difference of each groove, in order to the support glass lid, this glass cover is used to prevent to be loaded into this groove interior solution evaporation or loss.
Causing in the surface of modifying the polystyrene plastic slide glass, is under stronger alkaline condition, applies the polystyrene plastic slide glass with reagent solution, and this reagent solution comprises no NH
4 +The damping fluid of group and this damping fluid contain the polymkeric substance (for example polylysine) that positive charge is provided.This no NH
4 +The damping fluid of group can be carbonate buffer solution, phosphate buffered saline buffer or citrate buffer.Above-mentioned stronger alkaline condition can be pH9-11.The advantage for preparing the treated polystyrene plastic slide glass in surface of the present invention only need to be a step, and promptly simple reagent solution applies.
The DNA chip of above-mentioned gained can utilize label probe to detect under hybridization conditions whether to contain required DNA in the specimen spot.This detection method can comprise the following steps: that the solution that will contain label probe is added in the above-mentioned DNA chip and carry out hybridization (wherein, each groove covers with sheet glass to prevent to contain the solution evaporation of label probe), and with related device (for example laser scanner) develop and identify can with this label probe bonded sample point.As previously mentioned, the marker that is used for for label probe can be dyestuff or radioactive substance.
With regard to effect, utilize a little less than the biochip that biochip that the treated plastic slide in the present invention surface makes and its fluorescent background of DNA chip make than glass slide manyly.
The present invention will further be explained and illustrate to following examples, but it should be noted that, scope of the present invention is not exceeded with following embodiment.
Embodiment
Utilize fluorophorre labelled protein or peptide
According to the treatment process that manufacturers advises, utilize Fluori Link Cy3 difunctionality radical reaction dyestuff (Amersham) and FluoriLink Cy5 difunctionality radical reaction dyestuff (Amersham) to prepare the Cy3-SA streptavidin (SA) of Cy3 mark (that is through) and the Cy5-SA streptavidin of Cy5 mark (that is through) respectively.Use is equilibrated Bio-Gel P2 (Bio-Rad) chromatography column (0.5cm * 10cm) carry out gel permeation chromatography, to remove the fluorescent residue of not coupling in TBST damping fluid (50mM Tris HCl, pH7.35 contains 0.15M NaCl).The estimation ultimate density is 0.5mg/ml.
The pre-treatment of plastic slide and through the preparation of coating plastic slide glass
Molded plastics slide glass and smooth plastic slide are made by styrene polymer.The molded plastics slide glass has and microarray device or the suitable size of the employed habitual glass slide of laser scanner, and has two grooves, and the degree of depth of groove all is 0.05mm.Smooth plastic slide also has and microarray device or the suitable size of the employed habitual glass slide of laser scanner.
The all submergences 4 hours in the aqueous solution (pH5.0) of 0.4% glutaraldehyde of molded plastics slide glass and smooth plastic slide, water flushing subsequently, again in 60 ℃ at 3M NH
4Submergence is 4 hours in the OH aqueous solution (pH11.0).In 37 ℃, with 1 of 100mM, 4-butanediol diglycidyl ether (pH11.0) is handled the plastic slide that is generated all night.The plastic slide of gained is used 0.1M NaHCO again
3The aqueous solution (pH8.0) washes once, and then washes four times with redistilled water.Deposited in 4 ℃ redistilled water 4～16 hours subsequently, this step can be omitted.Before facing usefulness, gained slide glass drying at room temperature in safety cage.
Automatically dispose Cy3-SA and Cy5-SA
The automatic gear MicroGrid II (available from BioRobotics) that utilization is equipped with 0.4mm solid needle tubing is disposed at specific position on above-mentioned molded plastics slide glass and the smooth plastic slide with diluted Cy3-SA and Cy5-SA.Use 30% (10～60%) DMSO/0.1M carbonate buffer solution, pH9.5 (pH7.5～11), 2 times of serial dilution Cy3-SA and Cy5-SA protein, making Cy3-SA and the concentration of Cy5-SA protein in each diluent is 20 μ g/ml to 39ng/ml.This solid needle tubing is used to inject the solution that contains protein compound and forms spot, and therefore, before each sample of configuration, this solid needle tubing is with redistilled water flushing 2 seconds, again with 70%EtOH flushing 2 seconds, with being placed in the hot blast dry 2 seconds.After finishing the configuration sample, this molded plastics slide glass and smooth plastic slide were all at room temperature cultivated 2 hours, were immersed in subsequently in the 1M thanomin.Utilize subsequently the TBST damping fluid (0.05%Tween20) flushing gained biochip is 3 times for 50mM Tris HCl, 0.15M NaCl, again with redistilled water fine laundering 3 times, then place 37 ℃ dry 5 minutes down.Then with GenePix 4000A slide glass scanner (Axon Instruments) scanning, and with the image of GenePix 3.0 software analysis gained.The results are shown among Fig. 2 A and the 2B, wherein, be configured sample from left to right by the 1st to the 10th hurdle 2 times of dilutions successively shown in Fig. 2 A and the 2B.
Concentration during each sample is expert at is as follows from left to right, wherein, in each row to the operation of secondary series with to first be listed as identical:
(A) with (B) go:
Cy5-SA：20，10，5，2.5，1.25，0.625，0.312，0.156，0.078，0.039μg/ml
(C) with (D) go:
Cy3-SA：20，10，5，2.5，1.25，0.625，0.312，0.156，0.078，0.039μg/ml
Claims (22)
1. plastic slide, its surface is used in fixing protein, peptide or micromolecular compound on this surface through chemical treatment, and this plastic slide has at least one groove, and the degree of depth of each groove is identical or different, is 0.01-0.5mm.
2. plastic slide according to claim 1, the material of described plastic slide is a polycarbonate, or homopolymer or the multipolymer made by one or more monomers, and described monomer is selected from ethene, halogen ethene, propylene, halogen propylene, acrylate, methacrylic ester, divinyl, vinyl cyanide, norbornene and vinylbenzene.
3. plastic slide according to claim 1, the material of described plastic slide are cinnamic polymkeric substance.
4. plastic slide according to claim 1, described plastic slide has at least two grooves.
5. plastic slide according to claim 4, wherein the degree of depth of each groove is identical or different, is 0.01-0.5mm.
6. according to each described plastic slide in the claim 1 to 5, described plastic slide can be used for making biochip.
7. according to each described plastic slide in the claim 1 to 5, its surface is what to be handled, and described processing is: with multiple functional radical aldehyde plastic slide is carried out pre-treatment, plastic slide is immersed in provides NH again
2In the precursor solution of group, then apply this plastic slide again to form a coating.
8. plastic slide according to claim 7, described multiple functional radical aldehyde is glutaraldehyde.
9. plastic slide according to claim 7, the described NH that provides
2The precursor of group is NH
4OH.
10. plastic slide according to claim 7, described coating is made of the multiple functional radical molecule.
11. plastic slide according to claim 7, described multiple functional radical molecule are the multiple functional radical epoxide that each end contains at least one epoxy group(ing).
12. plastic slide according to claim 11, the epoxy group(ing) that wherein is positioned at described multiple functional radical epoxide one end can be reacted with the lip-deep amido of plastic slide after this pre-treatment.
13. plastic slide according to claim 11, wherein be positioned at this multiple functional radical epoxide the other end epoxy group(ing) can with the free hydroxyl of protein, peptide or micromolecular compound, the reaction of sulfydryl and/or amido.
14. plastic slide according to claim 11, described multiple functional radical epoxide are the long-chains that contains 6 to 24 carbon atoms.
15. plastic slide according to claim 1, described protein, peptide or micromolecular compound are homogeneous phase or heterogeneous.
16. a polystyrene slide glass that is used for fixing oligonucleotide or polynucleotide, described slide glass has at least one groove, and the degree of depth of each groove is identical or different, is 0.01-0.5mm.
17. polystyrene slide glass according to claim 16, its surface is to be what to handle, and described being treated to: in alkaline condition, apply a kind of reagent solution on the plastic slide surface, this reagent solution comprises no NH
4 +The damping fluid of group, and this damping fluid contains the polymkeric substance that positive charge can be provided.
18. polystyrene slide glass according to claim 17, described the polymkeric substance of positive charge is provided is polylysine.
19. polystyrene slide glass according to claim 17, described no NH
4 +The damping fluid of group is selected from carbonate buffer solution, phosphate buffered saline buffer and citrate buffer.
20. polystyrene slide glass according to claim 17, described alkaline condition are pH9-11.
21. polystyrene slide glass according to claim 16, described slide glass has at least one groove.
22. as the polystyrene slide glass of claim 21, wherein the degree of depth of each groove is identical or different, is 0.01-0.5mm.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA021473366A CN1490414A (en) | 2002-10-14 | 2002-10-14 | Plastic carriers for producing biochips |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA021473366A CN1490414A (en) | 2002-10-14 | 2002-10-14 | Plastic carriers for producing biochips |
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| Publication Number | Publication Date |
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| CN1490414A true CN1490414A (en) | 2004-04-21 |
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|---|---|---|---|
| CNA021473366A Pending CN1490414A (en) | 2002-10-14 | 2002-10-14 | Plastic carriers for producing biochips |
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| CN (1) | CN1490414A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105652000A (en) * | 2014-12-31 | 2016-06-08 | 天津东亚生物技术有限公司 | Novel protein chip |
-
2002
- 2002-10-14 CN CNA021473366A patent/CN1490414A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105652000A (en) * | 2014-12-31 | 2016-06-08 | 天津东亚生物技术有限公司 | Novel protein chip |
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