(5) embodiment
The present invention discloses the surface through chemically treated plastic slide, be used for directly fixing protein, peptide or micromolecular compound to its treated surface with the manufacturing biochip.Utilize surface of the present invention to can be used for useing as the platform of subject matter screening (shotgunscreening) through the biochip of chemically treated plastic slide manufacturing, this subject matter screening can filter out biologically active substance in the mode that the subject matter guiding reaches efficient result (highthroughput).
The material of plastic slide of the present invention can be homopolymer or multipolymer, is prepared by a kind or multiple monomer, and this monomer is to be selected from ethene, halogen ethene, propylene, halogen propylene, acrylate, methacrylic ester, divinyl, propylene
Nitrile
, norbornene or vinylbenzene, wherein cinnamic polymkeric substance suits.
The material of plastic slide of the present invention also can be polycarbonate.The size of this plastic slide is to be equivalent to the slide glass size that little array device or laser scanner are habitually practised.
In the present invention, the advantage of using plastic slide is to be to use various chemical agent to handle or to modify the surface of plastic slide, make and be not only macromolecular cpd (such as protein and DNA), also comprise that micromolecular compound (such as the metabolite of plant or herbal medicine) can be fixed on the surface of this plastic slide.This advantage seems important especially if the habitual glass slide of contrast only can be used for fixing the fact of macromolecular cpd (such as protein and DNA).In addition, plastic slide can be easy to be molded as needed shape, and also has the low advantage of cost.In preferable system of the present invention, plastic slide be through molded to have at least 2 grooves, make and in the groove of same plastic slide, can carry out at least 2 different reactions simultaneously.The degree of depth of each groove can be identical or different, and is between being lower than O.03MM extremely up to 0.5MM.In addition, the relative both sides of each groove can be covered to be used for support glass by molded respectively railing, and wherein this glass cover is to be used to avoid evaporating or lose the solution that is loaded in this groove.
In preparing surface of the present invention in chemically treated plastic slide, be to utilize multi-functional aldehyde and be immersed in subsequently in the precursor solution that the NH2 group is provided, make that the plastic slide through handling in advance contains the active amido of tool on its surface to handle above-mentioned plastic slide in advance.This provides NH
2The precursor of group can be organism or inorganics, and can be selected from NH
40H, primary amine, secondary amine or tertiary amine, this primary amine wherein, the aliphatics of secondary amine and tertiary amine and/or aromatic series partly can be used for useing as extra wall.Provide NH in this
2In the precursor of group, directly provide free NH
2The NH of group
4OH and primary amine suit.
Further, make this plastic slide apply one deck and use the multi-functional molecule (for example, multi-functional epoxide) of wall as to prepare surface of the present invention through chemically treated plastic slide through handling in advance.On function, the effect of this multi-functional epoxide is to be connected and fixed on needed composition in the specimen (pattern that is little array aptly) of this surface on chemically treated plastic slide.The active epoxy base of one end of this multi-functional epoxide is and lip-deep amido reaction of this plastic slide through handling in advance, and the active epoxy base of the other end of this multi-functional epoxide be adsorb in the specimen needed composition or with its reaction.Specifically, contain free hydroxyl in the specimen, the composition of thiohydroxy and/or amido can with the active epoxy radical reaction of the other end of this multi-functional epoxide forming at least 1 covalent linkage, thereby this composition can be connected to this surface on chemically treated plastic slide.This multi-functional epoxide is the chemical chain that contains 6 to 24 carbon atoms aptly, make needed composition can be not directly in conjunction with or be adsorbed on this plastic slide through handling in advance.This needed composition is firm with surface of the present invention through combining of chemically treated plastic slide, even also is like this after severe disengaging flushing.In the present invention, be not only macromolecular cpd (such as protein and polypeptide), also comprise micromolecular compound (such as the metabolite of plant or herbal medicine) all can homogeneous phase or not the homogeneous mode be fixed in surface of the present invention on the surface of chemically treated plastic slide.
Therefore, prepare surface of the present invention and comprise the following step through chemically treated plastic slide: obtain plastic slide (containing fluted aptly), utilizing multi-functional aldehyde and being immersed in subsequently provides NH
2The precursor solution of group (is NH aptly
4OH solution) handling this plastic slide in advance, and utilize multi-functional molecule (being multi-functional epoxide aptly) in to apply the surface of this plastic slide through handling in advance.
Surface of the present invention can be used for preparing biochip through chemically treated plastic slide, wherein homogeneous phase or not the little array sample point of homogeneous can be fixed in surface of the present invention on the surface of chemically treated plastic slide.Resulting biochip can be used for filtering out needed composition in the specimen spot in the mode of subject matter guiding.Above-mentioned screening method can comprise the following step: the solution that will contain label probe loads in the above-mentioned biochip carrying out chemical reaction (but wherein each groove cover glass sheet contains the evaporation of the solution of label probe to prevent this), and can react or the bonded sample point with this label probe to develop and to identify by use device (for example laser scanner).The marker of using for label probe can be dyestuff or radioactive substance.In addition, the probe that is used for hybridization can be based on the branch handset that has defined then is homogeneous phase or homogeneous known mark thing not, its be for, for example, antagonism is such as cell, acceptor, enzyme or proteinic micromolecular compound, competitive part or antibody through selecting.
On the other hand, the present invention also discloses the plastic slide of the treated polystyrene material in surface, be used on its treated surface with the mode of suitable little array fixedly oligonucleotide or polynucleotide with manufacturing DNA chip.Utilize the DNA chip of the plastic slide manufacturing of the treated polystyrene material in surface of the present invention to can be used for useing the platform that detection DNA uses as, this platform can detect under hybridization conditions for whether there being needed DNA in the sample point by utilizing label probe.
The size of the plastic slide of polystyrene material used in the present invention also is equivalent to the slide glass size that little array device or laser scanner are habitually practised.As previously mentioned, the plastic slide of this polystyrene material also can make and can carry out 2 different hybridizations simultaneously in the groove of same plastic slide through molded to have 2 grooves aptly at least at least.The degree of depth of each groove can be identical or different, and is between being lower than 0.03MM between up to 0.5MM.In addition, the relative both sides of each groove can be covered to be used for support glass by molded respectively railing, and wherein this glass cover is to be used to avoid evaporating or lose the solution that is loaded in this groove.
For the surface of the plastic slide of modifying the polystyrene material, be under stronger alkaline condition, utilize reagent solution to apply the original plastic slide glass of polystyrene material, this reagent solution comprises and does not contain NH
4 +The damping fluid of group and this damping fluid contain the polymkeric substance (for example poly-from amino acid) that positive charge is provided.This does not contain 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 between between the PH9 to 11.The advantage for preparing the plastic slide of the treated polystyrene material in surface of the present invention is only need to be one step to apply simple reagent solution.
Above-mentioned resulting DNA chip can detect under hybridization conditions for whether there being needed DNA in the specimen spot by utilizing label probe.This detection method can comprise following step: the solution that will contain label probe loads in the above-mentioned DNA chip carrying out hybridization (but wherein each groove cover glass sheet contains the evaporation of the solution of label probe to prevent this), and by use device (for example laser scanner) with development and identify can with this label probe bonded sample point.As previously mentioned, the marker of using for label probe can be dyestuff or radioactive substance.
On effect, utilize the biochip of the treated plastic slide manufacturing in surface of the present invention and DNA chip all to show extremely weak fluorescent background than the biochip of glass slide manufacturing.
The present invention further explained and illustrated with following embodiment, but what must understand is that scope of the present invention is not exceeded with following embodiment.
Embodiment:
Embodiment 1
The surface of chemically modified polystyrene material slide glass be used for fixing protein,
Peptide or small moleculesization
Compound
Use polystyrene material slide glass, its size is to be equivalent to little array device or the employed habitual glass slide of laser scanner, and this polystyrene material slide glass has 2 grooves, and O.05MM the degree of depth of this groove is all.Utilize redistilled water to wash this polystyrene material slide glass, under room temperature, utilize 95% ethanol (ETOH) flushing to reach at least 2 hours subsequently.The polystyrene material slide glass that utilizes redistilled water to wash once more to have cleaned 3 times, in addition dry subsequently.Under the room temperature, the polystyrene material slide glass of above-mentioned drying is immersed in the 0.1M phosphate buffered liquor (PH5.0) of 0.4% glutaraldehyde reaches 4 hours, washed with redistilled water subsequently, under 60 ℃, be immersed in 3M NH again
4Reach 4 hours in the OH solution (PH11.0).Under 37 ℃, utilize 100mM 1,4 one butanediol diglycidyl ether (PH11.0) the polystyrene material slide glass that processing overnight generated.Utilize redistilled water to wash above-mentioned treated polystyrene material slide glass, in addition dry subsequently.The treated polystyrene material slide glass that this generated promptly can be used for making biochip, but wherein fixing protein, peptide or micromolecular compound.
Embodiment 2
The surface of modifying polystyrene material slide glass is to be used for fixing DNA
Use embodiment 1 employed polystyrene material slide glass, utilize redistilled water to be washed, under room temperature, utilize the 95%ETOH flushing to reach at least 2 hours subsequently with 2 grooves.The polystyrene material slide glass that utilizes redistilled water to wash once more to have cleaned 3 times, in addition dry subsequently.Under the room temperature, the polystyrene material slide glass that is generated is immersed in reaches 30 minutes in the reagent solution, it is 1.12% poly-from amino acid (Sigma) that this reagent solution contains, the 50mM carbonate buffer solution of 0.03% diformazan fragrant-flowered garlic sulfone (DMSO), PH9.5.Take out treated polystyrene material slide glass, and with redistilled water flushing 4 times, dry with being placed in the laminar flow device (Laminar Flow).The treated polystyrene material slide glass that is generated promptly can be used for making the DNA chip.
Embodiment 3
The manufacturing fixing protein,
The biochip of peptide or micromolecular compound
Utilization is equipped with the automatic gear MicroGrid II (available from BioRobotics) of 0.4MM solid needle tubing, and protein, peptide or micromolecular compound are disposed on the prepared treated polystyrene material slide glass of embodiment 1.Make in the water-soluble dissolubility damping fluid of protein, and make peptide and micromolecular compound be dissolved in respectively among the DMSO,, make the stock solution that contains protein, peptide and micromolecular compound respectively be diluted to 30%DMSO/ subsequently to generate stock solution
0.1M carbonate buffer solution, PH9.5 (its details will be described in more detail in following " result " partly).This solid needle tubing is to be used to deliver the solution that contains protein, peptide or micromolecular compound respectively and to form spot, therefore before each sample of load, utilize redistilled water to wash this solid needle tubing and reach 2 seconds, utilize 70%ETOH to wash this solid needle tubing again and reach 2 seconds, drying reaches 2 seconds in the hot blast with being placed on.Through utilizing this solid needle tubing to finish the configuration sample after on this treated polystyrene material slide glass, under room temperature, cultivate the biochip that is generated and reach 2 hours, subsequently it is immersed in the 1M thanomin to block residual unreacted epoxide group.Utilize subsequently the TBST damping fluid (50mM TrisHCl, 0.15M NaCl, 0.05%Tween20) flushing resulting biochip 3 times, again with redistilled water fine laundering 3 times, and then place 37 ℃ dry down.
Embodiment 4
The system of DNA chip respectively reaches processing
Utilize the above-mentioned O.4MM automatic gear MicroGrid II (available from BioRobotics) of solid needle tubing that is equipped with, to be dissolved in widow among the 30%DMSO/SSC examines on the groove that poly-thuja acid or polynucleotide sample ligand place the prepared treated polystyrene material slide glass of embodiment 2 with preparation DNA chip, wherein a row oligonucleotide or polynucleotide sample were to utilize FluoriLink in advance before being disposed on this treated polystyrene material slide glass
TMCy3
TMBi-functional reactive dye (Amersham) is mark in addition.
(1.5M NaCl, 0.5M NaOH) loads in the groove of this DNA chip with denaturing soln, and cultivates this DNA chip 2 minutes under room temperature.Remove this denaturing soln from this groove, (1.5MNaCl, 0.5M TrisHCl, PH7.2,1mM EDTA) loads in this groove with neutralization solution, and cultivates this DNA chip 1 minute under room temperature.Dry this DNA chip in 37 ℃ of baking ovens.Little joule descends in 600 (* 100) to utilize the crosslinked device of Stratalinker UV (Stratagene), and the oligonucleotide or the crosslinked of polynucleotide that carry out the array arrangement reach 5 minutes.Utilize redistilled water to wash this DNA chip reached 4 times in 1 minute.This DNA chip is immersed in reaches 1 minute among the 95%ETOH, dry with being placed in 37 ℃ of baking ovens.Resulting DNA chip is submerged through succinyl oxide/dobell's solution (fresh obtain solution: succinyl oxide (5g) is dissolved in the 1-Methyl-2-Pyrrolidone (20ML) of gentle agitation, and only before this DNA chip of submergence, add the 0.2M dobell's solution, 300ml reaches 15 minutes in PH8.0).As previously mentioned, utilize redistilled water and 95%ETOH to wash this DNA chip, and in addition dry subsequently.
Embodiment 5
Utilize fluorophorre labelled protein or peptide
According to the treatment process that manufacturers advises, utilize Fluori Link
TMCy3
TMThe streptoavidin of bi-functional reactive dye (Amersham) preparation Cy3 mark (strepavidin, SA), rabbit anti-mouse igg and glucocorticoid receptor peptide.Similarly, according to the processing mode that manufacturers advises, utilize FluoriLink
TMCy5
TMThe SA of bi-functional reactive dye (Amersham) preparation Cy5 mark.
Embodiment 6
Utilize the fluorophorre marker DNA
According to the treatment process that manufacturers advises, utilize FluoriLink
TMCy3
TMThe DNA of the Cy3 mark of probe is useed in-dCTP (Amersham) preparation as.
Embodiment 7
Utilize the protein or the peptide of fluorophorre mark to survey biochip
With prepared the loading in the groove of the prepared biochip of embodiment 3 of embodiment 5 through the protein of fluorophorre mark or the TBST diluting soln of peptide (about 20 to 25 μ l), under room temperature, cultivate this biochip subsequently and reach 2 hours, wherein utilize 22 * 22MM sheet glass to cover this groove.Utilize this biochip of TBST solution fine laundering 1 time, utilize TBST solution low to wash this biochip 3 times again, subsequently again with redistilled water flushing 4 times.In 37 ℃ of dry down biochips, utilize Gene Pix 400A slide glass scanner (Axon Instruments) to be scanned or under room temperature, be stored in the darkroom subsequently through surveying.
Embodiment 8
Utilize the DNA of fluorophorre mark to survey the DNA chip
Preceding hybridization buffer (25% methane amide, 5 * SSC, 0.1%SDS and 1%BSA) is loaded in the groove of the prepared DNA chip of embodiment 4, and cultivate these DNA chips down in 42 ℃ and reach 1 hour, wherein utilize 22 * 22mm sheet glass to cover this groove.Utilize redistilled water and 95%ETOH to wash this groove.Utilize damping fluid (to contain 25% methane amide subsequently, 5 * SSC, 0.1%SDS, the poly-adenosine blocker of 0.5mg/ml and 0.5mg/ml intestinal bacteria or the whole RNA of yeast) 2 times sequentially dilute the prepared DNA through the fluorophorre mark (0.4 μ g/ml) of embodiment 6.Through in 95 ℃ down heating this contain solution through the DNA of fluorophorre mark and reach 5 minutes and be placed on subsequently after cooled on ice reaches 1 minute, this solution (about 20 to 25 μ l) that contains through the DNA of fluorophorre mark is loaded in this groove.Under 42 ℃ and wet condition, this DNA chip of cultivation overnight is to carry out hybridization.Remove the sheet glass of covering subsequently, and utilize 2 * SSC to wash this groove.Utilize 0.1 * SSC to wash this groove 5 minutes under the room temperature.Repeat above-mentioned flushing flow process once, reached in 5 minutes 4 times with this groove of redistilled water fine laundering again.Dry resulting DNA chip through surveying, and scan with aforesaid method.
Embodiment 9
Biochip and the DNA chip of scanning through surveying
Utilize GenePix 4000A slide glass scanner (Axon Instruments) scanning embodiment 8 and 9 prepared biochip and DNA chips through surveying, and utilize GenePix 3.0 softwares to be analyzed subsequently, wherein passing ratio intermediate value (medium of ratios) is calculated fluorescence intensity, and it the results are shown in the accompanying drawing.
The result
Array sample shown in Figure 1A, 1B and the 1C is sequentially from 2 times of dilutions of hurdle A to J (from left to right).The concentration of the 1st sample of each row is as described below:
The 1st row: B-G (biotinylation gelatin), 100 μ g/ml
The 2nd row: vitamin H (Sigma B-4501), 400 μ g/ml
The 3rd row: B-ACH (biotin amido group hexanoyl hydrazine (biotinamidocaproyl hydrazide) (Sigma B-3770), 400 μ g/ml
The 4th row: B-ACHSE (vitamin H acid amides caproic acid N-hydroxyl succinyl-Asia amine ester (biotinamidocaproate N-hydroxysuccinimide ester) (Sigma B-2643), 400 μ g/ml
The 5th row: B-WGA (biotinylation wheat tooth lectin, Vector Lab B1025 S), 100mg/ml
The 6th row: IgG, 100 μ g/ml
The 7th row: DIG-HSE (digoxin (digoxigenin) 3-O-methyl carbonyl-amidcaproic acid-N-hydroxy-succinamide ester, BM1333054), 100 μ g/ml
The 8th row: rabbit igg, 100 μ g/ml
The 9th row: hydrocortisone, 100 μ g/ml
The 10th row: peptide PQGIAGQR, 100 μ g/ml
The employed protein concn of prepared product is as described below in the accompanying drawing:
Fig. 1 (a): 1 μ G/ml Cy5 streptoavidin
Fig. 1 (b): 0.5 μ g/mlCy3 streptoavidin
Fig. 1 (c): 1/5000 individual plant mouse-anti DIG, 0.5 μ g/ml Cy3 rabbit anti-mouse igg
The sequence of Cy3 mark peptide shown in Figure 2 is CKPLIPDTKPKIKD.The concentration of array peptide is to start from 2 μ g/ml, 2 times of dilutions sequentially from hurdle A to J (from left to right).
Employed DNA is by the PCR preparation, and (High Pure PCR ProductPurification Kit BM) follows the indication of manufacturers to carry out purifying by utilizing the group of products cover.
Fig. 3 (a): be dissolved in the DNA of the Cy3 mark of 30%DMSO/SSC,, sequentially dilute 4 times from hurdle A to J without any modification.After group blocking-up and flushing are handled, the screen dna chip.The 1st sample concentration is 0.2 μ g/ml.
Fig. 3 (b): sequentially dilute 4 times from hurdle A to J as the DNA that will be dissolved in damping fluid as above-mentioned.Initial concentration is 0.4 μ g/ml.After blocking-up and flushing processing, utilize the dna probe of Cy3 mark to cultivate the DNA chip.The DNA concentration of this Cy3 mark is 0.1 μ g/ml.
Certainly, those of ordinary skill in the art will be appreciated that, above embodiment is used for illustrating the present invention, and be not to be used as limitation of the invention, as long as in connotation scope of the present invention, all will drop in the scope of claims of the present invention variation, the modification of the above embodiment.