CN1431321A - Assembled type compound array chip and its preparing method - Google Patents
Assembled type compound array chip and its preparing method Download PDFInfo
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- CN1431321A CN1431321A CN 03112772 CN03112772A CN1431321A CN 1431321 A CN1431321 A CN 1431321A CN 03112772 CN03112772 CN 03112772 CN 03112772 A CN03112772 A CN 03112772A CN 1431321 A CN1431321 A CN 1431321A
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Abstract
An assembled compound array chip is composed of supporter, several carriers assembled on said supporter, and polymer moleculae (oligonucleotide, polypeptide, polyose and peptide nucleic acid) synthesized at known positions on relative carrier. After the polymer moleculae act on particular fluid, the change of its natural can be detected optically.
Description
Technical field
What assembly type chemical array chip of the present invention and preparation method thereof related to is a kind of assembly type compound molecule detection chip and preparation technology's method thereof.
Background technology
Combinatorial chemistry is a kind of fast method that obtains a large amount of different compounds.For resembling the macromolecular cpd that oligonucleotide, polypeptide class are made of the few small molecule monomer of number, can obtain a large amount of similar different compounds within a short period of time by combinational chemistry.These different compounds can be built into array bio-chip by assembling mode.Biochip mainly refers to the microarray that the biologically active substance (comprising nucleic acid, protein, cell and small tissue etc.) in surface of solid phase carriers assembling constitutes, with realize to compound (comprising medicine), protein, nucleic acid, cell and other biological components accurately, fast, the screening or the detection of large information capacity.
Biochip has extremely important meaning on biological detection, medical test and medical diagnosis on disease, drug screening and gene sequencing.The preparation method of biochip has synthetic and from synthetic two kinds of sheet (point sample) at sheet (original position).The synthetic superchip of original position is to utilize combinational chemistry to pass through mask plate (or virtual mask plate) will synthesize the site gradation and activate and then base A, G, C, T gradation are coupled at corresponding site.Original position synthetic main drawback is the pollution of coupling reagent causes synthetic product in the building-up process mistake, also is difficult to guarantee that the reaction conditions when all sites is synthetic is in full accord in the coupling process simultaneously, thereby cause producing difference between the quality and quantity of compound molecule on the synthetic site, the information errors rate height that finally draws (if synthetic identical oligonucleotide sequence, then the result who provides by hybridization understands different).In addition, though the synthetic mass production cost of original position is low, but all there is the deficiency of very flexible in existing in-situ synthetic method, and the manpower and financial resources that preparing any chip all needs to cost a lot of money is carried out the design and the production of a cover mask plate (the mask plate number is generally four times of base number in the synthetic oligonucleotide); It is earlier that oligonucleotide sequence is synthetic, purifying that the point sample legal system is equipped with low density chip, is connected with active group on the substrate corresponding position by synthesizing at the active group on the oligonucleotide sequence then.Its main drawback is for buying the expense height of probe, fixes the price that its price in on-chip active group (as amino etc.) is equivalent to the oligonucleotide sequence of synthetic 50 bases at present for synthetic one on the probe.Secondly, the difference of the difference of sample size, reaction conditions also can cause the inconsistent of purpose compound molecule number on each position in the point sample process.
Summary of the invention
The present invention is directed to the weak point that present conventional biochip exists in research and development, a kind of assembly type chemical array chip and preparation method thereof has been proposed, be combined in sheet synthetic in the directly advantage and the handiness in sheet synthesizes of synthetic (fix) probe, use a spot of reagent, on the identical solid phase carrier of coding, press a collection of or multiple batches of synthetic a large amount of different compounds by combinational chemistry, random then solid phase carrier (corresponding compound has been synthesized by combination in the surface) structure chemical array with different coding.The assembly type preparation method of the low chemical array chip of a kind of chip information accuracy rate height and cost of manufacture is provided.
Assembly type chemical array chip and preparation method thereof takes following scheme to realize: assembly type chemical array chip is characterized in that structure comprises upholder, carrier and polymer compound molecule; Be assembled with some carriers on the upholder, synthesizing on the carrier separately has corresponding polymer compound molecule, and the pairing polymer compound molecule of carrier and this position of polymer compound on array chip are one to one on the assembly type chemical array chip.Assembly type chemical array chip is characterized in that upholder adopts substrate, Glass tubing, plastics tubing, plastic coil etc.
In the assembly type chemical array chip production method: (1) is according to the requirement of desiring synthetic polymer compound molecule on the chemical array chip to be prepared, synthesizing corresponding polymer compound molecule on the encoded carrier separately respectively by following operation: the monomer molecule solution that carrier in heaps is corresponding with the first location of polymer compound molecule carries out first set reaction earlier, the corresponding monomer molecule solution of the second position that will synthesize first monomeric carrier in heaps and polymer compound molecule more carries out the reaction second time, later on still according to the requirement of desiring synthetic polymer compound sequence, reactant and the corresponding monomer molecule solution of the next position that to work as one by one on time carrier react, until synthesized required complete polymer compound molecule on carrier.(2) after the first step operation has obtained polymer molecule required on the chemical array chip to be prepared, the polymer molecule of carrier together with correspondence together is assembled on the upholder.The polymer compound molecule here is meant that (monomer is not limited to) is the compound molecule that monomer constitutes by the few small molecules of number; Mainly comprise biomacromolecules such as oligonucleotide, polypeptide, polysaccharide and peptide nucleic acid(PNA); Solid support material is by inorganic materials: glass, quartz, pottery, crystal, metal; Organic materials: macromolecular materials such as rubber, plastics, gel, nylon, biomacromolecule, conductive polymers, or by the mixture of above-mentioned inorganic materials and organic materials; The coding of carrier is the direct mark on solid phase carrier, or on the solid phase carrier indirectly or virtual tag, the purpose of the coding of solid phase carrier is to make things convenient for the entrained compound molecule in synthetic back on the synthetic and identification surface of solid phase carriers of the combination of different compound molecules on the solid phase carrier.
Behind the compound molecule and fluid interaction in the assembly type chemical array chip, the change of its character can detect by methods such as optics, electricity, magnetics, acoustics or calorifics; The different polymer molecules of carrier surface synthetic can not move under the fluid effect.
Beneficial effect (1) is because the present invention is that the carrier that can concentrate concentrates in together, be formed into the preloading body, this both can make the carrier of the synthetic identical polymer molecule of desire concentrate the synthesized polymer molecule, in also can be identical at the polymer monomer molecule of certain position of the process that is used for synthetic different polymer molecules, concentrate these carriers or preceding primary first-order equation gains in heaps, place reaction solution to react simultaneously, therefore, the cost of synthesized polymer molecule of the present invention is low, and the manufacturing cost of its chip is also low; And the present invention synthesizes the polymer molecule respectively on encoded carrier separately, and then with these synthetic carrier arrangement that the polymer molecule is arranged on substrate, thereby the chip that the present invention is made has the high advantage of chip information accuracy rate.(2) the present invention take according to current position desire synthetic polymer monomer molecule whether identical principle concentrate classify in heaps, current position be will be used for and the carrier or the concentrated preloading body that is formed into of preceding primary first-order equation gains of the identical polymer monomer molecule of synthetic desired, and carrier concentrated reaction, thereby avoided the mutual pollution in the building-up process, further improve the chip manufacturing accuracy rate, reduced cost.
Description of drawings
Below in conjunction with accompanying drawing the specific embodiment of the present invention is described in further detail:
Fig. 1 is the assembly type chemical array chip structure synoptic diagram that the present invention proposes.
The synoptic diagram of the synthetic different compounds of the combinatorial chemistry that Fig. 2 the present invention proposes.
The different compounds with coding that Fig. 3 the present invention proposes make up chemical array chip synoptic diagram.
The analyzing and testing result schematic diagram that provides behind solid-state carrier array that Fig. 4 the present invention proposes and the specific fluid interaction.
Embodiment
With reference to accompanying drawing 1, assembly type chemical array chip structure comprises polymer compound molecule 1 (amplifying signal), carrier 2, upholder 3 Glass tubings or upholder 4 substrates.Upholder adopts substrate, Glass tubing, plastics tubing, plastic coil.
Fig. 2 is the synoptic diagram of the synthetic different compounds of combinatorial chemistry that the present invention proposes: with big area solid phase carrier coding, the solid phase carrier number of each coding can be 1 also can be very big.The coding solid phase carrier is classified in heaps on request, carries out identical chemical reaction with the heap solid phase carrier then.This classification can only be carried out once, makes the surface of solid phase carriers with the heap coding have identical compound; Also can carry out many times repeatedly, make the solid phase carrier of different coding have different compounds at last.After the surface of solid phase carriers corresponding compounds is synthetic, solid phase carrier is cut into numerous still less solid phase carrier, solid phase carrier still less had at least on the identification face and contained the synthetic compound molecule this moment.
The different compounds with coding that Fig. 3 the present invention proposes make up chemical array chip synoptic diagram.From the solid phase carrier with respective compound of each coding, take out one or many and be filled in the one-dimensional array that just constitutes compound in the small straight line open tube; From the solid phase carrier with respective compound of each coding, take out one or many and be distributed in the flat two-dimensional arrays that just constitutes compound on the flat carrier.
The analyzing and testing result schematic diagram that provides behind solid-state carrier array that Fig. 4 the present invention proposes and the specific fluid interaction.The fluid that contains material to be detected interacts by some compound in the solid-state carrier array under certain condition, obtains taking place results of interaction under specific detector.Result by the analyzing and testing effect determines that effect has taken place the material in those compounds (corresponding its coding of coordinate position) and the fluid.
Further specify the present invention below in conjunction with specific embodiment:
Embodiment one: the preparation of the synthetic and chemical array chip of oligonucleotide combination of compounds.After glass sheet surface cleaning, drying, the benzole soln of putting into APTS (aminopropyltriethoxywerene werene) respectively reacted 2 hours, and the benzole soln of putting into succsinic acid in benzene after the rinsing again reacted 1 hour, thereby formed hydroxyl at glass surface.The sheet glass that these surfaces is had hydroxyl is encoded then.The sheet glass of all codings is divided into four heaps by certain way; the first heap sheet glass carries out even reaction in containing first reaction tank of anhydrous acetonitrile that 5 '-OH used the dAdp of two pairs of methoxies, three stupid methyl (DMT) protections and tetrazolium (catalyzer); the hydroxyl of glass sheet surface is base A in the coupling just; the second heap sheet glass carries out even reaction in containing second reaction tank of anhydrous acetonitrile that 5 '-OH used the dGdp of two pairs of methoxies, three stupid methyl (DMT) protections and tetrazolium (catalyzer); the hydroxyl on surface, glass section of jurisdiction is bases G in the coupling just; the 3rd heap sheet glass carries out even reaction in containing the 3rd reaction tank of anhydrous acetonitrile that 5 '-OH used the dCdp of two pairs of methoxies, three stupid methyl (DMT) protections and tetrazolium (catalyzer); the hydroxyl on surface, glass section of jurisdiction is base C in the coupling just; the 4th heap sheet glass carries out even reaction in containing the 4th reaction tank of anhydrous acetonitrile that 5 '-OH used the dTdp of two pairs of methoxies, three stupid methyl (DMT) protections and tetrazolium (catalyzer), the hydroxyl of glass sheet surface is base T in the coupling just.After linked reaction is finished, the not participation reactant that sticks to glass sheet surface is cleaned with acetonitrile solvent.Then with above-mentioned four heap sheet glass and become a pile, and in the 5th reaction tank respectively with acetic acid/acetic anhydride solution carry out capping, iodine reagent carries out oxidizing reaction and trichoroacetic acid(TCA)/dichloromethane solution carries out deprotection reaction.The hydroxyl that glass sheet surface is reacted is closed, and the phosphorus on the Nucleotide becomes stable pentavalent phosphorus and makes the blocking group DMT on 5 '-OH be deviate to obtain activation.After carrying out aforesaid operations, synthesized first base on all glass sheet surface, this base is A, G, C, among the T one of them.Sheet glass with the synthetic base of above-mentioned coding glass sheet surface requires to be divided into again four heaps according to synthetic then.The quantity of every heap sheet glass is not quite similar in two subseries.Two different sheet glass can belong to same a pile in secondary classification, also can not belong to same a pile.Then, in the same way, first piles up and carries out even reaction in first reaction tank, make on the sheet glass coupling A on first base of synthetic, second piles up in second reaction tank and carries out even reaction, make on the sheet glass coupling G on first base of synthetic, the 3rd piles up in the 3rd reaction tank and carries out even reaction, make on the sheet glass coupling C on first base of synthetic, the 4th piles up in the 3rd reaction tank and carries out even reaction, makes on the sheet glass coupling T on first base of synthetic.After linked reaction is finished, the not participation reactant that sticks to the solid phase material surface is cleaned with solvent.With above-mentioned four heaps and one-tenth a pile, in the 5th reaction tank, carry out capping, oxidizing reaction and deprotection reaction then.After carrying out aforesaid operations, all synthesized the oligonucleotide that contains 2 bases on the glass sheet surface of all codings, the form of oligonucleotide is a kind of in the form in 16.Form comprises AA in 16, AG, AC, AT, GA, GG, GC, GT, CA, CG, CT, CA, TA, TG, TC, TT.According to above-mentioned same method; require all sheet glass are divided into four heaps according to synthetic at every turn; same stack is carried out identical linked reaction, finishes corresponding sealing, oxidation and deprotection reaction simultaneously, and then the oligonucleotide on all coding glass sheet surface has just increased a base.So desiring the oligonucleotide of 0 base of Synthetic 2 just carries out 20 subseries and carries out corresponding number of times chemical reaction.How much haveing nothing to do of different oligonucleotide sequences with the number of times of classifying, but relevant with the workload of classification.Oligonucleotide sequence many more, the workload of classification is big more.
The sheet glass of all codings is finished oligonucleotide sequence and is carried out aftertreatment after synthetic, and the blocking group of base and phosphorus is deviate from, and has so just had biological activity.
The sheet glass that synthesizes the oligonucleotide of biologically active is cut into few glass block, and the glass gob after the cutting has the synthetic oligonucleotide that correspondence is arranged of an identification face at least.A lot of glass gob that the sheet glass of same-code cuts into all have the identical coding and the oligonucleotide of identical sequence.
One of taking-up or polylith are arranged and are fixed on the planar substrates from the glass gob (oligonucleotide with corresponding sequence) of each coding, because glass gob has been synthesized corresponding oligonucleotide and the position that is fixed on the planar substrates is known, thereby these are fixed on the array chip that on-chip glass gob has just constituted oligonucleotide sequence.Oligonucleotide arrays chip and specific back detected result that under specific detector, provides of reactant interaction (hybridization).
Claims (10)
1, a kind of assembly type chemical array chip is characterized in that structure comprises upholder, carrier and polymer compound molecule; Be assembled with some carriers on the upholder, separately the synthetic polymer compound molecule that correspondence is arranged on the carrier.
2, assembly type chemical array chip according to claim 1 is characterized in that upholder adopts substrate, Glass tubing, plastics tubing, plastic coil.
3, assembly type chemical array chip production method according to claim 1 is characterized in that: (1) is according to the requirement of desiring synthetic polymer compound molecule on the chemical array chip to be prepared, synthesize corresponding polymer compound molecule respectively by following operation on carrier separately: the monomer molecule solution that carrier in heaps is corresponding with the first location of polymer compound molecule carries out first set reaction earlier, the corresponding monomer molecule solution of the second position that will synthesize first monomeric carrier in heaps and polymer compound molecule more carries out the reaction second time, later on still according to the requirement of desiring synthetic polymer compound sequence, reactant and the corresponding monomer molecule solution of the next position that to work as one by one on time carrier react, until synthesized required complete polymer compound molecule on carrier; (2) after the first step operation has obtained polymer molecule required on the chemical array chip to be prepared, the polymer molecule of carrier together with correspondence together is assembled on the upholder.
4, assembly type chemical array chip preparation method according to claim 3, it is characterized in that requirement, the polymer compound molecule of carrier together with correspondence together is assembled on the upholder according to the position on chemical array chip to be prepared of the polymer compound molecule on the carrier.
5, assembly type chemical array chip preparation method according to claim 3, the carrier that it is characterized in that being used for polymer compound molecule synthetic inequality is encoded, it is corresponding with the carrier of different coding respectively to desire synthetic polymer compound molecule inequality again, to desire the identical pairing carrier of polymer compound molecule of synthetic first location monomer then and concentrate in together the corresponding carrier in heaps of formation, and again carrier in heaps be placed the corresponding monomer molecule solution of first location of polymer compound molecule to react; After, the pairing carrier of polymer compound molecule that the next position monomer is identical concentrates in together again and forms corresponding carrier in heaps, and carrier in heaps and pairing monomer molecule solution are reacted; By that analogy, be set to the heap reaction by turn, until on carrier, having synthesized required complete polymer compound molecule.
6, assembly type chemical array chip preparation method according to claim 3 is characterized in that the polymer compound molecule is meant that by the few small molecules of number be the compound molecule that monomer constitutes; Mainly comprise biomacromolecules such as oligonucleotide, polypeptide, polysaccharide and peptide nucleic acid(PNA).
7, assembly type chemical array chip preparation method according to claim 3 is characterized in that solid support material is by inorganic materials: glass, quartz, pottery, crystal, metal; Organic materials: macromolecular materials such as rubber, plastics, gel, nylon, biomacromolecule, conductive polymers, or by the mixture of above-mentioned inorganic materials and organic materials.
8, assembly type chemical array chip preparation method according to claim 5, the coding that it is characterized in that carrier is the direct mark on solid phase carrier, or on the solid phase carrier indirectly or virtual tag, the purpose of the coding of solid phase carrier is to make things convenient for the entrained compound molecule in synthetic back on the synthetic and identification surface of solid phase carriers of the combination of different compound molecules on the solid phase carrier.
9, assembly type chemical array chip according to claim 1, it is characterized in that compound molecule and fluid interaction wherein after, the change of its character can detect by methods such as optics, electricity, magnetics, acoustics or calorifics.
10, assembly type chemical array chip according to claim 1 is characterized in that the different polymer molecules of carrier surface synthetic can not move under the fluid effect.
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| CN 03112772 CN1431321A (en) | 2003-01-28 | 2003-01-28 | Assembled type compound array chip and its preparing method |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101317093B (en) * | 2005-12-20 | 2012-09-19 | 夏普株式会社 | Novel capture agents for binding a ligand |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101317093B (en) * | 2005-12-20 | 2012-09-19 | 夏普株式会社 | Novel capture agents for binding a ligand |
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