CN101309896B - Novel iminecalixarene derivatives and aminocalixarene derivatives, method of preparation thereof, self-assembled monolayer prepared by the method, fixing method of oligo-DNA by using the self-asse, and oligo-DNA chip prepared by the method - Google Patents

Novel iminecalixarene derivatives and aminocalixarene derivatives, method of preparation thereof, self-assembled monolayer prepared by the method, fixing method of oligo-DNA by using the self-asse, and oligo-DNA chip prepared by the method Download PDF

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CN101309896B
CN101309896B CN200680038071.4A CN200680038071A CN101309896B CN 101309896 B CN101309896 B CN 101309896B CN 200680038071 A CN200680038071 A CN 200680038071A CN 101309896 B CN101309896 B CN 101309896B
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oligomer dna
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金泰善
宋锦秀
金正勋
金亨燮
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Biometrix Technology Inc
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Abstract

The invention provides novel iminecalixarene derivatives, a method for preparation thereof, and a self-assembled monolayer prepared by the method, a method for fixing oligo-DNA by using the self-assembled monolayer, and an oligo-DNA chip prepared by the method. Additionally, the invention provieds novel aminocalixarene derivatives, a method for preparation thereof, and a self-assembled monolayer prepared by the method, method for fixing oligo-DNA in which the oligo-DNA is voluntarily fixed by molecular recognition on said self-assembled monolayer in a liquid phase, and an oligo-DNA chip prepared by the method.

Description

Imines Calixarene Derivatives and amino Calixarene Derivatives, its preparation method, the self-assembled monolayer of being prepared by the method, uses this self-assembled monolayer to fix the method for oligomer DNA, and the oligomer DNA chip of being prepared by the method
Technical field
The present invention relates to novel imines Calixarene Derivatives, its preparation method, and the self-assembled monolayer of being prepared by the method, use this self-assembled monolayer to fix the method for oligomer DNA, and the oligomer DNA chip of being prepared by the method.
The present invention more specifically relate to a kind of can by the oligomer DNA that contains continuous guanine base by molecular recognition be fixed to the imines calixarene compound of the solid substrate surface in individual layer in substrate of glass or gold substrate and by use self-assembled monolayer prepare the method for oligomer DNA individual layer (being oligomer DNA chip).
In addition, the present invention relates to novel amino Calixarene Derivatives, its preparation method, and the self-assembled monolayer of being prepared by the method, irreversible by oligomer DNA in described self-assembled monolayer, the method for the fixing oligomer DNA of molecular recognition, and the oligomer DNA chip of being prepared by the method automatically.
The present invention more specifically relate to a kind of can by the oligomer DNA that contains continuous guanine base by polybase base irreversible/automatically molecular recognition is fixed to the amino calixarene compound of solid substrate surface, thereby a kind of technology that is prepared as the substrate of individual layer biochip on as the solid substrate such as glass or gold substrate and a kind of oligomer DNA is fixed on using high-density as individual layer by as described in prepare the method for oligomer DNA chip in the biochip substrate prepared of technology of preparing.
Background technology
In the National Human Genome Research Institute and Sai Leila genome company, competitiveness under the Human Genome Project is explored and is studied after human genome, Human genome information pattern completed in 2000, thereby promote use DNA functional study, abnormal dna research, and information is wherein carried out the exploitation of diagnostic oligomer DNA chip.This oligomer DNA that is used for exploring monokaryon glycosides polymorphism (SNP) can be expected the genetic diseases for diagnosing defectiveness DNA to cause especially, or cancer in commitment etc.In addition, explore and study and become worldwide trend for the production of the technology of oligomer DNA chip (wherein a series of oligomer DNA is fixed in substrate), this oligomer DNA chip provides the information of the accurate virus infection approach based on Virus Type, the prediction of the generation for harmful or harmless etc. analysis and the somatotype based on one group of oncovirus to cancer etc.In fact the oligomer DNA chip that, can be used for measuring human papillomavirus (HPV) type Korea S by korean foods drug administration (KFDA) in the world authorization first for for predicting diagnosis and the preventive medicine biochip of cervical cancer possibility occurrence.In addition, the oligomer DNA chip of this diagnosing cervical is authorized by KFDA, and comes into operation.
Oligomer DNA chip has comprised and is fixed on suprabasil 15-50mer short dna s (oligomer DNA s).Therefore,, although conventionally adopt simple physisorphtion for the c-DNA chip of analyzing DNA variant, this oligomer DNA chip still adopts the method for exploitation decades ago, for example legal fixing means of imine linkage based on by chemical bond between aldehyde and amine; Or after chemical bonding based between aldehyde and amine functions group, carry out reduction reaction and improve the fixing means of the amine bonding method of bonding.This is to develop owing to also failing at present the biochip substrate that is better than aldehyde-chip on reproducibility and convenience.But in aldehyde-chip base, the amine functions group that is connected to oligomer DNA end by synthetic technology is passed imine linkage connection chemical bonding to this aldehyde-chip; Therefore, fixed dna quantity and can and the theoretical maximum density of the quantity of the fixing oligomer DNA that combines in actual hybridization of c-DNA between exist huge difference.In addition,, owing to being subject to the great effect of rigid condition, the density of fixing oligomer DNA often changes, and is therefore difficult to successfully develop the chip with reproducibility.
A kind of technology of the bonding method that adopts streptavidin-vitamin H is used widely, and described technology is a kind of method of preparing oligomer DNA, its by by streptavidin-vitamin H by physical adsorption, the methods such as chemical combination bonding are fixed in solid substrate, then make the identification of this streptavidin-biotin molecule connect vitamin H functional group (Science, 1993 of the oligomer DNA of this vitamin H; Vol.262, pp 1706-1708).But the technology of this employing chemical bonding or streptavidin-vitamin H bonding method still lags behind aldehyde-chip base in performance.
Meanwhile, in protein chip, the avtive spot of antibody is present on a position that is fixed on lip-deep albumen, if while therefore only having the described position of energy conjugated antigen to be positioned, should obtain result.But, in oligomer DNA chip, part base at least most oligomer DNA s must with c-DNAs phase bonding approaching in hybridization, and the quantity of this bonding base should be maximized by make this cDNAs approach the fixing substrate of this oligomer DNA s as far as possible.For this reason, the European association suggestion of 2003 retains some spaces to allow c-DNAs freely to enter between fixing oligomer DNA s, and the technology that obtains now this kind of space has become the hot research object of this area.Be fixed on lip-deep oligomer DNA s with molecular level and be easy to spontaneous fitting together, therefore this technology will be applied to relatively difficulty of chemical bonding oligomer DNA chip.Accordingly, can by 20-25-mer just the oligomer DNA of enough oligomer DNAs extend to 50-mer, thereby make approaching c-DNA without falling to bottom, and the base quantity that has improved bonding in hybridization is to reappear the result of diagnosis.But, make that this result is clear readablely at least needs several hours to exceeding one day, therefore this technology is outstanding unlike traditional diagnostic techniques of analyzing about bird flu etc.
Summary of the invention
Technical problem
In as the traditional method of the fixing oligomer DNA s such as chemical bonding and streptavidin-vitamin H method, find following problem:
1. the homogeneity of constant density: chemical bonding is the most widely used method, and known in the time that oligomer DNA s is fixed, between aldehyde functional group group (CHO) and amine functions group (NH), chemical reaction has occurred, thereby chemistry is fixed oligomer DNA s; In this reaction, take off a H 2o so that oligomer DNA be fixed with the form of imine linkage (C=N-).But, because this reaction betides in the aqueous solution, this constant density heterogeneity, that is, not reproducible.In addition, the density of this oligomer DNA is difficult to maintain homogeneity, because easily there is imine linkage and revert to the reversed reaction of original functional group in the aqueous solution.In addition, for reducing by this kind of reversed reaction, can adopt as NaBH 4deng reductive agent, imine linkage is carried out to reduction reaction, but this kind of reduction reaction can affect the bonding of oligomer DNA s, thereby be difficult to remain the length of fixing oligomer DNA s.Due to the discordance of each production result, the exploitation of novel oligomer DNA chip will be a long process, and this is counted as the huge obstacle in oligomer DNA chip production.Meanwhile, this fixing oligomer DNA can for example, by the proportional c-DNAs that has been connected fluorescent substance (, Cy-3) of bonding and described oligomer DNA density, then detects the existence of c-DNAs and concentration thereof to diagnose.Therefore, if cannot continue to maintain the density of oligomer DNA, this oligomer DNA cannot be produced as commerical prod, and in view of same reason, many companies that dropped into fund still fail to obtain their expected result.
2. between oligomer DNA s, obtain the necessity (ultra-high speed hybridization can not occur) that space enters for c-DNAs: the templet gene of pathogenic agent must finally be connected to oligomer DNA chip by the c-DNAs of polymerase chain reaction (PCR) amplification.But as shown in Figure 7, if the distance between fixed dna s is too short, c-DNAs cannot arrive the bottom of oligomer DNA s easily.Current many oligomer DNA chips need a few hours to the hybridization that exceedes a day, and this is mainly to allow c-DNAs to approach easily the space of the base of bonding with it, fixing oligomer DNA s bottom owing to lacking.Therefore the technology that obtains the space of proper level between oligomer DNA s becomes a kind of needs, but people do not obtain effective solution to this.For addressing this problem, be necessary that exploitation makes c-DNAs can approach easily the technology of DNA chip by the surface bond of continuous base with the space that obtains proper level.
3. the connection of functional group: should only just may exist in the time that vitamin H or amine functions group are connected to oligomer DNA by technology of the fixing oligomer DNA of chemical bond or streptavidin-vitamin H bonding.But the cost that connects again this kind of functional group after oligomer DNA is synthetic is three to ten times of the oligomer DNA prior synthesizing method that has been connected of a small amount of functional group.In addition, even if only fixed tens oligomer DNA s oligomer DNA chips that next life, production-supply-marketing was sold, still need to detect hundreds of sample, and its required cost (disregarding the cost of producing finished product) just needs hundreds of or millions of dollar, and this becomes another obstacle of exploitation oligomer DNA chip.For reducing by this kind of problem, be necessary to develop a kind of by the technology that the oligomer DNA s of linkage function group does not produce oligomer DNA chip.
4. with three kinds of base A, chemical bonding between amine functions group and the functional group of aldehyde-chip base that C and G connect: this aldehyde-chip base can be produced oligomer DNA chip, wherein this oligomer DNA s is fixed with the reaction of the oligomer DNA s that has been connected amine functions group by surperficial aldehyde functional group group.But, this amine functions group has been connected in the described numerous bases that are connected with this oligomer DNA and has accounted for three kinds of bases of 3/4ths, and when the amine functions group being connected with oligomer DNA end faster when having fixed base at the oligomer DNA s at middle part during with aldehyde reaction, therefore can be used for and approach, the c-DNA that fluorescence connects carries out the base quantity not sufficient of many hydrogen bondings.Aldehyde can also be by hydrogen bond the base bonding with in addition approaching c-DNA, thereby removed, after oligomer DNA s is fixed, should carry out chemical treatment, knownly generated copying of DNA chip and become more difficult.
5. diagnose the technology of SNP: oligomer DNA chip is conducive to analyzing DNA s most so that all kinds of DNA that are present in multiple virus (as a bird flu, swine fever epidemic disease, cancer diffusion virus etc.) are carried out to somatotype in various technology.But this kind of virus major part is the new form generating from the form variation of protovirus class, same, the base sequence between viral initial form and variant thereof does not make a big difference as a rule.More difficultly many virus groups only show difference on one or two base sequences.Accordingly, in exploitation oligomer DNA chip, most important part is to prepare a kind of oligomer DNA chip that can differentiate in switch level the variation (, can diagnose SNP) of single base.Many investigators agree to adopt the oligomer DNA chip of current the most frequently used aldehyde-chip base production between fixing oligomer DNA s, not differentiate the necessary sufficient space of this list base variant, and therefore cannot produce the diagnostic oligomer DNA chip for differentiating single base variant.
6. produce the technology of chip at a low price: if by connecting as amine, the functional groups such as vitamin H are fixed oligomer DNA, the cost of this oligomer DNA can rise more than ten doubly, and the DNAs that this chip must cover high density is to stop surperficial base bonding.To become high so produce the cost of this kind of oligomer DNA chip.
Technical scheme
The object of the present invention is to provide the imines cup derivative can molecular recognition with the oligomer DNA of continuous guanine group, and preparation method thereof, thereby solve the problem of above-mentioned traditional oligomer DNA fixing means, as the homogeneity problem of constant density, between oligomer DNA s, obtain the problem of sufficient space, the connectivity problem of functional group etc.
Another object of the present invention is to provide a kind of oligomer DNA individual layer, wherein the combination of all kinds of oligomer DNA s by described imines Calixarene Derivatives and substrate of glass (a kind of amine slide) or gold substrate (au film coating or gold) can be without any processing by intensive filling and retain the space of proper level; A kind of imines Calixarene Derivatives individual layer that can make oligomer DNA chip is namely provided.
The present invention more specifically target for a kind of oligomer DNA s individual layer is provided, the oligomer DNA s wherein with at least seven continuous guanines by intensive filling and retained the space of proper level, namely provides a kind of imines Calixarene Derivatives individual layer that can make oligomer DNA chip without any processing.
The object of the present invention is to provide on the other hand a kind of oligomer DNA chip, its simultaneously solution must not carry out ultra-high speed hybridization, can not guarantee to diagnose SNP technology, with three base A, chemical bonding between amine functions group and the functional group of aldehyde-chip base that C and G connect, the problem that is difficult to homogeneous density fixes all traditional oligomer DNA chips such as oligomer DNA s on chip.That is to say, the object of the present invention is to provide can the continuous guanine group of irreversible molecular recognition amino Calixarene Derivatives, a production method for the substrate of the oligomer DNA chip of preparing with its self-assembly form, and a kind of oligomer DNA chip of producing by the irreversible fixing oligomer DNA s of the spontaneous molecular recognition on chip base surface.
Another object of the present invention is to provide a kind of oligomer DNA s individual layer, wherein by using above-claimed cpd as individual layer with the solid substrate such as substrate of glass (amine slide) with amine functions group, or be connected with gold substrate (au film coating or gold), all kinds of oligomer DNA s can the unprocessed space of fixing and retain proper level with maximum density, that is to say, a kind of chip base that can make oligomer DNA chip of producing with amino Calixarene Derivatives is provided.
Another object of the present invention is to provide a kind of oligomer DNA individual layer, wherein this continuous guanine base is fixed on this surface by linear, thereby make the unprocessed space of fixing and retain proper level with maximum density of this oligomer DNA s, a kind of technology for the production of oligomer DNA chip that provides is provided.
Accompanying drawing explanation
Fig. 1 is the schematic diagram in the preparation process of the upper imines Calixarene Derivatives self-assembled monolayer of the present invention of substrate of glass (amine slide).
Fig. 2 is the schematic diagram of the preparation process of imines Calixarene Derivatives self-assembled monolayer of the present invention in gold substrate.
Fig. 3 is the fixing means schematic diagram of the present invention's oligomer DNA with continuous guanine base.
Figure 4 and 5 have shown the selectivity fixing means of the oligomer DNA only with continuous guanine base, and have shown the result of analyzing fixing oligomer DNA density with the c-DNA with fluorescence.
At least 7 continuous guanine functional groups of needs when Fig. 6 has shown to the fixing oligomer DNA of imines Calixarene Derivatives self-assembled monolayer of the present invention.
Fig. 7 has shown for verifying that this guanine base is identified by selectivity by molecular recognition when to the fixing oligomer DNA of imines Calixarene Derivatives self-assembled monolayer of the present invention, the result that uses the imidazoles functional group with similar structures to carry out competitive reaction.
Fig. 8 is the schematic diagram of the preparation process of amino Calixarene Derivatives self-assembled monolayer of the present invention in substrate of glass.
Fig. 9 is the schematic diagram of the preparation process of amino Calixarene Derivatives self-assembled monolayer of the present invention in gold substrate.
Figure 10 and 11 has method schematic diagram and the fixing actual tests result of maximum density that the oligomer DNA of continuous guanine base is fixed by automatic molecular recognition for the present invention.
Figure 12 detects the test-results of carrying out the continuous guanine base number of the fixing the best of maximum density.
Figure 13 is the schematic diagram that the space between the fixing oligomer DNA that obtains by the suitable space of guaranteeing between continuous base can be by fixing oligomer DNA time of c-DNA approaches chip bottom.
Figure 14 and 15 is for showing that hybridizing SNP by high speed differentiates the actual tests result that may reach switch level.
Figure 16 is the actual competitive reaction result that shows guanine base and other base stationary phase than carrying out selectivity and fix at a high speed.
Invention pattern
Novel imines Calixarene Derivatives of the present invention has the structure of following formula 1 or 2.Described imines Calixarene Derivatives is the key compound with the self-assembled monolayer adopting in the fixing means of oligomer DNA of at least 7 continuous guanine bases.
[formula 1]
Figure DEST_PATH_G200680038071401D00011
(wherein R 1, R 2, R 3and R 4independently be selected from combination-H ,-CH 3,-C 2h 5,-C 3h 7,-OCH 3,-Cl ,-C 6h 5with-COOR, and in described-COOR, R representative-CH 3or-C 2h 5).
[formula 2]
(wherein R 1, R 2, R 3and R 4independently be selected from combination-H ,-CH 3,-C 2h 5,-C 3h 7,-OCH 3,-Cl ,-C 6h 5with-COOR, and in described-COOR, R representative-CH 3or-C 2h 5; In addition described Y, 1, Y 2, Y 3and Y 4independently be selected from combination-H ,-(CH 2) n-CH=O ,-(CH 2) n-SH ,-(CH 2cH 2o) m-CH 2cH 2-CH=O ,-(CH 2cH 2o) m-CH 2cH 2-SH ,-(CH 2) m-C 6h 4-(CH 2) c-Z and-CO-(CH 2) m-1-C 6h 4-(CH 2) c-Z (wherein n=2-15, m=1-10, c=0-10, Z=-SH ,-CHO ,-COOH or-NH 2)).
Figure S2006800380714D00111
The present invention also provides the method for the imines calixarene derivative compound of the described formula 1 and 2 of preparation.
Have following formula 3 this 5,11,17,23-tetramino cup [4] aromatic hydroxy compound can be by being converted into 5,11,17 with reference to the method described in the following reference of quoting 1 by cup [4] aromatic hydrocarbons, after 23-tetranitro cup [4] aromatic hydrocarbons, synthesize and obtain [the reference 1:Journal of Organic Chemistry quoting through reduction reaction, 1990, VoI 55, pp 5639-5643; Journal of Organic Chemistry, 1979, VoI 44, pp 1233-1238].
[formula 3]
Figure DEST_PATH_G200680038071401D00031
The imines calixarene derivative compound of formula 1 of the present invention can pass through with 5 of described formula 3,11,17,23-tetramino cup [4] aromatic hydroxy compound, as starting raw material, reacts the amine functions group of formula 3 synthetic obtain (reaction 2 vide infra in embodiment) with the aromatic aldehyde with following formula 4.
[formula 4]
Figure DEST_PATH_G200680038071401D00032
(wherein R is selected from combination-H ,-CH 3,-C 2h 5,-C 3h 7,-OCH 3,-Cl ,-C 6h 5with-COOR ', and in described-COOR ', R ' representative-CH 3or-C 2h 5).
[formula 4]
In addition, this imines calixarene derivative compound of formula 2 of the present invention can obtain with reacting of aldehyde cpd is synthetic by through type 1 compound, wherein halogen is connected to an end group, thereby aldehyde or mercaptan are connected to first and three-OH group position (referring to the reaction 3-5 of the following example) of formula 1 in this end group.
Described halogenation aldehyde is selected from following combination X-(CH 2) n-CH=O, X-(CH 2) n-SH, X-(CH 2cH 2o) m-CH 2cH 2-CH=O, X-(CH 2cH 2o) m-CH 2cH 2-SH, X-(CH 2) m-C 6h 4-(CH 2) c-Z and X-CO-(CH 2) m-1-C 6h 4-(CH 2) c-Z, wherein n=2-15, m=1-10, c=0-10, X=-Cl ,-Br ,-I or-OSO 2c 6h 4cH 3, Z=-SH ,-CHO ,-COOH or-NH 2.
In addition, the invention provides a kind of to gold substrate connect a kind of self-assembled monolayer solid substrate preparing with the imines Calixarene Derivatives of thiol group.
In addition, the invention provides one is connected to imines Calixarene Derivatives as glass by imine linkage, the self-assembled monolayer solid substrate that the solid substrate such as silicon wafer or crystal prepares, and a kind of passing through as ester, the chemical bond such as ether and amido linkage is connected to imines Calixarene Derivatives as glass, the self-assembled monolayer solid substrate that the solid substrate such as silicon wafer or crystal prepare.
In addition, the invention provides a kind of oligomer DNA individual layer (, a kind of oligomer DNA chip), and preparation method thereof, wherein all kinds of oligomer DNAs by by the imines calixarene derivative compound of described formula 2 be connected the gold substrate of amine functions group or substrate of glass and be connected and fixed by high-density.
Fig. 1 illustrates the preparation process of imines Calixarene Derivatives self-assembled monolayer of the present invention in substrate of glass.
The concrete grammar of preparing imines Calixarene Derivatives self-assembled monolayer in the substrate of glass that has connected amine functions group is as follows:
First, the reference 2:Langmuir that this substrate of glass (glass slide) that has connected described amine functions group can be quoted with reference to the following reference 2[quoting, 1997, VoI 13, pp 4305-4308; Langmuir, 1996, Vol 12, pp 5338-5342] form of the substrate of glass with amine end group (amine slide or amine chip) that obtains by the chemical reaction in the substrate of glass having sufficient silanol (Si-OH) functional group is prepared.Connect the substrate of glass of amine functions group and formula 2 compounds have been added into chloroform and THF mixing solutions (CHCl with the concentration of 0.1-5mM thereby prepare 3: THF=9: 1).After 1-5 hour, with chloroform, the order of acetone and ethanol is washed and is dried, and then completes imines calixarene self-assembled monolayer as shown in Figure 1.The formation of described individual layer can be confirmed by infrared rays external reflection spectrum.All kinds of slides on market all can be used as described substrate of glass.For imine linkage used in bonding, fracture when this key can be deposited for a long time in water.But, because the active materials such as oligomer DNA are dissolved in pH in the preparation conventionally in the buffered soln of 7-8, do not have problems in the time forming imine linkage immediately with it through confirmation.
Fig. 2 illustrates the preparation process of imines Calixarene Derivatives self-assembled monolayer of the present invention in gold substrate.
The concrete grammar of preparing imines Calixarene Derivatives self-assembled monolayer in gold substrate is as follows:
The compound of the formula 2 that has connected mercaptan is dissolved in as chloroform (CHCl with the concentration of 0.1-5mM 3) etc. prepare solution in organic solvent.Gold substrate is placed into prepared solution and places after 1-5 hour, by its taking-up and with chloroform, and the order washing of acetone and water, and dry, then complete imines Calixarene Derivatives self-assembled monolayer as described in Figure 2.Described gold substrate can be the gold thin film of any type; But, generally speaking, preferably at glass, molten silicon, silicon wafer, plastic-substrates etc. after Vacuum Deposition is with the material such as chromium (Cr) or titanium (Ti) of 2-10nm with the substrate of the thickness vacuum metallizing of 50-200nm.Generally speaking, prepared gold substrate is placed in Piranha solution (strength sulfuric acid: 30% hydrogen peroxide=3: 1) approximately 1 minute, then use after dry with pure water washing nitrogen blowing before being about to use.The formation of described individual layer can be confirmed by infrared rays external reflection spectrum.
In addition, the invention provides a kind of oligomer DNA chip of preparing the method for oligomer DNA chip and being prepared by the method by fixing oligomer DNA, wherein identify continuous guanine base is fixed to described solid substrate by polymolecular.
More specifically, the invention provides a kind of method of the fixing oligomer DNA of polymolecular identification of four continuous guanine functional groups of nitrogen-atoms molecular recognition by described imines Calixarene Derivatives.
Oligomer DNA fixing means of the present invention has formed the basis of the analytical procedure of all employing oligomer DNAs, and this is that the whole world has no the novel method that similar research is reported so far.
Fig. 3 is the fixing means schematic diagram of the present invention's oligomer DNA with continuous guanine group.
This has for the oligomer DNA of fixing continuous guanine group is 5 '-GGGGGG GGG AAA TCA ACC CAC AGC TGC A-3 ' (SEQ ID NO.1).By with the c-DNA with fluorescence of its combination be 5 '-Cy3-GT GCA GCT GTG GGTTGA TT-3 ' (SEQ ID NO.2), it has and the DNA sequence dna of fixing oligomer DNA complementation.Now, the constant density of this oligomer DNA can by by fluorescent substance Cy-3 (Telechem, the U.S.) thus being connected to this end group only shows that in this connection site fluorescence is measured.After in oligomer DNA is dissolved in to the solution 500mM KCl that contains 15% glycerol with the concentration of 54 μ g/ml (6.75pmol/ml), this oligomer DNA can be fixed by being coated on the imines calixarene individual layer of using the diameter of preparing purchased from the microarray of Proteogen Co. (Korea) to be about 150um.After about 1-4 hour, with 2X SSC (the 1X SSC=Trisodium Citrate 15mM+NaCl 150mM) solution washing that contains 0.1%SDS (sodium laurylsulfonate), with 0.1X SSC solution washing 3 minutes dry preparation.Prepare through determining this that oligomer DNA is one-sided does not demonstrate any variation in the measurement of at least 6 months.
Figure 4 and 5 are the schematic diagram having shown in this oligomer DNA density of oligomer DNA individual layer of the present invention fluorometric analysis.
This oligomer DNA with the continuous guanine group for being fixed to oligomer DNA individual layer prepared by Fig. 3 is 5 '-GGG GGG GGG AAA TCA ACC CACAGC TGC A-3 ' (SEQ ID NO.1).By with the c-DNA with fluorescence of its combination be 5 '-Cy3-GT GCA GCT GTG GGT TGA TT-3 ' (SEQ ID NO.2), it has and the DNA sequence dna of fixing oligomer DNA complementation.In addition, the constant density of this oligomer DNA can by by fluorescent substance Cy-3 (Telechem, the U.S.) thus being connected to this end group only shows that in this connection site fluorescence is measured.This individual layer is coated with the 60 μ l 4x SSC+0.002%SDS+50% glycerol mixing solutionss of the c-DNA being connected with fixing oligomer DNA fluorescence containing 0.17nmol/ml, then at 50 ℃, DNA is hybridized 30 minutes.First at room temperature wash with the 4x SSC solution containing 0.1%SDS subsequently, then with 4x SSC solution washing, and dry.Products obtained therefrom can use scanner (GSI lite, the U.S.) to confirm.
For the oligomer DNA s for fixing, can adopt the oligomer DNA respectively with following DNA sequence dna: there are 9 continuous A, T, C, G base sequence, one have connected oligomer DNA and a kind of oligomer DNA without continuous base sequence of vitamin H (functional group of similar guanine).Accordingly, confirm that through test only continuous guanine base is fixed by selectivity.It is as follows that this is used for fixing DNA sequence dna relatively:
9G 5′-GGG GGG GGG AAA TCA ACC CAC AGC TGCA-3′(SEQ ID NO.1)
9T 5′-TTT TTT TTT TAA TCA ACC CAC AGC TGC A-3′(SEQ ID NO.3)
9A 5′-AAA AAA AAA TAA TCA ACC CAC AGC TGCA-3′(SEQ ID NO.4)
9C 5′-CCC CCC CCC CAA TCA ACC CAC AGC TGC A-3′(SEQ ID NO.5)
Vitamin H 5 '-vitamin H-T ATA TAA TCA ACC CAC AGC TGCA-3 ' (SEQ ID NO.6)
no 5′-AA TCA ACC CAC AGC TGC A-3′(SEQ ID NO.7)
Should can there is complementary base sequence through synthesizing by the c-DNA of hybridizing combination with it.
5′-Cy3-GT GCA GCT GTG GGT TGA TT-3′(SEQ ID NO.2)
Can by the relative density of analyzing the c-DNA connecting of connection with fluorescent scanning instrument by fluorescent substance (Cy3), that is, connect the density of the fixing oligomer DNA of c-DNA simultaneously.According to actual tests result, in above-mentioned condition, apply in the same manner and fix after each oligomer DNA, this c-DNA applies with three times of can be fixed on surperficial oligomer DNA maximum, thereby makes c-DNA that abundant fluorescence connects and all fixing oligomer DNAs in this combination.Then, wash and be dried and analyze with fluorescent scanning instrument.
Actual tests result is the middle figure of Fig. 5 that shows result with 6X6, and this result carries out with numerical value that fluorometric analysis obtains and this analysis is shown in the histogram on Fig. 5 right side.Actual tests result shows to have fluorescence that the oligomer DNA 9G of 9 continuous guanine groups presents approximately than having connected different continuous bases, or has connected vitamin H, or the high 10-40 of oligomer DNA that does not connect continuous base doubly.Described result shows that while only there is continuous guanine base in the time of fixing oligomer DNA, this oligomer DNA could be with on the analyzable surface that is horizontally fixed on imines calixarene.
Fig. 6 must have at least 7 continuous guanine functional groups while having shown to the fixing oligomer DNA of imines Calixarene Derivatives self-assembled monolayer of the present invention.
This oligomer DNA is fixed under the condition shown in Fig. 3 and carries out, and this c-DNA can use and hybridize for the band fluorescence oligomer DNA of Fig. 4 equally according to above-mentioned condition.
1G 5′-GAA TCA ACC CAC AGC TGC A-3′(SEQ ID NO.8)
4G 5′-GGG GAA TCA ACC CAC AGC TGC A-3′(SEQ IDNO.9)
7G 5′-GGG GGG GAA TCA ACC CAC AGC TGC A-3′(SEQ ID NO.10)
9G 5′-GGG GGG GGG AAA TCA ACC CAC AGC TGCA-3′(SEQ ID NO.1)
12G 5′-GGG GGG GGG GGG GAA TCA ACC CAC AGCTGC A-3′(SEQ ID NO.11)
15G 5′-GGG GGG GGG GGG GGG GAA TCA ACC CACAGC TGC A-3′(SEQ ID NO.12)
C-DNA 5 '-Cy3-GT GCA GCT GTG GGT TGA TT-3 ' (SEQID NO.2) (Cy-3; Fluorescent substance)
Under above-mentioned the same terms, apply washing dry oligomer DNA.According to actual tests result, in above-mentioned condition, apply in the same manner and fix after each oligomer DNA, this c-DNA applies with three times of can be fixed on surperficial oligomer DNA maximum, thereby makes the c-DNA being connected with all fixing oligomer DNA fluorescence in this combination.Then it is washed and is dried analyze by fluorescent scanning instrument.
Actual tests result is the middle figure of Fig. 6 that shows result with 6X6, and this result carries out with numerical value that fluorometric analysis obtains and this analysis is shown in the histogram on Fig. 6 right side.In the time that continuous guanine base sequence is about 1 or 4, its result is not for almost having surface fixing.In addition, in the time that it has 7 continuous guanines (7G), show intense fluorescence, and demonstrate the strongest fluorescence in the time thering are 9 continuous guanines (9G).Meanwhile, in the time that oligomer DNA has 12 (12G) and 15 (15G) continuous guanine base of being longer than 9 continuous guanine bases, this fluorescence sensitivity starts to decline.This result shows that 9 guanine bases are the levels that are enough to be fixed by molecular recognition, and in theory in the time using longer guanine base, the oligomer DNA fixedly with 12G need to have more approximately 1/3 space than the oligomer DNA fixedly with 9G, thereby this fluorescence sensitivity drops to approximately 1/3 level.The oligomer DNA with 15G need to be more than the space of 9G approximately extra 2/3, and the quantity being therefore fixed in theory drops to approximately 3/5 level (60%), and in fact fluorescence sensitivity has declined approximately 55% compared with 9G.Comprehensive described result, is appreciated that fixing required continuous guanine quantity is at least 7.The quantity of described continuous guanine is preferably 7-15, more preferably 9-12, selects 9 most.
Fig. 7 has shown for verifying that this guanine base is identified by selectivity by molecular recognition when to the fixing oligomer DNA of imines Calixarene Derivatives self-assembled monolayer of the present invention, the result that uses the imidazoles functional group with similar structures to carry out competitive reaction.
Fig. 7 has shown the result that continuous guanine base is fixed by molecular recognition, and this result comprises a kind of can carrying out and obtains with the research of the imidazoles of the similar molecular recognition of guanine base in fixing by one.Because imidazoles has the molecular recognition relatively more weak than guanine base in imines calixarene, therefore can be at the competitive reaction higher than treating to carry out under the concentration of fixing oligomer DNA molecular recognition.Under the rigid condition identical with Fig. 3, be fixed and adopt the c-DNA in Fig. 4 same procedure used to carry out the measurement of fluorescence sensitivity.The left figure of Fig. 7 has shown actual tests result, and right figure has shown the decline of the fluorescence sensitivity that depends on imidazole concentration used in fluorescence sensitivity competitive reaction.In fact, result shows that this decline originates in the level of about 20mM, and drops to half in the level of 30mM, and 100mM and when above oligomer DNA be fixed hardly.This result shows that oligomer DNA is fixed by the polymolecular identification of 9 continuous guanine bases in imines Calixarene Derivatives.
As above result demonstration imines Calixarene Derivatives self-assembled monolayer is identified and is carried out the irreversible fixing of 9 continuous guanine bases by polymolecular.The fixing oligomer DNA quantity representing by fluorometric analysis has shown the constant density of 5-30 times higher than conventionally known method especially.
Similarly, the invention provides the novel oligomer DNA fixing means that one can obtain the space with its length (2.5-3.5nm) in the time of the fixing continuous guanine base in individual layer surface, thereby the c-DNA that hundreds of base sequences are at least provided by providing when have the gene of specific base sequence with diagnosis or the goal analysis such as research drops to oligomer DNA bottom the enough space (2.5-3.5nm) required with fixing oligomer DNA strong bonding, c-DNA is hybridized to the required time and significantly dropped in 1-2 hour by original at least 12 hours.
As another aspect of the present invention, novel amino Calixarene Derivatives of the present invention has the structure with following formula 5-8.Described amino Calixarene Derivatives is the key compound with self-assembled monolayer used in the fixing means of the oligomer DNA of fixing continuous guanine group.
[formula 5]
Figure DEST_PATH_G200680038071401D00041
(wherein R 1, R 2, R 3, R 4, R 5, R 6, R 7, R 8, R ' 1, R ' 2, R ' 3, R ' 4, R ' 5, R ' 6, R ' 7, R ' 8independently be selected from combination-H ,-CH 3,-C 2h 5,-C 3h 7,-OCH 3,-Cl ,-C 6h 5,-OH ,-OCH 2cH 3,-Br ,-CF 3,-OCH 2c 6h 5,-OC 6h 5,-OC 6h 4cH 3,-OC 6h 4c (CH 3) 3,-OC 6h 4cF 3,-OC 6h 4cl ,-OCOCH 3,-NHCOCH 3,-CONHCH 3,-CN, COOH and-COOR, and in described-COOR, R representative-CH 3or-C 2h 5).
[formula 6]
Figure DEST_PATH_G200680038071401D00051
(wherein R 1, R 2, R 3, R 4, R 5, R 6, R 7, R 8, R ' 1, R ' 2, R ' 3, R ' 4, R ' 5, R ' 6, R ' 7, R ' 8independently be selected from combination-H ,-CH 3,-C 2h 5,-C 3h 7,-OCH 3,-Cl ,-C 6h 5,-OH ,-OCH 2cH 3,-Br ,-CF 3,-OCH 2c 6h 5,-OC 6h 5,-OC 6h 4cH 3,-OC 6h 4c (CH 3) 3,-OC 6h 4cF 3,-OC 6h 4cl ,-OCOCH 3,-NHCOCH 3,-CONHCH 3,-CN, COOH and-COOR, and in described-COOR, R representative-CH 3or-C 2h 5.In addition described Y, 1, Y 2, Y 3and Y 4independently be selected from combination-H ,-(CH 2) n-CH=O ,-(CH 2) n-SH ,-(CH 2cH 2o) m-CH 2cH 2-CH=O ,-(CH 2cH 2o) m-CH 2cH 2-SH ,-(CH 2) m-C 6h 4-(CH 2) c-Z, and-CO-(CH 2) m-1-C 6h 4-(CH 2) c-Z (wherein n=2-15, m=1-10, c=0-10, Z=-SH ,-CHO ,-COOH ,-NH 2, and-C 6h 4-and C 6h 5be defined as phenyl group)).
[formula 7]
Figure DEST_PATH_G200680038071401D00061
(wherein R 1, R 2, R 3, R 4, R 5, R 6, R 7, R 8, R ' 1, R ' 2, R ' 3, R ' 4, R ' 5, R ' 6, R ' 7, R ' 8independently be selected from combination-H ,-CH 3,-C 2h 5,-C 3h 7,-OCH 3,-Cl ,-C 6h 5,-OH ,-OCH 2cH 3,-Br ,-CF 3,-OCH 2c 6h 5,-OC 6h 5,-OC 6h 4cH 3,-OC 6h 4c (CH 3) 3,-OC 6h 4cF 3,-OC 6h 4cl ,-OCOCH 3,-NHCOCH 3,-CONHCH 3,-CN, COOH and-COOR, and in described-COOR, R representative-CH 3or-C 2h 5).
[formula 8]
Figure DEST_PATH_G200680038071401D00071
(wherein R 1, R 2, R 3, R 4, R 5, R 6, R 7, R 8, R ' 1, R ' 2, R ' 3, R ' 4, R ' 5, R ' 6, R ' 7, R ' 8independently be selected from combination-H ,-CH 3,-C 2h 5,-C 3h 7,-OCH 3,-Cl ,-C 6h 5,-OH ,-OCH 2cH 3,-Br ,-CF 3,-OCH 2c 6h 5,-OC 6h 5,-OC 6h 4cH 3,-OC 6h 4c (CH 3) 3,-OC 6h 4cF 3,-OC 6h 4cl ,-OCOCH 3,-NHCOCH 3,-CONHCH 3,-CN, COOH and-COOR, and in described-COOR, R representative-CH 3or-C 2h 5.But, get rid of R 1, R 2, R 3, R 4, R 5, R 6, R 7, R 8, R ' 1, R ' 2, R ' 3, R ' 4, R ' 5, R ' 6, R ' 7, R ' 8be-situation of H simultaneously.In addition described Y, 1, Y 2, Y 3and Y 4independently be selected from combination-H ,-(CH 2) n-CH=O ,-(CH 2) n-SH ,-(CH 2cH 2o) m-CH 2cH 2-CH=O ,-(CH 2cH 2o) m-CH 2cH 2-SH ,-(CH 2) m-C 6h 4-(CH 2) c-Z, and-CO-(CH 2) m-1-C 6h 4-(CH 2) c-Z (wherein n=2-15, m=1-10, c=0-10, Z=-SH ,-CHO ,-COOH ,-NH 2, and-C 6h 4with-C 6h 5be defined as phenyl group)).
[formula 8]
The invention provides the method for the amino calixarene derivative compound of the described formula 5-8 of preparation.
[formula 9]
Figure DEST_PATH_G200680038071401D00081
Tetramino cup [4] aromatic hydrocarbons of described formula 9 can be converted into cup [4] aromatic hydrocarbons to carry out that reduction reaction is synthetic to be obtained after tetranitro cup [4] aromatic hydrocarbons by the method with reference to the reference 1 that quote.
The invention provides the method for the amino calixarene derivative compound of the described formula 5-8 of preparation.
The amino calixarene derivative compound of formula 5 and 7 of the present invention can be by using tetramino cup [4] aromatic hydroxy compound by the synthetic formula 9 of currently known methods as starting raw material, the amine functions group of formula 9 is reacted to synthesize with benzyl chloride in embodiment 13 below or benzyl bromide derivatives obtain.Alternatively, after the functional group of formula 9 and aromatic aldehyde reacts with reference to the method for embodiment 16 below, use reductive agent carries out reduction reaction, is the amino Calixarene Derivatives of reacting synthesis type 5 and 7 with benzyl chloride or benzyl bromide derivatives.
(wherein aldehyde or mercaptan are by following-OH for the amino calixarene derivative compound of formula 6 and 8 of the present invention,, first in alcohol functional group is connected with alkyl group end with triol functional group) can be by the compound of formula 5 and 7 be reacted and is obtained with the compound (wherein halogen is connected to end group with reference to the following example 17-19) with functional groups such as aldehyde.
In addition, the invention provides a kind of for the preparation of can be by the amino Calixarene Derivatives of described formula 6 or 8 being connected to the gold substrate of the various functional groups such as amine or if the solid substrate such as glass are with the preparation method of the oligomer DNA chip of the oligomer DNA individual layer of the fixing various types of oligomer DNAs of high-density.
In addition, the invention provides one is connected amino Calixarene Derivatives (wherein aldehyde functional group group is connected to the end group of the amino Calixarene Derivatives of described formula 6 or 8) and is selected from glass, the self-assembled monolayer solid substrate that the solid substrate with amine functions group of silicon wafer and crystal prepares with amido linkage by imines.
In addition, the invention provides a kind of by being selected from ester, the chemical bond of ether or amido linkage connects amino Calixarene Derivatives (wherein aldehyde functional group group is connected to the end group of the amino Calixarene Derivatives of described formula 6 or 8) and is selected from glass, the self-assembled monolayer solid substrate that the solid substrate with amine functions group of silicon wafer and crystal prepares.
In addition, the invention provides and a kind ofly by irreversible molecular recognition, the undressed oligomer DNA with continuous base is fixed to described oligomer DNA chip base and prepares a kind of oligomer DNA individual layer, i.e. a kind of method of oligomer DNA chip.
Fig. 8 has shown as glass, and silicon wafer is prepared the process of amino Calixarene Derivatives self-assembled monolayer of the present invention in the solid substrate such as molten silicon.
The concrete grammar of preparing amino Calixarene Derivatives self-assembled monolayer in the substrate of glass that has connected amine functions group is as follows:
The form of the substrate of glass with amine end group (amine slide or amine chip) that the substrate of glass (glass slide) of the amine functions group having used before having connected can obtain by the chemical reaction on the glass basic surface having sufficient silanol (Si-OH) functional group with reference to the following reference of quoting 2 is prepared.The mixing solutions that the substrate of glass that is connected with amine functions group of so preparation is added to chloroform and THF, the compound of its Chinese style 6 or 8 is dissolved in wherein (CHCL with the concentration of 0.1-5mM 3: THF=9: 1).After 1-5 hour, with chloroform, the order washing of acetone and ethanol, and dry, then complete amino Calixarene Derivatives self-assembled monolayer as shown in Figure 8, i.e. A type BMT DNA chip base.The formation of described individual layer can be confirmed by infrared rays external reflection spectrum.All kinds of slides on market or glass all can be used as described substrate of glass.For imine linkage used in bonding, fracture when this key can be deposited for a long time in water.But because the active materials such as oligomer DNA are dissolved in pH in the preparation conventionally in the buffered soln of 7-8, results verification does not have problems in the time forming imine linkage immediately with it after deliberation.
If as shown in Figure 8 this being prepared to A type BMT DNA chip base puts into containing just like BH 3-THF or 1-5%NaBH 4deng 1-10 minute in the organic solvent of reductive agent, this imines is reduced into stronger amine, and therefore can be long-term preservation or within the scope of different pH, preparing DNA chip provides outstanding stability.
Fig. 9 has shown the preparation process of amino Calixarene Derivatives self-assembled monolayer of the present invention in gold substrate.
The concrete grammar of preparing amino Calixarene Derivatives self-assembled monolayer in gold substrate is as follows:
The compound of the formula 6 or 8 that has connected mercaptan is dissolved in as chloroform (CHCl with the concentration of 0.1-5mM 3) etc. prepare solution in organic solvent.Gold substrate is placed into prepared solution and places after 1-5 hour, by its taking-up and with chloroform, and the order washing of acetone and water, and dry, then complete amino Calixarene Derivatives self-assembled monolayer as described in Figure 9.Described gold substrate can be the gold thin film of any type; But, generally speaking, preferably at glass, molten silicon, silicon wafer, plastic-substrates etc. after Vacuum Deposition is with the material such as chromium (Cr) or titanium (Ti) of 2-10nm with the substrate of the thickness vacuum metallizing of 50-200nm.Preferably prepared gold substrate is prepended to Piranha solution (strength sulfuric acid: 30% hydrogen peroxide=3: 1) approximately 1 minute, then dry with pure water washing nitrogen blowing being about to use.The formation of described individual layer can be confirmed by infrared rays external reflection spectrum.
In addition, the invention provides and a kind ofly identify and connect the fixing oligomer DNA of continuous guanine base and described solid substrate to prepare the method for oligomer DNA chip by polymolecular, and the oligomer DNA chip being prepared by the method.
The present invention more specifically provides a kind of method of the fixing oligomer DNA of polymolecular identification of four continuous guanine functional groups of nitrogen-atoms molecular recognition by described amino Calixarene Derivatives.
Fixing means of the present invention is the technology that uses oligomer DNA the to prepare different kind organism chip technology that provides the foundation, and this is the novel method that a kind of whole world up to now has no similar result of study.
Figure 10 and 11 is for prepare the technology schematic diagram of high-density oligomer DNA chip by automatic molecular recognition, and the Theoretical Calculation schematic diagram of the amino Calixarene Derivatives quantity of composition individual layer.It is by actual oligomer DNA concentration with obtain by Theoretical Calculation be fixed on amino Calixarene Derivatives individual layer maximum oligomer DNA concentration ratio compared with the actual tests result obtaining.
The theoretical maximum of fixing oligomer DNA can be passed through below Equation for Calculating.The diameter of the amino calixarene obtaining by x-ray method is 2.1-2.2nm.In the time that it carries out self-assembly as individual layer from the teeth outwards, the quantity of desired molecule is identical with two leg-of-mutton areas that the straight line by connecting three amino Calixarene Derivatives centers forms.Therefore the area that, Calixarene Derivatives occupies from the teeth outwards can obtain by following equation:
[equation]
2.1n×2.1nm×sin60°＝3.82×10 -14cm 2
Therefore, every 1cm 2the quantity of the amino Calixarene Derivatives of middle existence is 1/3.82 × 10 -14cm 2=2.62 × 10 13=43.5pmol/cm 2.Because 6-7 can be used for fixing an oligomer DNA in conjunction with the area of amino Calixarene Derivatives, fixable maximum oligomer DNA density is 43.5/ (6-7) pmol/cm 2=6.2-7.25pmol/cm 2.With reference to the method for embodiment 24, oligomer DNA for fixing 5 '-GGG GGG GGG AAA TCA ACCCAC AGC TGC A-3 ' (SEQ ID NO.16) and the c-DNA5 '-cy3-GTGCA GCT GTG GGT TGA TT-3 ' (SEQ ID NO.19) with fluorescence are hybridized and be connected to after oligomer DNA chip with maximum density, be shown in the results of hybridization 2 of Figure 11 in the measuring result of washing and the dry rear fluorescence sensitivity recording by fluorescent scanning instrument (GSI, the U.S.) according to the method for embodiment 13) in.As using fluorescent scanning instrument in the dry identical rear result of measuring similar fluorescence sensitivity of the c-DNA 5 '-cy3-GT GCA GCT GTG GGT TGATT-3 ' (SEQ ID NO.19) for hybridizing under different concns, after applying and be dried with 3.8pmol/cm concentration, can obtain the fluorescence sensitivity of similar level, this level is the half of calculated value.This results are shown in Figure 11 1) in the result of dry fluorescence oligomer DNA.This result shows with reference to the BMT oligomer DNA chip base of the present invention's exploitation can prepare oligomer DNA chip by fixedly approaching the oligomer DNA of theoretical maximum, and can obtain 50% of about maximum fluorescence after 30 minutes in hybridization, and this is the first oligomer DNA chip technology of preparing of supporting ultra-high speed hybridization simultaneously and realizing high-density technique for fixing in the world.
Figure 12 is that the oligomer DNA with identical base sequence connecting continuously by guanine base with reference to embodiment 22 is fixed oligomer DNA (6.75nmol/mL with the concentration of 5 times, 1nL), hybridize with reference to embodiment 22, wash and be dried to find after best base quantity continuously, the result that uses fluorescent scanning instrument (GSI, the U.S.) to analyze.In the test of Figure 12, adopt the base sequence shown in table 4, in hybridization, adopted the fluorescence c-DNA shown in table 4.
Actual tests result is to show the scan-data shown in the middle figure of Figure 12 of result with 6X6, and this result carries out with numerical value that fluorometric analysis obtains and this analysis is shown in the histogram of Figure 12 bottom.In the time that continuous guanine base sequence is about 1 or 4, its result is not for almost having surface fixing.But, in the time that it has 7 continuous guanines (7G), show intense fluorescence, and show the fluorescence level that doubles 7 continuous guanines (7G) in the time thering are 9 continuous guanines (9G).In the time that use has the oligomer DNA of 12 (12G) and 15 (15G) continuous guanine base of being longer than 9 continuous guanine bases, the level of 1/2,1/3 when fluorescence sensitivity drops to 9 continuous guanine bases according to the show.This shows that 9 continuous guanine bases are to be enough to fix the level of oligomer DNA and when the longer guanine base of use, the oligomer DNA of 12G need to be greater than the space of 9G in theory by molecular recognition, and therefore fluorescence sensitivity drops to 1/2 level.The oligomer DNA of 15G need to approximately 2/3 compared with 9G exceptional space, the oligomer DNA being therefore fixed drops to approximately 30% level, and fluorescence sensitivity is lowered by approximately 1/3 the level to 9G.Comprehensive these results, can see that the quantity of the fixing required continuous guanine of the best is 9.If the quantity of guanine is less than 9G continuously, possibly cannot reach the abundant level by the irreversible fixing oligomer DNA of molecular recognition.In addition,, can also see that from these results the quantity of connection guanine base is greater than 9G time, treat that fixing area (fixing required area) will be greater than 9G, thereby this has caused the decline of actual fixing oligomer DNA s quantity.
Figure 13 is that space between the fixing oligomer DNA guaranteed of suitable space that c-DNA obtains by continuous base can be by fixing oligomer DNA time approaches chip bottom and maintained ultra-high speed the schematic diagram that approaches required sufficient space level.Meanwhile, surface area when it has also shown actual the fixing of the amino Calixarene Derivatives of 6-7 can be used for fixing oligomer DNA.
In Figure 14 and 15, the base sequence shown in table 5 has been fixed respectively different A with reference to embodiment 23, C, G, T-shaped oligomer DNA.Then, with reference to embodiment 23 for after detecting DNA (Cy3-DNA) with fluorescence and with the hybridization that c-DNA carries out, the complementary c-DNA that can be combined with it being hybridized, as described in embodiment 23, the oligomer DNA of this table 5 with fluorescence is used in the fluorescence connection described in embodiment 23.After this can wash and be dried to carry out fluorescence sensitivity analysis by fluorescent scanning instrument (GSI, the U.S.).Figure 14 and 15 shown this be a kind of even can as the short hybridization time of about 30 minutes in carry out distinguishing and analysis of fluorescence sensitivity in on-off level, distinguish the oligomer DNA chip of the ultra-high speed hybridization of SNP in on-off level simultaneously.In addition, this result demonstration is a kind of new technique of supporting ultra-high speed hybridization and copying easy analysis list nucleosides polymorphism (SNP) with height as the technology of preparing of the oligomer DNA chip of target of the present invention.
Figure 16 is the actual competitive reaction result that shows independent guanine base and other base stationary phase than carrying out selectivity and fixing at a high speed.Use to have connected the oligomer DNA (9G-1) of 9G as embodiment 25 same concentrations, it is not combined with the oligomer DNA of the table 6 with fluorescence, by with oligomer DNA (9G-1) 1,2, the concentration of 4 times applies the base sequence that mixing 4 classes contain and band fluorescence oligomer DNA (Cy-3-DNA) is complementary and is connected 9A, 9G, 9T, the oligomer DNA mixture of 9C is with the order fixed dna shown in Figure 16.Simultaneously, if 9A, 9C, 9T, fixing faster than oligomer DNA (9G-1) of the oligomer DNA that 9G mixes with same concentrations, can be combined and show fluorescence by the band fluorescence oligomer DNA in the crossover process of carrying out with reference to embodiment 25, and if the oligomer DNA (9G-1) of this connection 9G is fixing faster, obviously can not show fluorescence.Can determine on the site of fixing 9G according to the actual experiment result of Figure 16, when fixing, fluorescence sensitivity rises with the rising of oligomer DNA concentration, thereby as 9G-1: 9G=1: 1 time, can show the fluorescence sensitivity that is about 50%, and at 1: 4 o'clock, can show approximately 80% fluorescence sensitivity.For 9T, it is few that 9C can determine that this fluorescence sensitivity rises, and even in the time of 9A, only shown the slight fluorescence sensitivity level of 4 times of amounts to 16G.Therefore, can see and in the time of actual fixing, having used while connecting the oligomer DNA of 9G, by the base for hybridizing (, by the base for being combined with c-DNA) partly do not participate in fixing, that is to say, only can prepare can be by the oligomer DNA copying with maximum density and amino Calixarene Derivatives single layer of the present invention in the site of 9G selective binding.
Generally speaking, thereby this result show can by with according to the present invention the amino Calixarene Derivatives self-assembled monolayer polymolecular of exploitation identify continuous guanine base irreversible fixing oligomer DNA on individual layer surface and prepare oligomer DNA chip, and this be a kind of can be by supporting all the time fixing so that the amount of the fixing oligomer DNA that fluorometric analysis shows approaches basic technology prepared by the reproducible oligomer DNA chip of theoretical maximum density.
In addition, in the present invention, continuously guanine base be fixed on individual layer surface and be the fixing oligomer DNA shown in Figure 13 and guarantee to have and the firm space of described base equal length.This space can make at least to have tens when have the DNAs of special base sequence with diagnosis or research purpose analysis can freely arrive chip base surface with ultra-high speed like a cork to the c-DNA of up to a hundred base sequences, thereby can be in the utmost point short period of time as 10 minutes-1 hour by making the base complementrity of maximum quantity in conjunction with carrying out ultra-high speed hybridization.In addition, thus even if the present invention proposes and a kind ofly prepare the technology of oligomer DNA individual layer and combine between fixed dna s the technology that space little monokaryon glycosides polymorphism also can simply be diagnosed that fully obtains, that is, and a kind of basic technology for the preparation of oligomer DNA chip.
The present invention more specifically describes by following embodiment.But the present invention is not limited only to this.
Embodiment
Embodiment 1
The preparation of 5,11,17,23-tetramino cup [4] aromatic hydrocarbons
[reaction formula 1]
Figure DEST_PATH_GSB00000673486500021
Light yellow solid product 5,11,17,23-tetramino cup [4] aromatic hydrocarbons (TACA) can 5,11,17, the reference 3:Van Wagenigen that 23-tetranitro cup [4] aromatic hydrocarbons (TNCA) is quoted with reference to following reference 3[as starting raw material, A.M.A.; Snip, E.; Verboom.W.; Reinhoudt, D.N.; Boerrigter, H.; Liebigs Ann/Recueil 1997.pp2235-2245] described synthetic method is with 75% synthetic the obtaining of productive rate.Described reaction table is shown reaction formula 1.
Embodiment 2
The preparation of 5,11,17,23-tetrabenzyl imines-cup [4] aromatic hydrocarbons
[reaction formula 2]
Figure DEST_PATH_RE-G200680038071401D00101
By the anhydrous MgSO of the TACA of 500mg (1.03mmol) and 200mg 4put into dry round-bottomed flask, add the acetonitrile of 150ml and the phenyl aldehyde of 0.84ml (8.24mmol) simultaneously, then under room temperature and nitrogen exchange, mix two hours.After reaction, filter and remove solid product, then to solution decompression and remove residual solvent.Then at the CH that is dissolved in 5ml completely 2cl 2after, it is lightpink solid that interpolation 30ml normal hexane makes 5,11,17,23-tetrabenzyl imines-cup [4] aromatic hydrocarbons (TBICA) crystallization.Filtration under diminished pressure decompression are removed residual solvent and are obtained TBICA (822mg, productive rate 95.7%).Described reaction table is shown reaction formula 2.
1H NMR(300MHz，CDCl 3)：10.15(s，4H，OH)，8.34(s，4H，N＝CH)，7.82-7.78(m，8H，Ar)，7.40-7.38(m，12H，Ar)，7.01(s，8H，Ar)，4.31(d，4H，ArCH 2Ar，J＝13Hz)，3.63(d，4H，ArCH 2Ar，J＝13Hz)
13C NMR(300MHz，CDCl 3)：159.26，146.28，136.40，131.32，tl28.86，121.90，32.47
embodiment 3
The preparation of 5,11,17,23-tetrabenzyl imines alkoxyl group-cup [4] aromatic hydrocarbons
[reaction formula 3]
Figure DEST_PATH_RE-G200680038071401D00111
By the TBICA of 500mg (0.6mmol), anhydrous K 2cO 3(830mg, 6mmol) and sodium iodide (810mg, 5.4mmol) are put into dry round-bottomed flask, add the acetonitrile of 150ml, then under nitrogen exchange, mix.After 10 minutes, add 6-bromine n-hexyl aldehyde (644mg, 3.6mmol), then the temperature of reaction in reaction vessel is risen to 80 ℃.Then mixture is stirred 24 hours.After reaction, acetonitrile is removed in decompression, and uses 150ml CH 2cl 2solubilizing reaction product.Due to the KBr obtaining in reaction process, KI, K 2cO 3do not dissolve Deng material, they can be removed by filtration under diminished pressure.Filtered soln decompression is removed to solvent and is dissolved in completely in the ethyl acetate of 5ml.If then add the normal hexane of 40ml, 5,11,17,23-tetrabenzyl imines alkoxyl group-cup [4] aromatic hydrocarbons (TBICOCA) can be used as yellow solid and is extracted.Use CHCl 3/ normal hexane makes TBICOCA crystallization obtain light yellow solid product (570mg, productive rate 90%).Described reaction table is shown reaction formula 3.
1H NMR(300MHz，CDCl 3)：9.81(t，2H，CHO)，8.46(s，2H，N＝CH)，8.24(s，2H，N＝CH)，7.86-7.82(m，4H，Ar)，7.73-7.70(m，4H，Ar)，7.43-7.31(m，12H，Ar)，7.07(s，4H，Ar)，6.83(s，4H，Ar)，4.33(d，4H，ArCH 2Ar，J＝13Hz)，4.04(t，4H，OCH 2)，3.46(d，4H，ArCH 2Ar，J＝13Hz)，2.57(t，4H，CH 2CHO)，2.10(t，4H，OCH 2CH 2)，1.88-1.70(m，8H，CH 2CH 2)
13C NMR(300MHz，CDCl 3)：202.07，197.11，159.61，158.90，148.21，147.19，145.93，143.10，H36.07，130.59-129.57，128.83-128.08，122.10，121.54，43.84，31.73，25.57
embodiment 4
The preparation of 5,11,17,23-tetrabenzyl imines-bromine butoxy-cup [4] aromatic hydrocarbons
[reaction formula 4]
By TBICA (500mg, 0.6mmol), anhydrous K 2cO 3(830mg, 6mmol) and sodium iodide (1.08g, 7.2mmol) are put into dry round-bottomed flask, add the acetonitrile of 150ml, then at room temperature mix 10 minutes.To reaction vessel interpolation Isosorbide-5-Nitrae-dibromobutane (1.3g, 0.72ml, 6mmol) and by the temperature rise to 80 in reaction vessel ℃.Then by mixture reaction 24 hours.Be cooled to after room temperature at reaction vessel, solvent is removed in decompression, and bottom product is dissolved in to the CH of 150ml 2cl 2in.Due to the KBr obtaining in reaction process, KI, K 2cO 3do not dissolve Deng material, they can be removed by filtration under diminished pressure, and filtered soln decompression is removed to solvent.After ethyl acetate/n-hexane extraction, filtration under diminished pressure obtains yellow solid product 5,11,17,23-tetrabenzyl imines-bromine butoxy-cup [4] aromatic hydrocarbons (TBBCA).Use CHCl 3/ normal hexane makes solid recrystallization to obtain light yellow TBBCA (480mg, productive rate 72%).Described reaction table is shown reaction formula 4.
1H NMR(300MHz，CDCl 3)：8.47(s，2H，N＝CH)，8.24(s，2H，N＝CH)，7.98(s，2H，ArOH)，7.86-7.83(dd，4H，ArH)，7.73-7.70(dd，4H，ArH)，7.41-7.31(m，12H，ArH)，7.08(s，4H，Ar)，6.83(s，4H，Ar)，4.33(d，4H，ArCH Ar，J＝13Hz)，4.06(t，4H，OCH)，3.49(s，4H，ArCH Ar，J＝13Hz)，3.45(t，4H，CH Br)，2.37-2.29(m，OCH 2CH 2)2.24-2.18(m，CH 2CH 2Br)
Embodiment 5
The preparation of 5,11,17,23-tetrabenzyl imines sulfydryl butoxy cup [4] aromatic hydrocarbons
[reaction formula 5]
Figure DEST_PATH_G200680038071401D00131
TBBCA (500mg, 0.45mmol) and thioacetic acid potassium (114.21mg, 1mmol) are put into dry round-bottomed flask, and be dissolved in 60ml acetone, sound wave reaction 90 minutes under room temperature and nitrogen exchange.After reaction, solvent is removed in decompression, and is dissolved in the CH of 30ml 2cl 2in.Then the undissolved throw out of filtration under diminished pressure, with water washing filtered soln twice, separates organic layer with MgSO 4dry.Organic layer filtration under diminished pressure is obtained to solid then by filtered soln drying under reduced pressure and use ethyl acetate/normal hexane recrystallization, and filtration under diminished pressure obtains light yellow solid crystal.The solid crystal of gained is put into round-bottomed flask, and be dissolved in CH 2cl 2: methyl alcohol=5: in 1 mixing solutions, sound wave reaction under room temperature and nitrogen exchange.After 1 minute, add 1.0M KOH (1ml, 1mmol), sound wave reaction 30 minutes.After reaction, solvent is removed in decompression, is then dissolved in the CH of 5ml 2cl 2in and with 0.1M HCl solution washing once.Separate organic layer with MgSO 4after dry, filtration under diminished pressure, the solvent that filtered soln is removed in decompression is faint yellow 5,11,17 to obtain, 23-tetrabenzyl imines sulfydryl butoxy cup [4] aromatic hydrocarbons (TBIMBCA).With the TBIMBCA of ethyl acetate/normal hexane recrystallization gained to obtain white solid crystal TBIMBCA (450mg, productive rate 73%).Described reaction table is shown reaction formula 5.
1H NMR(300MHz，CDCl 3)：8.48(s，2H，N＝CH)，8.17(s，2H，N＝CH)，7.84(m，4H，ArOH)，7.68(m，4H，ArOR)，7.38(m，6H，ArH)，7.28(m，6H，ArOR)，7.10(s，4H，ArH)，6.81(s，4H，ArH)，4.34(d，4H，ArCHA 2r)，4.04(t，4H，ArOCH 2)，3.47(d，4H，ArCH 2Ar，J＝13Hz)，3.03(t，4H，SHCH 2)，2.38(m，4H，ArOCH 2CH 2)，2.11(m，4H，CH 2CH 2)
Embodiment 6
The preparation of imines Calixarene Derivatives individual layer
The synthetic TBICOCA obtaining in embodiment 3 is dissolved in as CHCl 3deng preparing the solution that concentration is 0.1-5mM in organic solvent.As shown in Figure 1, the slide (amine chip) that has connected amine functions group is put into the solution 1-24 hour preparing, then take out and with chloroform, acetone and water washing, then dry, to prepare imines calixarene individual layer of the present invention.Other imines Calixarene Derivatives individual layer is with reference to identical method preparation.
Embodiment 7
The preparation of imines Calixarene Derivatives individual layer in gold substrate
The synthetic TBIMBCA obtaining in embodiment 5 is dissolved in as CHCl 3deng preparing the solution that concentration is 0.1-5mM in organic solvent.As shown in Figure 2, gold substrate is put into the solution 1-24 hour preparing, then taken out and with chloroform, acetone and water washing, then dry, to prepare imines calixarene individual layer of the present invention.Other imines Calixarene Derivatives individual layer is with reference to identical method preparation.Described gold substrate can be used by various forms, but generally speaking, preferably at glass, molten silicon, silicon wafer, plastic-substrates etc. after Vacuum Deposition is with the material such as chromium (Cr) or titanium (Ti) of 5-20nm with the substrate of the thickness vacuum metallizing of 100-300nm.By prepared gold substrate approximately 10 second-1 minute in being prepended to Piranha solution (strength sulfuric acid: 30% hydrogen peroxide=3: 1 mixing solutions) by use, then with water washing dry under nitrogen exchange, exist side by side and use the formation of described individual layer to analyze by infrared rays external reflection spectrum (FTIR-ERS).
Embodiment 8
Identify the method for fixing oligomer DNA by polymolecular
In the oligomer DNA shown in Fig. 3 is fixing, the microarray equipment of Genetics (Britain) and the microarray equipment of Proteogen Co. (Korea S) are adopted.The oligomer DNA with 9 continuous guanine bases is dissolved in BMT spotting solution to prepare fixed solution with 6.75pmol/ml.Use array contact pin to apply imines Calixarene Derivatives single-glass substrate (slide) with described fixed solution as shown in Figure 1 and use with described in embodiment described in 6 identical method take the amount of 1-5nL, oligomer DNA is fixed on to diameter in the point of 150-180 μ m.After 1-4 hour, with the BMT Wa-A-1 solution washing slide of 500ml one minute, and with BMT Wa-A-2 solution washing twice, then dry.For sealing other the fixing site of oligomer DNA, the slide after washing is put into the BMT lock solution of 250ml and processed 30 minutes.Then with the BMT Wa-A-1 solution washing slide of 500ml 3 minutes, and with twice of BMT Wa-A-2 solution washing.Then water is removed to be dried.
Embodiment 9
Identify the only method of the irreversible oligomer DNA fixedly with 9 continuous guanine bases by polymolecular
In the oligomer DNA shown in Fig. 4 is fixing, adopt the microarray equipment of Genetics (Britain).For determining as 6 kinds of different fixed function group 9A of table 1 below, 9G, 9C, 9T, no, the fixed efficiency of vitamin H etc., is dissolved in each oligomer DNA in BMT spotting solution to prepare fixed solution with 6.75pmol/ml.Use array contact pin to apply imines Calixarene Derivatives single-glass substrate (slide) with described fixed solution as shown in Figure 1 and use with described in embodiment described in 6 identical method take the amount of 1-5nL, oligomer DNA is fixed on to diameter in the point of 150-180 μ m.After 1-4 hour, with the BMT Wa-A-1 solution washing slide of 500ml one minute, and with BMT Wa-A-2 solution washing twice, then dry.For sealing other the fixing site of oligomer DNA, the slide after washing is put into the BMT lock solution of 250ml and processed 30 minutes.Then the BMT Wa-A-1 solution washing slide 3 minutes of 500ml, and with twice of BMT Wa-A-2 solution washing.Then remove moisture to be dried, and prepare the oligomer DNA chip shown in Fig. 4.Then the cell that, a kind of diameter of stable connection is 0.8cm is to hybridize.
Table 1
Figure S2006800380714D00421
In order to hybridize with the following target DNA with fluorescence, can in 1.5ml pipe, prepare the target DNA of 2 μ l with 2pmol/ μ l fluorescence and the mixing solutions of 58 μ l BMT hyb-mixA.Mixing solutions is heated to 3 minutes in 100 ℃ of water, and be placed on ice 3 minutes cooling to carry out, the mixing solutions of 60 μ l is put into the slide that has connected hybridization chamber.Then, in the baking oven of fixed temperature and humidity, slide is hybridized 30 minutes at 50 ℃.In the thermostatted of 30 ℃ with BMT Wa-B-1 (4xSSC, 0.1%SDS) solution washing the slide 2 minutes through hybridization, and at room temperature wash 2 times with BMT Wa-B-2 (4xSSC) solution.After dry, with microarray scanner (GSI Lumonics, the U.S.) quantitative analysis fluorescence sensitivity.Actual result is presented in Fig. 5.Described result shows that the oligomer DNA with 9 guanines fixes with high-density, and that other base does not affect is fixing.
Embodiment 10
Efficiency comparative approach when identify the irreversible oligomer DNA fixedly with 1-5 continuous guanine base by polymolecular
The oligomer DNA showing at Fig. 6 has adopted the microarray equipment of Genetics (Britain) in fixing.For determining as 6 kinds of different fixed function group 1G of table 2 below, 4G, 7G, 9G, 12G, the fixed efficiency of 15G etc., is dissolved in each oligomer DNA in BMT spotting solution to prepare fixed solution with 6.75pmol/ml.Use array contact pin to apply imines Calixarene Derivatives single-glass substrate (slide) with described fixed solution as shown in Figure 1 and use with described in embodiment described in 6 identical method take the amount of 1-5nL, oligomer DNA is fixed on to diameter in the point of 150-180 μ m.After 1-4 hour, with the BMT Wa-A-1 solution washing slide of 500ml one minute, and with BMT Wa-A-2 solution washing twice, then dry.For sealing other the fixing site of oligomer DNA, the slide after washing is put into the BMT lock solution of 250ml and processed 30 minutes.Then with the BMT Wa-A-1 solution washing slide of 500ml 3 minutes, and with twice of BMT Wa-A-2 solution washing.After dry, prepare the oligomer DNA chip shown in Fig. 4.Then the cell that, a kind of diameter of stable connection is 0.8cm is to hybridize.
Table 2
Figure S2006800380714D00431
In order to hybridize with identical band fluorescent DNA used in embodiment 9, can in 1.5ml pipe, prepare the target DNA of 2 μ l with 2pmol/ μ l fluorescence and the mixing solutions of 58 μ l BMThyb-mixA.Mixing solutions is heated to 3 minutes in 100 ℃ of water, and be placed on ice 3 minutes cooling to carry out, the mixing solutions of 60 μ l is put into the slide that has connected hybridization chamber.Then, in the baking oven of fixed temperature and humidity, slide is hybridized 30 minutes at 50 ℃.In the thermostatted of 30 ℃ with BMT Wa-B-1 (4xSSC, 0.1%SDS) solution washing the slide 2 minutes through hybridization, and at room temperature wash 2 times with BMT Wa-B-2 (4xSSC) solution.After dry, with microarray scanner (GSI Lumonics, the U.S.) quantitative analysis fluorescence sensitivity.Actual result as shown in Figure 6.Described result is shown as to be fixed at least needs 7 continuous guanine bases.In addition, they show that 9 continuous guanine bases demonstrate the highest constant density.
Embodiment 11
By verifying that with the competitive reaction of imidazoles the polymolecular identification of oligomer DNA is fixing
The oligomer DNA showing at Fig. 7 has adopted the microarray equipment of Genetics (Britain) in fixing.The oligomer DNA that is 5 '-GGG GGG GGG AAA TCA ACCCAC AGC TGC A-3 ' (SEQ ID NO.1) by base sequence is dissolved in BMT spotting solution to prepare fixed solution with 6.75pmol/ml.Use array contact pin to apply imines Calixarene Derivatives single-glass substrate (slide) with described fixed solution as shown in Figure 1 and use with described in embodiment described in 6 identical method take the amount of 1-5nL, oligomer DNA is fixed on to diameter in the point of 150-180 μ m.Different concns imidazoles with the composition of concentration shown in Fig. 7 applies this fixed solution.After 3 hours, with the BMT Wa-A-1 solution washing slide of 500ml 1 minute, with twice of BMT Wa-A-2 solution washing dry.For sealing other the fixing site of oligomer DNA, the slide after washing is put into the BMT lock solution of 250ml and processed 30 minutes.Then with the BMT Wa-A-1 solution washing slide of 500ml 3 minutes, and with twice of BMT Wa-A-2 solution washing.Remove after moisture is also dried and prepare the oligomer DNA chip shown in Fig. 4.Then the cell that, a kind of diameter of stable connection is 0.8cm is to hybridize.
In order to hybridize with identical band fluorescent DNA used in embodiment 9, can in 1.5ml pipe, prepare the target DNA of 2 μ l with 2pmol/ μ l fluorescence and the mixing solutions of 58 μ l BMThyb-mixA.Mixing solutions is heated to 3 minutes in 100 ℃ of water, and be placed on ice 3 minutes cooling to carry out, the mixing solutions of 60 μ l is put into the slide that has connected hybridization chamber.Then, in the baking oven of fixed temperature and humidity, slide is hybridized 30 minutes at 50 ℃.In the thermostatted of 30 ℃ with BMT Wa-B-1 (4xSSC, 0.1%SDS) solution washing the slide 2 minutes through hybridization, and at room temperature wash 2 times with BMT Wa-B-2 (4xSSC) solution.After dry, with microarray scanner (GSI Lumonics, the U.S.) analysis of fluorescence sensitivity.Actual result as shown in Figure 7.Described result shows that imidazoles identify and suppressed continuous guanine and fix by polymolecular, and in the time having comprised at least imidazoles of 100mM, carries out hardly fixing of oligomer DNA.That is to say, they have shown that imidazoles is at war with by molecular recognition, and the fixed rate with the oligomer DNA of 9 continuous guanine bases declines to some extent.
In embodiment 8-11, for fixing, the solution composition of the purposes such as cleaning is as follows:
BMT spotting solution (4xSSC, 15% glycerol, 1x PBS)
BMT Wa-A-1(2xSSC，0.1％SDS)
BMT Wa-A-2(0.1x SSC)
BMT lock solution (5% milk casein solution)
BMT hyb-mixA (4 × SSC, 0.1%SDS, 50% glycerol, 1 × PBS)
BMT Wa-B-1(4×SSC，0.1％SDS)
BMT Wa-B-2(4×SSC)
Embodiment 12
Synthesizing of 5,11,17,23-tetramino cup [4] aromatic hydrocarbons
[reaction formula 6]
Figure DEST_PATH_RE-GSB00000673486500011
Light yellow solid product 5,11,17,23-tetramino cup [4] aromatic hydrocarbons (TACA) can 5,11,17,23-tetranitro cup [4] aromatic hydrocarbons (TNCA) synthesizes and obtains with 75% productive rate with reference to the synthetic method described in following reference 3 as starting raw material.
Embodiment 13
Synthesizing of the amino cup of 5,11,17,23-tetra-(2,4-dimethyl) dibenzyl [4] aromatic hydrocarbons
[reaction formula 7]
Figure 728521DEST_PATH_RE-G200680038071401D00151
By bar magnet, TACA (1.0g, 2.05mmol) and sodium iodide (3.0g, 20.5mmol) are put into dry round-bottomed flask, by mixture drying under reduced pressure.After dry, add the anhydrous acetonitrile of 50ml, then it is mixed under nitrogen exchange in well heater.After 5 minutes, 2,4-dimethyl benzyl bromine (16.2g, 82mmol) is put into reaction vessel, then heat up and stir.Then add pyridine (6.62ml, 82mmol), make mixture reaction 6 hours.After reaction, make reaction vessel be cooled to room temperature, solvent decompression is removed.Then, be dissolved in the CH of 150ml 2cl 2after, with twice of water washing organic layer.By dried organic layer filtration under diminished pressure, and filtered soln is removed in decompression.Then with CH 2cl 2/ MeOH recrystallization is to obtain the light gray solid product 5,11,17 of 2.1g, 23-tetra-(2,4-dimethyl) dibenzyl amino cup [4] aromatic hydrocarbons (2,4-DMTDBACA) (productive rate 80%).
1H NMR(300MHz，CDCl 3)；10.14(s，4H，ArOH)，7.287.12(m，24H，ArH)，6.03(s，8H，ArH)，4.24(s，16H，ArNCH 2)，4.11(d，4H，ArCH 2Ar，13Hz)，3.10(d，4H，ArCH 2Ar，13Hz)，2.43(s，12H，ArCH 3)，2.28(s，12H，ArCH 3)
Embodiment 14
Synthesizing of 5,11,17,23-tetra-(2,4-dimethyl) benzimide-cup [4] aromatic hydrocarbons
[reaction formula 8]
Figure 995555DEST_PATH_RE-G200680038071401D00161
TACA (100mg, 0.2mmol) and magnetic stripe are put into dry round-bottomed flask.Add the acetonitrile of 15ml and mix to reaction vessel.Add after 2,4-dimethylbenzaldehyde (322mg, 2.4mmol), under nitrogen exchange and room temperature, mix 2 hours.After reaction, by mixture filtration under diminished pressure, and solvent is removed in decompression.Then by reactants dissolved in the CH of appropriate amount 2cl 2in, then add the normal hexane of 15ml to obtain light brown solid product.This product is dissolved in to 3mlCH again 2cl 2in, and the normal hexane that adds 15ml is to obtain the bright light brown solid product 5,11,17 of 155mg, 23-tetra-(2,4-dimethyl) benzimide-cup [4] aromatic hydrocarbons (2,4-DMICA) (productive rate 81%).
1H NMR(300MHz，CDCl 3)；10.19(s，4H，ArOH)，8.54(s，8H，N＝CH)，7.79(d，4H，ArH)，7.02(s，4H，ArH)，7.01(d，4H，ArH)，6.96(s，8H，ArH)，4.30(d，4H，ArCH 2Ar，13Hz)，3.61(s，4H，ArCH 2Ar，13Hz)，2.43(s，12H，ArCH 3)，2.30(s，12H，ArCH 3)
Embodiment 15
Synthesizing of the amino cup of 5,11,17,23-tetra-(2,4-dimethyl) benzyl [4] aromatic hydrocarbons
[reaction formula 9]
Figure 194455DEST_PATH_RE-G200680038071401D00171
2,4-DMICA (100mg, 0.1mmol) and magnetic stripe are put into dry round-bottomed flask, and drying under reduced pressure.Then add the anhydrous THF of 20ml and mix under nitrogen exchange.After 10 minutes, add solution 1.0MBH 3/ THF solution (0.8ml, 0.8mmol), then makes mixture at room temperature react 3 hours.After reaction, solvent is removed in decompression, and the product remaining in flask is dissolved in to CH 2cl 2in.Again with after twice of 0.1M HCl solution washing, with twice of distilled water wash.Organic layer is separated and is dried, and filtered soln is removed in filtration under diminished pressure decompression.Then with CH 2cl 2/ hexane recrystallization is to obtain the light yellow solid product 5,11,17 of 72mg, 23-tetra-(2,4-dimethyl) benzyl amino cup [4] aromatic hydrocarbons (2,4-TDMBACA) (productive rate 75%).
1H NMR(300MHz，CDCl 3)；9.86(s，4H，ArOH)，7.33-7.18(m，12H，ArH)，6.21(s，8H，ArH)，4.21-4.09(m，16H，ArCH 2Ar，ArNHCH 2)，3.58(s，4H，NH)，3.22(d，4H，ArCH 2Ar)，2.43(s，12H，ArCH 3)，2.28(s，12H，ArCH 3)
Embodiment 16
Synthesizing of the amino cup of 5,11,17,23-tetra-(2,4-dimethyl tetrabenzyl) benzyl [4] aromatic hydrocarbons
[reaction formula 10]
Bar magnet and 2,4-TDMBACA (500mg, 0.5mmol) and sodium iodide (80mg, 0.5mmol) are put into dry round-bottomed flask, and drying under reduced pressure.After dry, add the anhydrous acetonitrile of 50ml, then it is mixed under nitrogen exchange in well heater.After 5 minutes, add bromotoluene (1.1ml, 10mmol) exchange mixing to reaction vessel.Add pyridine (0.8ml, 10mmol) to reaction vessel, and react 6 hours.After reaction, make reaction vessel be cooled to room temperature, then solvent is removed in decompression.Then be dissolved in the CH of 60ml 2cl 2in and with water washing, then dry organic layer filtration under diminished pressure.Then filtered soln is removed in decompression, and with CH 2cl 2/ MeOH recrystallization is to obtain the light gray solid product 5,11,17 of 521mg, 23-tetra-(2,4-dimethyl tetrabenzyl) benzyl amino-cup [4] aromatic hydrocarbons (TB (2,4-DMTB) ACA) (productive rate 79%).
1H NMR(300MHz，CDCl 3)10.01(s，4H，ArOH)，7.35-7.10(m，34H，ArH)，6.20-6.06(br，8H，ArH)，4.31-4.01(m，16H， ArCH 2Ar，ArNHCH 2)，3.14(d，4H，ArCH 2Ar)，2.45(s，12H，ArCH 3)，2.27(s，12H，ArCH 3)
Embodiment 17
The amino cup of 5,11,17,23-tetra-(2,4-dimethyl) dibenzyl [4]-aromatic hydrocarbons-1,3-hexanal synthetic
[reaction formula 11]
Figure 856697DEST_PATH_G200680038071401D00191
Successively by bar magnet and 2,4-DMTDBACA (500mg, 0.35mmol), K 2cO 3(487mg, 3.5mmol), sodium iodide (473mg, 3.15mmol) is put into dry round-bottomed flask, then adds the anhydrous acetonitrile of 130ml at nitrogen exchange downhill reaction container, and mixes in well heater.Add after 6-bromine hexanal (376mg, 2.1mmol), exchange mixes 16 hours.After reaction, reactant is cooled to room temperature, and solvent is removed in decompression.By reactants dissolved in the CH of 130ml 2cl 2after, its filtration under diminished pressure decompression are removed to filtered soln.Then use MeOH/CH 2cl 2recrystallization to be to obtain the light yellow solid product 5,11,17 of 432mg, the amino cup of 23-tetra-(2,4-dimethyl) dibenzyl [4]-aromatic hydrocarbons-1,3-hexanal (2,4-DMTDBACAHA) (productive rate 76%).
1H NMR(300MHz，CDCl 3)；9.78(s，2H，CHO)，7.31 7.04(m，12H，ArH)，6.11-6.02(br，8H，ArH)，4.41-4.13(m，2OH，ArNCH 2，ArCH 2Ar)，3.89(t，4H，OCH 2)，3.05(d，4H，ArCH 2Ar，13Hz)，2.57-2.49(t，4H，CHOCH 2)，2.45-2.39(m，12H，ArCH 3)，2.31-2.25(m，12H，ArCH 3)，2.12-1.98(t，OCH 2CH 2)，1.78-1.62(m，8H，CH 2CH 2)
Embodiment 18
Synthesizing of amino bromo-butoxy cup [4] aromatic hydrocarbons of 5,11,17,23-tetra-(2,4-dimethyl tetrabenzyl) benzyl
[reaction formula 12]
Figure DEST_PATH_G200680038071401D00201
By 2,4-TDMBACA (500mg, 0.5mmol) and anhydrous K 2cO 3(619mg, 5.0mmol) and sodium iodide (674mg, 4.5mmol) are put into dry round-bottomed flask.Add the anhydrous acetonitrile of 160ml and at room temperature mix 15 minutes to reaction vessel.Add Isosorbide-5-Nitrae-dibromobutane (1.03g, 0.6ml, 5.0mmol) exchange to mix 20 hours to reaction vessel.Reaction vessel is cooled to after room temperature, and solvent decompression is removed.Then be dissolved in CH 2cl 2in and filtration under diminished pressure.Filtered soln, recrystallization in EA/ hexane are removed in decompression.Then filtration under diminished pressure is to obtain the light brown solid product of 610mg, 5,11,17, amino bromine butoxy cup [4] aromatic hydrocarbons of 23-tetra-(2,4-dimethyl tetrabenzyl) benzyl (TB (2,4-DMTB) ABCA) (productive rate 77%).
1H NMR(300MHz，CDCl 3)；7.81(s，2H，ArOH)，7.297.04(m，34H，ArH)，6.06-6.02(d，8H，ArH)，4.38-4.11(m，2OH，ArNCH 2，ArCH 2Ar)，3.89(t，4H，OCH 2)，3.04(d，4H，ArCH 2Ar，13Hz)， 2.46-2.43(m，12H，ArCH 3)，2.41-2.40(m，12H，ArCH 3)2.31-2.27(t，4H，BrCH 2)，2.16-2.06(m，8H，CH 2CH 2)
Embodiment 19
Synthesizing of 5,11,17,23-tetra-(2,4-dimethyl tetrabenzyl) benzyl amino mercapto-butoxy cup [4] aromatic hydrocarbons
[reaction formula 13]
TB (2,4-DMTB) ABCA (500mg, 0.31mmol) and thioacetic acid potassium (212mg, 1.86mmol) are put into dry round-bottomed flask.Then dissolve therein the anhydrous propanone of 60ml and sound wave reaction 90 minutes under the exchange of room temperature and nitrogen.After reaction, solvent is removed in decompression, and with the CH of 30ml 2cl 2dissolve.After filtration under diminished pressure, filtered soln is also separated and dry organic layer for twice with water washing.By organic layer filtration under diminished pressure and by the ethyl acetate/normal hexane recrystallization for solid obtaining after filtered soln drying under reduced pressure, and filtration under diminished pressure obtains light yellow solid crystal.The solid crystal of gained is put into round-bottomed flask and is dissolved in CH 2cl 2: methyl alcohol=5: in 1 mixing solutions, sound wave reaction under room temperature and nitrogen exchange.After 1 minute, add 1.0M KOH (1.5ml, 1.5mmol), sound wave reaction 30 minutes.After reaction, solvent is removed in decompression, is dissolved in CH 2cl 2and with 0.1M HCl solution washing. after organic layer separates, be dried and filtration under diminished pressure.The solvent of filtered soln is removed in decompression.With CH 2cl 2after/MeOH recrystallization, can obtain the light yellow 5,11,17 of 320mg, 23-tetra-(2,4-methoxyl group) benzyl amino mercapto butoxy cup [4] aromatic hydrocarbons (TB (2,4-DMTB) AMBCA) (productive rate 73%).
1H NMR(300MHz，CDCl 3)；7.84(s，2H，ArOH)，7.29 7.01(m，34H，ArH)，6.25-6.19(br，8H，ArH)，4.36-4.11(m，2OH，ArNCH 2，ArCH 2Ar)，3.90(t，4H，OCH 2)，3.05(d，4H，ArCH 2Ar，13Hz)，2.30(t，4H，SHCH 2)，2.45-2.39(m，12H，ArCH 3)，2.31-2.25(m，12H，ArCH 3)2.05-2.02(m，CH 2CH 2)
Embodiment 20
The preparation of the amino Calixarene Derivatives individual layer shown in Fig. 8
By in embodiment 17 synthetic obtain 2,4-DMTDBACAHA is dissolved in as CHCl 3deng preparing the solution that concentration is 0.1-5mM in organic solvent.As shown in Figure 8, the slide (amine chip) that has connected amine functions group is put into solution 1-24 hour of preparing and taken out, respectively with chloroform, acetone and water washing are also dry.Then prepare the amino Calixarene Derivatives individual layer (A type BMT oligomer DNA chip) of Fig. 8.Above-mentioned A type is being put into containing BH 3-THF or NaBH 4meOH reductive agent in after 1-10 minute, water (deionized water) washing three times, the dry amino Calixarene Derivatives individual layer (Type B BMT oligomer DNA chip) with Preparation Example 8 in nitrogen atmosphere.
Embodiment 21
The preparation of amino Calixarene Derivatives individual layer in gold substrate shown in Fig. 9
Synthetic TB (2, the 4-DMTB) AMBCA obtaining in embodiment 19 is dissolved in as CHCl 3deng preparing the solution that concentration is 0.1-5mM in organic solvent.As shown in Figure 9, gold substrate is put into solution 1-24 hour of preparing and taken out, respectively with chloroform, acetone and water washing are also dry.Then prepare the amino Calixarene Derivatives individual layer shown in Fig. 9.Other amino Calixarene Derivatives individual layer is with reference to identical method preparation.Described gold substrate can be used by various forms, but generally speaking, preferably at glass, molten silicon, silicon wafer, plastic-substrates etc. after Vacuum Deposition is with the material such as chromium (Cr) or titanium (Ti) of 2-10nm with the substrate of the thickness vacuum metallizing of 50-200nm.By prepared gold substrate approximately 10 second-1 minute in being prepended to Piranha solution (strength sulfuric acid: 30% hydrogen peroxide=3: 1 mixing solutions) by use, then, with water washing dry under nitrogen exchange, exist side by side and use.The formation of described individual layer can be analyzed by infrared rays external reflection spectrum (FTIR-ERS).
The solution that embodiment 22-25 is used, oligomer DNA, with the oligomer DNA of fluorescence, and c-DNA base sequence
Table 3
The solution that embodiment 22-25 is used
BMT spotting solution 1 4XSSC, 15% glycerol, 1XPBS
BMT spotting solution 2 600mM NH 4Cl, 15% glycerol, 1XPBS
BMT spotting solution 3 500mM KCl, 15% glycerol, 1XPBS
BMT Wa-A-1 2X SSC，0.1％SDS
BMT Wa-A-2 0.1X SSC
BMT lock solution 5% milk casein solution
BMT hyb-mix A 4XSSC, 0.002%SDS, 15% glycerol, 1XPBS
BMT Wa-B-1 4XSSC，0.1％SDS
BMT Wa-B-2 4XSSC
Table 4
Best base number
Figure S2006800380714D00581
Table 5
For the SNP testing
Figure S2006800380714D00591
Table 6
For competitive reaction
Figure S2006800380714D00601
Table 7
For maximum density test
Sequence Remarks
9G
5′-GGG GGG GGG AAA TCA ACC CAC AGCTGC A-3′ (SEQ ID NO.16) Probe
Cy3-DN A 5′-cy3-GT GCA GCT GTG GGT TGA TT-3′(SEQ ID NO. 19) Target
Table 8
Be used for the composition (ul) of the solution of competitive reaction
1∶0 1∶1 1∶2 1∶4
9G-1 (100pmol/ul) 1.5 1.5 1.5 1.5
Be respectively 9A, 9T, 9C, 9G 0 1.5 3.0 6.0
2M NH4Cl 30 30 30 30
90% glycerol 17 17 17 17
Distilled water 51.5 50 48.5 45.5
Amount to 100ul 100ul 100ul 100ul
Embodiment 22
Measure the best guanine base quantity of identifying the amino Calixarene Derivatives individual layer of the irreversible Figure 12 of being fixed on by polymolecular
-oligomer DNA is identified in fixing on amino Calixarene Derivatives individual layer by polymolecular
For measuring, to be identified in sample solution and the concentration composition etc. of guanine quantity fixing on amino Calixarene Derivatives individual layer by polymolecular as shown in table 3.First, 10ul is had respectively to the below 1G of table 4,4G, 7G, 9G, 12G, continuous each oligomer DNA (100pmol/ul) of guanine base such as 15G and the mixing solutions of 148ul BMT spotting solution 3 use microarray (Genetix Qarray2) thereby with the amount of 1-5nL, solution are coated on the amino Calixarene Derivatives individual layer in order to fixing oligomer DNA and under room temperature, it are fixed respectively to 1 hour with the diameter of 150-180um in fixed chamber, 2 hours, 4 hours.After fixing, for taking out residual oligomer DNA, with BMT Wa-A-1 (2xSSC, 0.1%SDS) at room temperature wash amino Calixarene Derivatives individual layer 1 minute, then wash with BMT Wa-A-2 (0.1xSSC), and at room temperature seal 30 minutes with BMT lock solution (5% milk casein solution).With the at room temperature amino Calixarene Derivatives individual layer after carrying out washing treatment 3 minutes of BMT Wa-B-1 (4xSSC, 0.1%SDS), then wash and be dried with the wash bottle of BMT Wa-A-2 (0.1XSSC).
-hybridize with the DNA (Cy3-DNA) with fluorescence
In order hybridizing with the Cy3-DNA with fluorescence, hybridization chamber to be connected to and to have fixed oligomer DNA dried amino Calixarene Derivatives individual layer.Then the Cy3-DNA (5nmol/ml), 2ul table 4 being specified mixes in the water that is incorporated in 100 ℃ and heats 3 minutes with the BMT hyb-mixA of 58ul (4xSSC, 0.002%SDS, 50% glycerol, 1x PBS).It is placed on ice 3 minutes, apply 60ul with micropipet, then in the fixed temperature and humidity incubator of 50 ℃, hybridize 30 minutes.After hybridization, for clean amino Calixarene Derivatives individual layer, at 30 ℃, wash 2 minutes with BMT Wa-B-1 (4xSSC, 0.1%SDS).Then, at room temperature wash 2 minutes and use fluorescent scanning instrument (GSI Lumonics, the U.S.) analysis of fluorescence sensitivity with BMT Wa-B-2 (4xSSC) for twice.Actual result is shown in Figure 12.Described result shows when oligomer DNA to be identified by polymolecular while being fixed on amino Calixarene Derivatives individual layer and at least requires 7 guanine bases, and fixes with maximum density in the time of 9 guanine bases of use.
Embodiment 23
Determine monokaryon glycosides polymorphism (SNP by the optimal spatial layout of Figure 14 and Figure 15; Single Nucleotide Polymorphism) differentiate test
-definite SNP on amino Calixarene Derivatives individual layer
On the amino Calixarene Derivatives individual layer for fixing oligomer DNA, the test of definite SNP can, by first using microarray (Genetix Qarray2) that the oligomer DNA s (100pmol/ul) of a table 6 that base differs from one another of the only same loci of 9.1ul is coated with the thickness of 150-180um and the amount of 1-5nl with the mixing solutions of the BMT spotting solution 1 of 35.9ul, then at room temperature be fixed 3 hours in fixed chamber.After fixing, for taking out residual oligomer DNA, with BMT Wa-A-1 (2xSSC, 0.1%SDS) at room temperature wash amino Calixarene Derivatives individual layer 1 minute, then wash with BMT Wa-A-2 (0.1x SSC), and at room temperature seal 30 minutes with BMT lock solution (5% milk casein solution).With the at room temperature amino Calixarene Derivatives individual layer after carrying out washing treatment 3 minutes of BMT Wa-B-1 (4xSSC, 0.1%SDS), then wash and be dried with the wash bottle of BMT Wa-A-2 (0.1XSSC).
-hybridize to detect the DNA (Cy3-DNA) with fluorescence with C-DNA
For hybridizing, be connected to after individual layer BMT hyb-mixA (4xS SC, 0.002%SDS by the each C-DNA (5nmol/ul) specifying in 2ul table 6 with 28ul in hybridization chamber, 50% glycerol, 1xPBS) mix to be incorporated in 100 ℃ of water and heat.Place on ice 3 minutes and cooling, then use micropipet to apply 30ul, in 55 ℃ of Constant Temperature Incubators, hybridize 30 minutes.After hybridization, for clean amino Calixarene Derivatives individual layer, at 30 ℃, wash 2 minutes with BMTWa-B-1 (4xSSC, 0.1%SDS), then at room temperature wash 2 minutes with BMTWa-B-2 (4xSSC) for twice.Then be dried and prepare the oligomer DNA individual layer that can hybridize with band fluorescent DNA (Cy3-DNA).
-C-DNA and hybridization with the Cy3-DNA of fluorescence
For the Cy3-DNA with fluorescence and the hybridization that completes the individual layer of hybridization with C-DNA, first, connect hybridization chamber, then the BMT hyb-mixA (4xSSC with 28ul by the Cy3-DNA of 2ul table 6 (5nmol/ml), 0.002%SDS, 50% glycerol, 1x PBS) mix to be incorporated in 100 ℃ of water and heat 3 minutes.After placing on ice 3 minutes and being dried, use micropipet to apply 30ul, then at 45 ℃, in constant humidity incubator, hybridize 30 minutes.After hybridization, for clean amino Calixarene Derivatives individual layer, at 30 ℃, wash 2 minutes with BMT Wa-B-1 (4xSSC, 0.1%SDS), then at room temperature wash 2 minutes and be dried with BMT Wa-B-2 (4xSSC) for twice.Then use fluorescent scanning instrument (GSI Lumonics, the U.S.) to confirm that by fluorescence sensitivity SNP differentiates.This results are shown in Figure 14 and 15.This result is presented at has not combination in hybridization of other DNAs on the site that the oligomer DNA of different bases is fixed.Therefore, this result shows that technology can, by suitable space is set between the oligomer DNA being fixed, be differentiated thereby realize fast accurate SNP by the optimal spatial layout between this oligomer DNA s as described in the present invention.
Embodiment 24
Figure 10 and 11 high-density restraint test
-oligomer DNA is fixed
The method of fixing oligomer DNA on amino Calixarene Derivatives individual layer can, by using microarray (Genetix Qarray2) to apply the mixing solutions of the oligomer DNA (100pmol/ul) of 9.1ul table 7 and the MBT spotting solution 1 of 35.9ul with the amount of 1-5nl on the amino Calixarene Derivatives individual layer for fixing oligomer DNA, then at room temperature be fixed in fixed chamber 3 hours.After fixing, for taking out residual oligomer DNA, with BMT Wa-A-1 (2xSSC, 0.1%SDS) at room temperature wash amino Calixarene Derivatives individual layer 1 minute, then wash with BMT Wa-A-2 (0.1x SSC), and at room temperature seal 30 minutes with BMT lock solution (5% milk casein solution).With the at room temperature amino Calixarene Derivatives individual layer after carrying out washing treatment 3 minutes of BMT Wa-B-1 (4xSSC, 0.1%SDS), then wash and be dried with the wash bottle of BMT Wa-A-2 (0.1XSSC).
-hybridize with the Cy3-DNA with fluorescence
For hybridizing with the Cy3-DNA with fluorescence, first, connect hybridization chamber, then the BMT hyb-mixA (4xSSC with 28ul by the Cy3-DNA of 2ul table 7 (5nmol/ml), 0.002%SDS, 50% glycerol, 1x PBS) in 100 ℃ of water, heat after mixing and be positioned over cooled on ice.Then, use micropipet to apply 30ul, and hybridize 30 minutes in 50 ℃ of incubators.After hybridization, for clean amino Calixarene Derivatives individual layer, at 30 ℃, wash 2 minutes with BMT Wa-B-1 (4xSSC, 0.1%SDS), then at room temperature wash 2 minutes and be dried with BMT Wa-B-2 (4xSSC) for twice.Then use fluorescent scanning instrument that fluorescence sensitivity and theoretical fluorescence sensitivity are compared and analyzed.
-dry a certain amount of band fluorescence c-DNA in chip base, and itself and theoretical maximum are compared and analyzed
By microarray (Genetix Qarray2) by the band fluorescence c-DNA of table 7 with 1-5nl, 1/2 theoretical maximum (0.675fmol/nl) be coated to amino calixarene sub-biological and dry after, use fluorescent scanning instrument to analyze.The results are shown in Figure 10 and 11 of theoretical fluorescence sensitivity and the comparison of test fluorescence sensitivity, use the result with fluorescence Cy3-DNA of theoretical maximum half level it seems with by hybridizing coming to the same thing shown in fixing oligomer DNA.Therefore, this result shows that the oligomer DNA that approaches theoretical maximum is fixed.
Embodiment 25
By with the 9A of Figure 16,9T, 9G, the competitive reaction of 9C-DNA is to the only irreversible fixing validation test of 9 guanine bases
For the oligomer DNA of competitive reaction, the composition shown in can employing table 7 is prepared.Now, be put into table 8 and with 0 times for the different DNAs of competitive reaction, 1 times, the concentration of 2 times and 4 times is prepared.Can oligomer DNA be fixed by using micropipet that each oligomer DNA mixing solutions of preparing by method shown in table 7 respectively of 1ul is put respectively on the amino Calixarene Derivatives individual layer of thickness 2mm and at room temperature fix 1 hour in fixed chamber.After fixing, for taking out residual oligomer DNA, with BMT Wa-A-1 (2xSSC, 0.1%SDS) at room temperature wash amino Calixarene Derivatives individual layer 1 minute, then wash with BMT Wa-A-2 (0.1x SSC), and at room temperature seal 30 minutes with BMT lock solution (5% milk casein solution).With the at room temperature amino Calixarene Derivatives individual layer after carrying out washing treatment 3 minutes of BMT Wa-B-1 (4xSSC, 0.1%SDS), then wash and be dried with the wash bottle of BMT Wa-A-2 (0.1XSSC).
-hybridize with the Cy3-DNA with fluorescence
For hybridizing with the Cy3-DNA with fluorescence, first, connect hybridization chamber, then the BMT hyb-mixA (4xSSC with 590ul by the Cy3-DNA of 10ul table 8 (10nmol/ml), 0.002%SDS, 50% glycerol, 1x PBS) mix the rear micropipet coating 600ul that uses, then in 50 ℃ of incubators, hybridize 30 minutes.After hybridization, for clean amino Calixarene Derivatives individual layer, at 30 ℃, wash 2 minutes with BMT Wa-B-1 (4xSSC, 0.1%SDS), then at room temperature wash 2 minutes and be dried with BMT Wa-B-2 (4xSSC) for twice.Then use fluorescent scanning instrument (GSI) to confirm fluorescence sensitivity.Described the results are shown in Figure 16.No matter this result only shows 9 guanines and identify by irreversible fixing and whether other base continuously all can be to fixing generation considerable influence by polymolecular.This result has shown the technology of preparing that can obtain the oligomer DNA chip of reproducible results of hybridization as actual tests result, wherein for the base of hybridizing do not participate in fixing and only continuously specific base sequence participate in fixing, thereby maintain all the time the essential base quantity of hybridization.
Industrial usability
The present invention is different from the conventional in the world traditional oligomer DNA fixing means that passes through Chemical bond or physical adsorption completely, it is a kind of novel method that uses continuous guanine base irreversible fixing oligomer DNA in solid substrate by molecular recognition concept (, polymolecular identification).
The invention solves by high price oligomer DNA be connected traditional chemical combined techniques in chemical reaction between the aldehyde slide (aldehyde chip base) of conventional amine functions group and fix many problems that oligomer DNA produces when preparing oligomer DNA chip in aldehyde chip base, thereby make any oligomer DNA chip of preparing easily per capita.
The oligomer DNA that use has connected high price functional group is difficult to obtain reproducibility in fixing step while preparing oligomer DNA chip, therefore the cycle of research and development very long, and this point has stoped many biological products to turn to commercialization from research and development.In addition, while selecting to bring the oligomer DNA of best results of hybridization by testing hundreds and thousands of kinds of oligomer DNA base sequences, because the burden of development costs had once been abandoned the product in many exploitations.
As shown in Figure 3, because the continuous guanine base in the present invention is identified and is fixed on imines Calixarene Derivatives individual layer (BMT imines chip) by polymolecular, the density of fixing oligomer DNA is consistent all the time under the same conditions, thereby has shown high reproducibility.In addition,, in Fig. 3, by continuous guanine base is fixed on to the space that substrate surface forms, the c-DNA that should be combined with substrate surface can and be hybridized between the oligomer DNA being fixed by freely entering.Accordingly, it for the advantage of intensive traditional oligomer DNA chip of having fixed oligomer DNA be can several times or the speed of tens times hybridize, thereby support quick diagnosis.The oligomer DNA fixing by molecular recognition is identified in solution and is fixed to BMT imines chip by polymolecular, therefore can use oligomer DNA to prepare the fixing oligomer DNA chip of high-density, even the 1/3-1/10 that the density of this oligomer DNA is conventional levels.
In addition, approximately need in the present invention 2-4 hour fixing oligomer DNA, then wash and be dried.Therefore, it can make the preparation of oligomer DNA chip become easy and quick.
Especially, in Fig. 4 and 6, oligomer DNA is passed irreversible being fixed on imines Calixarene Derivatives self-assembled monolayer of polymolecular identification of 9 continuous guanine bases, and the fluorometric analysis result of hybridization shows 5 to 30 times that the constant density of fixing oligomer DNA is traditional method.
Therefore, the comparable technology of fixedly preparing oligomer DNA chip by traditional oligomer DNA of the present invention is prepared oligomer DNA chip at faster speed, and can reduce R&D costs by high reproduction level.In addition, it is a kind of epoch-making new technique of cost preparing product that can traditional preparation method 1/3-1/5.Correspondingly, it can be applicable to various based in DNA chip preparation of the present invention subsequently.
In addition,, as another aspect of the present invention, the present invention is that a kind of variety of issue presenting in existing DNA chip technology of preparing that can solve (for example, cannot carry out high speed hybridization according to conventional art; Cannot guarantee to diagnose the technology of monokaryon glycosides polymorphism (SNP); Owing to being difficult to maintain as A, C, the shown results of hybridization of chemical bonding of carrying out between the amine functions group in three bases such as G and the aldehyde functional group of aldehyde chip base group, is difficult to obtain the reproducibility of DNA chip; Being difficult to form oligomer DNA homogeneous is fixed on the uniform density of preparing on chip and fixes) new technique.
The invention provides a kind of amino Calixarene Derivatives that can the continuous guanine group of irreversible molecular recognition, the technology of the oligomer DNA chip base of preparing with its self-assembled monolayer form, oligomer DNA chip and the technology of preparing thereof prepared by the irreversible fixing oligomer DNA of the spontaneous molecular recognition on chip base surface.As described in as shown in embodiment and accompanying drawing, this technique for fixing can automatically suitably between base, maintain with continuous base by irreversible while being fixed on bottom this base be arranged suitable space, occupied space.This space is to be enough to make c-DNA in crossover process, freely to enter the space of chip base bottom, therefore can hybridize at a high speed.Meanwhile, this is used for can maintaining all the time maximum value in conjunction with the base quantity of base when hybridization; Therefore, this is a kind of new technique that can confirm by the hybridization of high degree of specificity independent base difference in on-off level.
In addition, the invention provides a kind of fixing oligomer DNA individual layer that there is maximum density level and maintain simultaneously suitable space level, wherein all kinds of oligomer DNAs are connected to solid substrate (for example having substrate of glass (amine slide) or the gold substrate (gold thin film or gold) of amine functions group) without any processing and are fixed, that is to say, imine derivative provides the chip base of producing as individual layer for fixing oligomer DNA (, producing oligomer DNA chip).The present invention thereby provide for the preparation of the necessary basic technology of oligomer DNA chip with identical reproducible product in all solids substrate.
In addition, according to the present invention, oligomer DNA can pass through a kind of new technique (, polymolecular identification) in the aqueous solution without fixing under the state of clearance spaces.Therefore the oligomer DNA s of maximum is fixed, that is, thereby the present invention a kind ofly makes the spontaneous assembling of oligomer DNA s can carry out at a high speed hybridization simultaneously with the new technique of the fixing oligomer DNA s of maximum density.Therefore, thus the invention provides a kind of there is maximum sensitivity level can be by identify the production technology of the oligomer DNA chip that c-DNA diagnoses under lower concentration.Meanwhile, because oligomer DNA is fixed with maximum density all the time, it provides the production technology that oligomer DNA chip is prepared as to the necessary oligomer DNA chip with reproducibility of product.The conventional chip base using can be produced by the oligomer DNA with high price reactor, and is difficult to obtain productivity in the stage of fixing oligomer DNA.Therefore, the cycle of these research and development is very long, and in addition, this becomes these research and development and cannot proceed to the reason of production phase.Can make to produce identification molecular engineering of the present invention more easily by adopting, and by the hundreds of oligomer DNA base sequence of checking, can select to bring the oligomer DNA of maximum results of hybridization to reduce the research and development burden in oligomer DNA chip development.In addition, the invention provides a kind of new technique that can immediately release fast best oligomer DNA chip by obtaining productivity.If adopt the basic technology of production chip provided by the invention, can carry out quick production and the commercialization of range gene chip.
In addition, the present invention can be only the oligomer DNA s of 3 to 5 times of the fixing oligomer DNA s amount in surface by using, thereby fixes oligomer DNA s production high-density oligomer DNA chip by molecular recognition.Therefore, the present invention makes the only cost production oligomer DNA s with traditional oligomer DNA chip production 1/10-1/100 become possibility, thereby has saved a large amount of production costs.
In addition, the present invention is different from the conventional in the world traditional oligomer DNA fixing means that passes through Chemical bond or physical adsorption completely, it is a kind of novel method that define (, polymolecular is identified) and used continuous guanine base irreversible fixing oligomer DNA in solid substrate by molecular recognition.
Figure IYZ000003997107300011
Figure IYZ000003997107300031
Figure IYZ000003997107300041
Figure IYZ000003997107300051
Figure IYZ000003997107300061

Claims (4)

1. the oligomer DNA chip preparing by fixing oligomer DNA, wherein at least 7 continuous guanine bases are identified and are connected to self-assembled monolayer solid substrate by polymolecular, wherein said self-assembled monolayer solid substrate by by suc as formula 6 or formula 8 described in amino Calixarene Derivatives in the end group amino Calixarene Derivatives that connects a kind of aldehyde functional group group be selected from glass, imine linkage the has been connected amine functions group solid substrate with amine key passed through of silicon wafer and crystal is connected and prepares:
[formula 6]
Figure FDA0000444096880000011
Wherein R 1, R 2, R 3, R 4, R 5, R 6, R 7, R 8, R' 1, R' 2, R' 3, R' 4, R' 5, R' 6, R' 7, R' 8independently be selected from combination-H ,-CH 3,-C 2h 5,-C 3h 7,-OCH 3,-Cl ,-C 6h 5,-OH ,-OCH 2cH 3,-Br ,-CF 3,-OCH 2c 6h 5,-OC 6h 5,-OC 6h 4cH 3,-OC 6h 4c (CH 3) 3,-OC 6h 4cF 3,-OC 6h 4cl ,-OCOCH 3,-NHCOCH 3,-CONHCH 3,-CN, COOH, and-COOR, wherein R Dai Biao – CH 3huo – C 2h 5, and described Y 1, Y 2, Y 3and Y 4independently be selected from combination-H ,-(CH 2) n-CH=O ,-(CH 2) n-SH ,-(CH 2cH 2o) m-CH 2cH 2-CH=O ,-(CH 2cH 2o) m-CH 2cH 2-SH ,-(CH 2) m-C 6h 4-(CH 2) c-Z, and-CO-(CH 2) m-1-C 6h 4-(CH 2) c-Z, wherein n=2-15, m=1-10, c=0-10, Z=-SH ,-CHO ,-COOH ,-NH 2， Qie – C 6h 4-and C 6h 5be defined as phenyl group;
[formula 8]
Wherein R 1, R 2, R 3, R 4, R 5, R 6, R 7, R 8, R' 1, R' 2, R' 3, R' 4, R' 5, R' 6, R' 7, R' 8independently be selected from combination-H ,-CH 3,-C 2h 5,-C 3h 7,-OCH 3,-Cl ,-C 6h 5,-OH ,-OCH 2cH 3,-Br ,-CF 3,-OCH 2c 6h 5,-OC 6h 5,-OC 6h 4cH 3,-OC 6h 4c (CH 3) 3,-OC 6h 4cF 3,-OC 6h 4cl ,-OCOCH 3,-NHCOCH 3,-CONHCH 3,-CN, COOH, and-COOR, wherein R Dai Biao – CH 3huo – C 2h 5, but R 1, R 2, R 3, R 4, R 5, R 6, R 7, R 8, R' 1, R' 2, R' 3, R' 4, R' 5, R' 6, R' 7, R' 8when different, be-H, and described Y 1, Y 2, Y 3and Y 4independently be selected from combination-H ,-(CH 2) n-CH=O ,-(CH 2) n-SH ,-(CH 2cH 2o) m-CH 2cH 2-CH=O ,-(CH 2cH 2o) m-CH 2cH 2-SH ,-(CH 2) m-C 6h 4-(CH 2) c-Z, and-CO-(CH 2) m-1-C 6h 4-(CH 2) c-Z, wherein n=2-15, m=1-10, c=0-10, Z=-SH ,-CHO ,-COOH ,-NH 2， Qie – C 6h 4-and C 6h 5be defined as phenyl group.
2. the oligomer DNA chip preparing by fixing oligomer DNA as claimed in claim 1, wherein by 5,11 of following formula 9, the sulfonamide derivatives of 17,23-tetramino cup [4] aromatic hydrocarbons is prepared self-assembled monolayer solid substrate, and wherein nitrogen is connected 5 of calixarene, on 11,17,23-position:
[formula 9]
Figure FDA0000444096880000031
3. prepare the method for oligomer DNA chip by fixing oligomer DNA for one kind, wherein at least 7 continuous guanine bases are identified and are connected to self-assembled monolayer solid substrate by polymolecular, wherein said self-assembled monolayer solid substrate by by suc as formula 6 or formula 8 described in amino Calixarene Derivatives in the end group amino Calixarene Derivatives that connects a kind of aldehyde functional group group be selected from glass, imine linkage the has been connected amine functions group solid substrate with amine key passed through of silicon wafer and crystal is connected and prepares:
[formula 6]
Wherein R 1, R 2, R 3, R 4, R 5, R 6, R 7, R 8, R' 1, R' 2, R' 3, R' 4, R' 5, R' 6, R' 7, R' 8independently be selected from combination-H ,-CH 3,-C 2h 5,-C 3h 7,-OCH 3,-Cl ,-C 6h 5,-OH ,-OCH 2cH 3,-Br ,-CF 3,-OCH 2c 6h 5,-OC 6h 5,-OC 6h 4cH 3,-OC 6h 4c (CH 3) 3,-OC 6h 4cF 3,-OC 6h 4cl ,-OCOCH 3,-NHCOCH 3,-CONHCH 3,-CN, COOH, and-COOR, wherein R Dai Biao – CH 3huo – C 2h 5, and described Y 1, Y 2, Y 3and Y 4independently be selected from combination-H ,-(CH 2) n-CH=O ,-(CH 2) n-SH ,-(CH 2cH 2o) m-CH 2cH 2-CH=O ,-(CH 2cH 2o) m-CH 2cH 2-SH ,-(CH 2) m-C 6h 4-(CH 2) c-Z, and-CO-(CH 2) m-1-C 6h 4-(CH 2) c-Z, wherein n=2-15, m=1-10, c=0-10, Z=-SH ,-CHO ,-COOH ,-NH 2， Qie – C 6h 4-and C 6h 5be defined as phenyl group;
[formula 8]
Figure FDA0000444096880000041
Wherein R 1, R 2, R 3, R 4, R 5, R 6, R 7, R 8, R' 1, R' 2, R' 3, R' 4, R' 5, R' 6, R' 7, R' 8independently be selected from combination-H ,-CH 3,-C 2h 5,-C 3h 7,-OCH 3,-Cl ,-C 6h 5,-OH ,-OCH 2cH 3,-Br ,-CF 3,-OCH 2c 6h 5,-OC 6h 5,-OC 6h 4cH 3,-OC 6h 4c (CH 3) 3,-OC 6h 4cF 3,-OC 6h 4cl ,-OCOCH 3,-NHCOCH 3,-CONHCH 3,-CN, COOH, and-COOR, wherein R Dai Biao – CH 3huo – C 2h 5, but R 1, R 2, R 3, R 4, R 5, R 6, R 7, R 8, R' 1, R' 2, R' 3, R' 4, R' 5, R' 6, R' 7, R' 8when different, be-H, and described Y 1, Y 2, Y 3and Y 4independently be selected from combination-H ,-(CH 2) n-CH=O ,-(CH 2) n-SH ,-(CH 2cH 2o) m-CH 2cH 2-CH=O ,-(CH 2cH 2o) m-CH 2cH 2-SH ,-(CH 2) m-C 6h 4-(CH 2) c-Z, and-CO-(CH 2) m-1-C 6h 4-(CH 2) c-Z, wherein n=2-15, m=1-10, c=0-10, Z=-SH ,-CHO ,-COOH ,-NH 2， Qie – C 6h 4-and C 6h 5be defined as phenyl group.
4. the method for preparing oligomer DNA chip by fixing oligomer DNA as claimed in claim 3, wherein by 5,11 of following formula 9, the sulfonamide derivatives of 17,23-tetramino cup [4] aromatic hydrocarbons is prepared self-assembled monolayer solid substrate, and wherein nitrogen is connected 5 of calixarene, on 11,17,23-position:
[formula 9]
CN200680038071.4A 2005-10-13 2006-05-11 Novel iminecalixarene derivatives and aminocalixarene derivatives, method of preparation thereof, self-assembled monolayer prepared by the method, fixing method of oligo-DNA by using the self-asse, and oligo-DNA chip prepared by the method Active CN101309896B (en)

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