CN101309896A - Novel iminecalixarene derivatives and aminocalixarene derivatives, method of preparation thereof, and self-assembled monolayer prepared by the method, fixing method of oligo-dna by using the self-asse - Google Patents

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

Novel imines Calixarene Derivatives and amino Calixarene Derivatives, its preparation method, the self-assembled monolayer by the preparation of this method uses the fixedly method of oligomer DNA of this self-assembled monolayer, and the oligomer DNA chip that is prepared by this method

Technical field

[1] the present invention relates to novel imines Calixarene Derivatives, its preparation method, and, use the fixedly method of oligomer DNA of this self-assembled monolayer, and the oligomer DNA chip for preparing by this method by the self-assembled monolayer of this method preparation.

[2] the present invention more specifically relates to a kind of oligomer DNA that contains continuous guanine base can being fixed to the imines calixarene compound of the solid substrate surface in the individual layer on substrate of glass or the gold substrate and preparing the method for oligomer DNA individual layer (being the oligomer DNA chip) by the use self-assembled monolayer by molecular recognition.

[3] in addition, the present invention relates to the novel amino Calixarene Derivatives, its preparation method, and by the self-assembled monolayer of this method preparation, irreversible by oligomer DNA in the described self-assembled monolayer, the fixing method of oligomer DNA of molecular recognition automatically, and the oligomer DNA chip for preparing by this method.

[4] the present invention more specifically relate to a kind of can with the oligomer DNA that contains continuous guanine base by the polybase base irreversible/molecular recognition is fixed to the amino calixarene compound of solid substrate surface automatically, thereby a kind of technology that is prepared as the substrate of individual layer biochip on as solid substrate such as glass or gold substrate and a kind of oligomer DNA is fixed on as individual layer with high-density by as described in prepare the method for oligomer DNA chip in the biochip substrate of technology of preparing preparation.

Background technology

[5] the National Human Genome Research Institute and Sai Leila genome company under the Human Genome Project competitive explore and the research human genome after, the Human genome information pattern was finished in 2000, thereby promoted 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 to explore monokaryon glycosides polymorphism (SNP) can be especially be used to the genetic diseases of diagnosing defectiveness DNA to cause by expection, or is in cancer of commitment etc.In addition, explore and technology that research is used to produce oligomer DNA chip (wherein a series of oligomer DNA is fixed on substrate) has become worldwide trend, this oligomer DNA chip provides the information based on the accurate virus infection approach of Virus Type, for harmful or harmless etc. analysis and based on the prediction to the generation of cancer etc. of the somatotype of one group of oncovirus.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 being used to predict the 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.

[6] the oligomer DNA chip has comprised and has been fixed on suprabasil 15-50mer short dna s (oligomer DNA s).Therefore, adopt the simple physics absorption method usually although be used for the c-DNA chip of analyzing DNA variant, this oligomer DNA chip still adopts the method for exploitation decades ago, for example based on the legal fixing means of imine linkage by chemical bond between aldehyde and the amine; Or improve the fixing means of the amine bonding method of bonding based on behind the chemical bonding between aldehyde and the amine functions group, carrying out reduction reaction.This is to develop the biochip substrate that is better than aldehyde-chip on reproducibility and convenience owing to also fail at present.Yet in aldehyde-chip base, the amine functions group that is connected to the oligomer DNA end by synthetic technology is connected chemical bonding to this aldehyde-chip by imine linkage; Therefore, fixed dna quantity and can and the theoretical maximum density of the quantity of the fixedly oligomer DNA that in actual hybridization, makes up of c-DNA between exist huge difference.In addition, owing to be subjected to the great effect of rigid condition, fixedly the density of oligomer DNA often changes, and therefore is difficult to successfully develop the chip with reproducibility.

[7] a kind of technology of the bonding method of streptavidin-vitamin H that adopts is used widely, and described technology is a kind of method for preparing oligomer DNA, its by with streptavidin-vitamin H by physical adsorption, methods such as chemical combination bonding are fixed on the solid substrate, make this streptavidin-biotin molecule identification connect vitamin H functional group (Science, 1993 of the oligomer DNA of this vitamin H then; Vol.262, pp 1706-1708).Yet the technology of this employing chemical bonding or streptavidin-vitamin H bonding method still lags behind aldehyde-chip base on performance.

[8] simultaneously, in protein chip, the avtive spot of antibody is present in and is fixed on the lip-deep proteic position, if therefore only have can conjugated antigen described position when being positioned then should obtain the result.Yet, in the oligomer DNA chip, at least the part base among most oligomer DNA s must with c-DNAs approaching in hybridization bonding mutually, and the quantity of this bonding base should obtain maximization near the substrate of this oligomer DNA s fixed by making this cDNAs far as possible.For this reason, European association suggestion in 2003 freely enters with permission c-DNAs fixedly keeping some spaces between the oligomer DNA s, obtains the hot research object that this kind spatial technology has become this area now.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, the oligomer DNA of oligomer DNA that can 20-25-mer is just enough extends to 50-mer, thereby makes approaching c-DNA need not to fall to the bottom, and has improved the result that the base quantity of bonding is diagnosed with reproduction in the hybridization.Yet, the clear readable several hrs that needs at least of this result was arrived above one day, so this technology is unlike outstanding about traditional diagnostic techniques of analyses such as bird flu.

Summary of the invention

Technical problem

[10] fixedly finding following problem in the traditional method of oligomer DNA s as chemical bonding and streptavidin-vitamin H method etc.:

[11]

[12] the 1. homogeneity of constant density: chemical bonding is the most widely used method, and it is known when oligomer DNA s is fixed, aldehyde functional group group (CHO) and the amine functions group (chemical reaction has taken place NH), thus chemical fixation oligomer DNA s; In this reaction, taken off a H ₂(form C=N-) is fixed O so that oligomer DNA is with imine linkage.Yet, owing to this reaction betides in the aqueous solution, this constant density and heterogeneity, that is, not reproducible.In addition, the density of this oligomer DNA is difficult to keep homogeneity, because the reversed reaction that imine linkage reverts to the primary functional group takes place in the aqueous solution easily.In addition, for reducing this kind reversed reaction, can adopt as NaBH ₄Deng reductive agent imine linkage is carried out reduction reaction, but this kind reduction reaction can influence the bonding of oligomer DNA s, thereby be difficult to remain the fixedly length of oligomer DNA s.Because each production result's discordance, the exploitation of novel oligomer DNA chip will be a long process, and this is counted as the huge obstacle in the oligomer DNA chip production.Meanwhile, this fixedly oligomer DNA can be connected fluorescent substance (for example, c-DNAs Cy-3) has detected the existence of c-DNAs and concentration thereof then to diagnose by bonding and described oligomer DNA density are proportional.Therefore, if can't continue to keep the density of oligomer DNA, then this oligomer DNA can't be produced as commerical prod, and in view of same reason, many companies that dropped into fund still fail to obtain their expected result.

[13]

[14] 2. obtain the necessity (the ultra-high speed hybridization can not take place) that the space enters for c-DNAs between oligomer DNA s: the templet gene of pathogenic agent must finally be connected to the oligomer DNA chip by the c-DNAs of polymerase chain reaction (PCR) amplification.Yet as shown in Figure 7, if the distance between the fixed dna s is too short, c-DNAs can't arrive the bottom of oligomer DNA s easily.Present many oligomer DNA chips need a few hours to arrive and surpass one day hybridization, and this mainly is owing to lack the space of the base of bonding with it, the approaching easily fixedly oligomer DNA s of permission c-DNAs bottom.Therefore a kind of spatial technology that obtains 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 to develop by continuous base surface bond with the space that obtains proper level make c-DNAs can be easily near the technology of DNA chip.

[15]

[16] the 3. connection of functional group: should by chemical bond or streptavidin-vitamin H bonding fixedly the technology of oligomer DNA only when vitamin H or amine functions group are connected to oligomer DNA, just may exist.Yet the cost that connects this kind functional group after oligomer DNA is synthetic again is three to ten times of the connected oligomer DNA prior synthesizing method of a small amount of functional group.In addition, even 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 cost of finished product (disregard produce) just needs hundreds of or millions of dollar, this becomes another obstacle of exploitation oligomer DNA chip.For reducing this kind problem, be necessary to develop a kind of not technology of the oligomer DNA s production oligomer DNA chip of linkage function group of passing through.

[17]

[18] 4. with three kinds of base A, chemical bonding between the amine functions group that C and G connect and the functional group of aldehyde-chip base: this aldehyde-chip base can be produced the oligomer DNA chip, and wherein this oligomer DNA s fixes with the reaction of the oligomer DNA s that has been connected the amine functions group by the aldehyde functional group group on surface.Yet, this amine functions group has been connected in described and the numerous bases that this oligomer DNA is connected and has accounted for three kinds of bases of 3/4ths, and it is faster when the oligomer DNA s at the middle part compares fixedly base during with aldehyde reaction with the terminal amine functions group that is connected of oligomer DNA, therefore can be used for approaching, the c-DNA that fluorescence connects carries out the base quantity not sufficient of many hydrogen bondings.Aldehyde can also be by the base bonding of hydrogen bond with in addition approaching c-DNA, thereby with its removal, should carry out chemical treatment after oligomer DNA s is fixed, and knownly generated duplicating of DNA chip and become more difficult.

[19]

[20] 5. technology of diagnosing SNP: the oligomer DNA chip helps analyzing DNA s most so that all kinds of DNA that are present in the multiple virus (as bird flu, swine fever epidemic disease, cancer diffusion virus etc.) are carried out somatotype in various technology.Yet this kind virus major part is the new form that generates from the form variation of protovirus class, and same, the base sequence between viral initial form and variant thereof does not have very big difference as a rule.More difficult is that many virus groups only show difference on one or two base sequence.Accordingly, most important parts is to prepare a kind of oligomer DNA chip that can differentiate the variation (that is, can diagnose SNP) of single base in switch level in the exploitation oligomer DNA chip.The oligomer DNA chip that many investigators agree to adopt current the most frequently used aldehyde-chip base to produce fixedly is not being differentiated this necessary enough space of list base variant between the oligomer DNA s, and so can't produce the diagnostic oligomer DNA chip that is used to differentiate single base variant.

[21]

[22] 6. produce the technology of chip at a low price: if by connecting as amine, functional groups such as vitamin H are oligomer DNA fixedly, and then the cost of this oligomer DNA can rise above ten times, and this chip must cover the DNAs of high density to stop surperficial base bonding.To become high so produce the cost of this kind oligomer DNA chip.

Technical scheme

[23] the object of the present invention is to provide the imines cup derivative that can molecular recognition has the oligomer DNA of continuous guanine group, and preparation method thereof, thereby solve the problem of above-mentioned traditional oligomer DNA fixing means, homogeneity problem as constant density, between oligomer DNA s, obtain enough spatial problems, the connectivity problem of functional group etc.

[24] another object of the present invention is to provide a kind of oligomer DNA individual layer, wherein all kinds of oligomer DNA s can be without any processing by the space of intensive filling and reservation proper level by described imines Calixarene Derivatives and combining of substrate of glass (a kind of amine slide) or gold substrate (au film coating or gold); A kind of imines Calixarene Derivatives individual layer that can make the oligomer DNA chip just is provided.

[25] the present invention more specifically target for a kind of oligomer DNA s individual layer is provided, the oligomer DNA s that wherein has at least seven continuous guanines by intensive filling and kept the space of proper level, just provides a kind of imines Calixarene Derivatives individual layer that can make the oligomer DNA chip without any processing.

[26] the object of the present invention is to provide a kind of oligomer DNA chip on the other hand, its can separate simultaneously the technology that must not carry out the ultra-high speed hybridization, can not guarantee to diagnose SNP, with three base A, chemical bonding between the amine functions group that C and G connect and the functional group of aldehyde-chip base is difficult to the fixedly problem of all traditional oligomer DNA chips such as oligomer DNA s of homogeneous density 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 kind of production method of substrate of the oligomer DNA chip with the preparation of its self-assembly form, and a kind of oligomer DNA chip of producing by the irreversible fixedly oligomer DNA of spontaneous molecular recognition s on the chip base surface.

[27] another object of the present invention is to provide a kind of oligomer DNA s individual layer, wherein by with above-claimed cpd as individual layer with have substrate of glass solid substrate such as (amine slides) of 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 keep proper level with maximum density, that is to say, a kind of chip base that can make the oligomer DNA chip of producing with amino Calixarene Derivatives is provided.

[28] 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, this oligomer DNA s is unprocessed to fix and keeps the space of proper level with maximum density thereby make, and a kind of technology that is used to produce the oligomer DNA chip that provides is provided.

[29]

Description of drawings

[30] Fig. 1 is a synoptic diagram of going up the preparation process of imines Calixarene Derivatives self-assembled monolayer of the present invention in substrate of glass (amine slide).

[32] Fig. 2 is the synoptic diagram of the preparation process of imines Calixarene Derivatives self-assembled monolayer of the present invention on gold substrate.

[32] Fig. 3 has the fixing means synoptic diagram of the oligomer DNA of continuous guanine base for the present invention.

[33] Figure 4 and 5 have shown the selectivity fixing means of the oligomer DNA that only has continuous guanine base, and have shown that the c-DNA with band fluorescence analyzes the fixedly result of oligomer DNA density.

[34] Fig. 6 has shown to fixing 7 the continuous guanine functional groups of needs during oligomer DNA of imines Calixarene Derivatives self-assembled monolayer of the present invention at least.

[35] Fig. 7 shown for checking to imines Calixarene Derivatives self-assembled monolayer of the present invention fixedly during oligomer DNA this guanine base discerned by selectivity by molecular recognition, use has the result that the imidazoles functional group of similar structures carries out competitive reaction.

[36] Fig. 8 is the synoptic diagram of the preparation process of amino Calixarene Derivatives self-assembled monolayer of the present invention on substrate of glass.

[37] Fig. 9 is the synoptic diagram of the preparation process of amino Calixarene Derivatives self-assembled monolayer of the present invention on gold substrate.

[38] Figure 10 and 11 oligomer DNAs that have a continuous guanine base for the present invention carry out fixed method synoptic diagram and maximum density fixed actual tests result by automatic molecular recognition.

[39] Figure 12 carries out the best test-results of guanine base number continuously of maximum density fixed for detecting.

[40] the fixedly space between the oligomer DNA that obtains by the suitable space of guaranteeing between the continuous base can be by fixing oligomer DNA the time for c-DNA of Figure 13 is near the synoptic diagram of chip bottom.

[41] Figure 14 and 15 differentiates the actual tests result that may reach switch level for showing by hybridizing SNP at a high speed.

[42] Figure 16 is for showing that guanine base and other base stationary phase are than the actual competitive reaction result that can carry out the selectivity high-speed fixed.

[43]

The invention pattern

[44] 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 that adopts in the fixing means of oligomer DNA of at least 7 continuous guanine bases.

[45] [formula 1]

[46]

[47] (R wherein₁，R ₂，R ₃And R₄Independently be selected from combination-H ,-CH₃，-C ₂H ₅，-C ₃H ₇， -OCH ₃，-Cl，-C ₆H ₅With-COOR, and in described-COOR, R representative-CH₃Or-C₂H ₅)。

[48] [formula 2]

[49]

[50] (R wherein₁，R ₂，R ₃And R₄Independently be selected from combination-H ,-CH₃，-C ₂H ₅，-C ₃H ₇， -OCH ₃，-Cl，-C ₆H ₅With-COOR, and in described-COOR, R representative-CH₃Or-C₂H ₅ In addition, described Y₁，Y ₂，Y ₃And Y₄Independently be selected from combination-H ,-(CH₂) _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-(CH₂) _m-1-C ₆H ₄-(CH ₂) _c-Z (n=2-15 wherein, m=1-10, c=0-10, Z=-SH ,-CHO ,-COOH or-NH₂))。

[51]

[52] the present invention also provides the method for the imines calixarenes derivative compound of the described formula 1 of preparation and 2.

[53] have following formula 3 this 5,11,17,23-tetramino cup [4] aromatic compound can be by being converted into 5,11,17 with reference to the method described in the following list of references of quoting 1 with cup [4] aromatic hydrocarbons, 23-tetranitro cup [4] aromatic hydrocarbons is by synthetic [the list of references 1:Journal of Organic Chemistry that quotes that obtains of reduction reaction, 1990, VoI 55, pp 5639-5643; Journal of Organic Chemistry, 1979, VoI 44, pp 1233-1238].

[54] [formula 3]

[55]

[56] the imines calixarenes 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 compound is as initiation material, obtains (reaction 2 among the embodiment that vide infra) with amine functions group and the aromatic aldehyde reaction with following formula 4 of formula 3 are synthetic.

[57] [formula 4]

[58]

[59] (wherein R is selected from combination-H ,-CH₃，-C ₂H ₅，-C ₃H ₇，-OCH ₃，-Cl，-C ₆H ₅With-COOR ', and in described-COOR ', R ' representative-CH₃Or-C₂H ₅)。

[60]

[61] in addition, obtain but this imines calixarene derivative compound through type 1 compound of formula 2 of the present invention and the reaction of aldehyde cpd are synthetic, wherein a kind of halogen is connected to end group, thereby aldehyde or mercaptan are connected to first and the 3rd-OH group position (referring to the reaction 3-5 of the following example) of formula 1 in this end group.

[62] 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, n=2-15 wherein, m=1-10, c=0-10, X=-Cl ,-Br ,-I or-OSO ₂C ₆H ₄CH ₃, Z=-SH ,-CHO ,-COOH or-NH ₂

[63] in addition, the invention provides a kind of self-assembled monolayer solid substrate for preparing to a kind of imines Calixarene Derivatives that has thiol group of gold substrate connection.

[64] in addition, the invention provides and a kind ofly the imines Calixarene Derivatives is connected to as glass by imine linkage, the self-assembled monolayer solid substrate that solid substrate such as silicon wafer or crystal prepares, and a kind of passing through as ester, chemical bond such as ether and amido linkage is connected to the imines Calixarene Derivatives as glass, the self-assembled monolayer solid substrate that solid substrate such as silicon wafer or crystal prepare.

[65] in addition, the invention provides a kind of oligomer DNA individual layer (promptly, a kind of oligomer DNA chip), and preparation method thereof, wherein all kinds of oligomer DNAs are connected with gold substrate that has been connected the amine functions group or substrate of glass by the imines calixarene derivative compound with described formula 2 and are fixed by high-density.

[66] Fig. 1 illustrates the preparation process of imines Calixarene Derivatives self-assembled monolayer of the present invention on substrate of glass.

[67] concrete grammar of preparation imines Calixarene Derivatives self-assembled monolayer is as follows on the substrate of glass that has connected the amine functions group:

[68] at 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[that quotes, 1997, VoI 13, pp 4305-4308; Langmuir, 1996, Vol 12, pp 5338-5342] by (Si-OH) form of the substrate of glass that has the amine end group (amine slide or amine chip) that obtains of the chemical reaction on the substrate of glass of functional group is prepared having sufficient silanol.Thereby prepare the substrate of glass that connected the amine functions group and formula 2 compounds are added into chloroform and THF mixing solutions (CHCl with the concentration of 0.1-5mM ₃: THF=9: 1).After 1-5 hour, with chloroform, acetone and alcoholic acid wash in proper order and are dry, finish imines calixarene self-assembled monolayer as shown in Figure 1 then.The formation of described individual layer can be confirmed by infrared rays external reflection spectrum.All kinds of slides on the market all can be used as described substrate of glass.For imine linkage used in the bonding, fracture when this key can be deposited in the water midium or long term.Yet,, when forming imine linkage immediately, do not have problems with it through affirmation because active materials such as oligomer DNA are dissolved in pH in the preparation usually in the buffered soln of 7-8.

[69] Fig. 2 illustrates the preparation process of imines Calixarene Derivatives self-assembled monolayer of the present invention on gold substrate.

[70] concrete grammar of preparation imines Calixarene Derivatives self-assembled monolayer is as follows on gold substrate:

[71] compound that will connect the formula 2 of mercaptan is dissolved in as chloroform (CHCl with the concentration of 0.1-5mM ₃) wait and prepare solution in the organic solvent.After gold substrate is placed into prepared solution and places 1-5 hour, with its taking-up and with chloroform, the washing of the order of acetone and water, and dry, finish imines Calixarene Derivatives self-assembled monolayer as described in Figure 2 then.Described gold substrate can be the gold thin film of any kind; Yet, generally speaking, preferably at glass, molten silicon, silicon wafer, plastic-substrates etc. after vacuum plating is with the chromium (Cr) of 2-10nm or titanium materials such as (Ti) with the substrate of the thickness vacuum metallizing of 50-200nm.Generally speaking, prepared gold substrate before be about to using, be placed in Piranha solution (strength sulfuric acid: 30% hydrogen peroxide=3: 1) about 1 minute, then to use after pure water washing and the nitrogen blowing drying.The formation of described individual layer can be confirmed by infrared rays external reflection spectrum.

[72]

[73] in addition, the invention provides and a kind ofly prepare the method for oligomer DNA chip and the oligomer DNA chip for preparing by this method, wherein continuous guanine base is fixed to described solid substrate by polymolecular identification by fixing oligomer DNA.

[74] more specifically, the fixing method of oligomer DNA of the polymolecular identification that the invention provides a kind of four continuous guanine functional groups of nitrogen-atoms molecular recognition by described imines Calixarene Derivatives.

[75] oligomer DNA fixing means of the present invention has formed all and has adopted the basis of the analytical procedure of oligomer DNA, and this is that the novel method that similar research is reported is not seen in the whole world so far.

[76] the fixing means synoptic diagram of the oligomer DNA of Fig. 3 continuous guanine group for the present invention has.

[77] this oligomer DNA with the continuous guanine group that is used for fixing be 5 '-GGGGGG GGG AAA TCA ACC CAC AGC TGC A-3 ' (SEQ ID NO.1).To be 5 with the c-DNA of its bonded band fluorescence '-Cy3-GT GCA GCT GTG GGTTGA TT-3 ' (SEQ ID NO.2), it has and fixing oligomer DNA complementary dna sequence dna.At this moment, the constant density of this oligomer DNA can by with fluorescent substance Cy-3 (Telechem, the U.S.) thus being connected to this end group only shows that in this connection site fluorescence obtains measuring.After being dissolved in the solution 500mM KCl that contain 15% glycerol with the concentration of 54 μ g/ml (6.75pmol/ml) oligomer DNA, this oligomer DNA can be about on the imines calixarene individual layer of 150um and is fixed by being coated on the diameter of using available from the microarray preparation of Proteogen Co. (Korea).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 and dry preparation.In at least 6 months measurement, do not demonstrate any variation through determining that this preparation oligomer DNA is one-sided.

[78]

[79] Figure 4 and 5 are the synoptic diagram that has shown in this oligomer DNA density of oligomer DNA individual layer of the present invention fluorometric analysis.

[80] this oligomer DNA with the continuous guanine group that is used for fixing the oligomer DNA individual layer for preparing to Fig. 3 be 5 '-GGG GGG GGG AAA TCA ACC CACAGC TGC A-3 ' (SEQ ID NO.1).To be 5 with the c-DNA of its bonded band fluorescence '-Cy3-GT GCA GCT GTG GGT TGA TT-3 ' (SEQ ID NO.2), it has and fixing oligomer DNA complementary dna sequence dna.In addition, the constant density of this oligomer DNA can by with fluorescent substance Cy-3 (Telechem, the U.S.) thus being connected to this end group only shows that in this connection site fluorescence obtains measuring.This individual layer is coated with the 60 μ l 4x SSC+0.002%SDS+50% glycerol mixing solutionss of the c-DNA that is connected with fixing oligomer DNA fluorescence that contains 0.17nmol/ml, then under 50 ℃ with DNA hybridization 30 minutes.At room temperature wash with the 4x SSC solution that contains 0.1%SDS earlier subsequently, again with 4x SSC solution washing, and dry.Products obtained therefrom can use scanner (GSI lite, the U.S.) to confirm.

[81] for the oligomer DNA s that is used for fixing, can adopt the oligomer DNA that has following dna sequence dna respectively: have 9 continuous A, T, C, G base sequence, a kind of oligomer DNA and a kind of oligomer DNA that does not have continuous base sequence that connects vitamin H (functional group of similar guanine).Accordingly, fixed by selectivity through the only continuous guanine base of test affirmation.This dna sequence dna that is used for fixing comparison is as follows:

[82]

[83]9G 5′-GGG GGG GGG AAA TCA ACC CAC AGC TGCA-3′(SEQ ID NO.1)

[84]9T 5′-TTT TTT TTT TAA TCA ACC CAC AGC TGC A-3′(SEQ ID NO.3)

[85]9A 5′-AAA AAA AAA TAA TCA ACC CAC AGC TGCA-3′(SEQ ID NO.4)

[86]9C 5′-CCC CCC CCC CAA TCA ACC CAC AGC TGC A-3′(SEQ ID NO.5)

[87] vitamin H 5 '-vitamin H-T ATA TAA TCA ACC CAC AGC TGCA-3 ' (SEQ ID NO.6)

[88]no 5′-AA TCA ACC CAC AGC TGC A-3′(SEQ ID NO.7)

[89]

[90] should can have a complementary base sequence through synthetic by hybridizing with it bonded c-DNA.

[91]5′-Cy3-GT GCA GCT GTG GGT TGA TT-3′(SEQ ID NO.2)

[92] can promptly, connect the density of the fixedly oligomer DNA of c-DNA by with the relative density of analyzing the c-DNA that is connected of fluorescent substance (Cy3) simultaneously with the connection of fluorescent scanning instrument.According to the actual tests result, after in above-mentioned condition, applying in the same manner and fixing each oligomer DNA, this c-DNA applies with three times of oligomer DNA maximum that can be fixed on the surface, thus make c-DNA that abundant fluorescence connects with all fixedly oligomer DNA combine at this.Then, wash and dry and analyze with the fluorescent scanning instrument.

[93] the actual tests result is the middle figure with Fig. 5 of 6X6 display result, 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 fluorescence that oligomer DNA 9G with 9 continuous guanine groups presented 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 only shows when having continuous guanine base when fixing oligomer DNA, and this oligomer DNA could be with on the analyzable surface that is horizontally fixed on the imines calixarene.

[94]

[95] Fig. 6 has shown to imines Calixarene Derivatives self-assembled monolayer of the present invention fixedly must have at least 7 continuous guanine functional groups during oligomer DNA.

[96] this oligomer DNA is fixed under the condition shown in Figure 3 and carries out, and this c-DNA can use the band fluorescence oligomer DNA that is used for Fig. 4 equally to hybridize according to above-mentioned condition.

[97]

[98]1G 5′-GAA TCA ACC CAC AGC TGC A-3′(SEQ ID NO.8)

[99]4G 5′-GGG GAA TCA ACC CAC AGC TGC A-3′(SEQ IDNO.9)

[100]7G 5′-GGG GGG GAA TCA ACC CAC AGC TGC A-3′(SEQ ID NO.10)

[101]9G 5′-GGG GGG GGG AAA TCA ACC CAC AGC TGCA-3′(SEQ ID NO.1)

[102]12G 5′-GGG GGG GGG GGG GAA TCA ACC CAC AGCTGC A-3′(SEQ ID NO.11)

[103]15G 5′-GGG GGG GGG GGG GGG GAA TCA ACC CACAGC TGC A-3′(SEQ ID NO.12)

[104] c-DNA 5 '-Cy3-GT GCA GCT GTG GGT TGA TT-3 ' (SEQID NO.2) (Cy-3; Fluorescent substance)

[105]

[106] under above-mentioned the same terms, apply washing and dry oligomer DNA.According to the actual tests result, after in above-mentioned condition, applying in the same manner and fixing each oligomer DNA, this c-DNA applies with three times of oligomer DNA maximum that can be fixed on the surface, thus make with all fixedly the c-DNA that is connected of oligomer DNA fluorescence in this combination.Then it is washed with dry to analyze by the fluorescent scanning instrument.

[107] the actual tests result is the middle figure with Fig. 6 of 6X6 display result, 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.When continuous guanine base sequence was about 1 or 4, its result was not for almost there being the surface fixing.In addition, when it has 7 continuous guanines (7G), show intense fluorescence, and when having 9 continuous guanines (9G), demonstrate the strongest fluorescence.Simultaneously, when oligomer DNA has 12 (12G) and 15 (15G) continuously during guanine base that is longer than 9 continuous guanine bases, this fluorescence sensitivity begins to descend.This result shows that 9 guanine bases are to be enough to carry out the fixed level by molecular recognition, and in theory when using longer guanine base, the oligomer DNA that fixedly has a 12G need have more about 1/3 space than the oligomer DNA that fixedly has 9G, thereby this fluorescence sensitivity drops to about 1/3 level.Oligomer DNA with 15G need be more than the space of 9G extra approximately 2/3, and therefore the quantity that is fixed in theory drops to about 3/5 level (promptly 60%), and in fact fluorescence sensitivity is compared with 9G and descended about 55%.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.

[108]

[109] Fig. 7 shown for checking to imines Calixarene Derivatives self-assembled monolayer of the present invention fixedly during oligomer DNA this guanine base discerned by selectivity by molecular recognition, use has the result that the imidazoles functional group of similar structures carries out competitive reaction.

[110] Fig. 7 has shown that continuous guanine base carries out the fixed result by molecular recognition, and this result comprises a kind of can carrying out by one and obtains with the research of the imidazoles of the similar molecular recognition of guanine base in fixing.Because imidazoles has the molecular recognition more weak relatively than guanine base in the imines calixarene, therefore the competitive reaction that carries out molecular recognition under the concentration for the treatment of fixing oligomer DNA can be higher than.Under the rigid condition identical, fix and adopt the c-DNA in the used same procedure of Fig. 4 to carry out the measurement of fluorescence sensitivity with Fig. 3.The left figure of Fig. 7 has shown the actual tests result, and right figure has shown the decline of the fluorescence sensitivity that depends on imidazole concentration used in the fluorescence sensitivity competitive reaction.In fact, the 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 fix hardly.This result shows that the polymolecular identification of oligomer DNA by 9 continuous guanine bases in the imines Calixarene Derivatives fixes.

[111] as above the result shows irreversible the fixing of imines Calixarene Derivatives self-assembled monolayer by 9 continuous guanine bases of polymolecular identification carrying out.The fixedly oligomer DNA quantity of representing by fluorometric analysis has shown the 5-30 constant density doubly that is higher than conventionally known method especially.

[112]

[113] similarly, obtain the novel oligomer DNA fixing means of spatial when the invention provides a kind of can be on individual layer surface fixing continuous guanine base with its length (2.5-3.5nm), thereby when having the gene of specific base sequence with diagnosis or goal analysis such as research by provide the c-DNA that has hundreds of base sequences at least drop to the oligomer DNA bottom and with fixing required enough spaces (2.5-3.5nm) of the strong bonding of oligomer DNA, c-DNA is hybridized the required time significantly dropped in 1-2 hour by original at least 12 hours.

[114]

[115] 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.

[116] [formula 5]

[117]

[118] (R wherein 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).

[119] [formula 6]

[120]

[121] (R wherein 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 ₅In 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 (n=2-15 wherein, m=1-10, c=0-10, Z=-SH ,-CHO ,-COOH ,-NH ₂, and-C ₆H ₄-and C ₆H ₅Be defined as phenyl group)).

[122] [formula 7]

[123]

[124] (R wherein 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).

[125] [formula 8]

[126]

[127] (R wherein 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 ₅Yet, 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 simultaneously-situation of H.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 (n=2-15 wherein, m=1-10, c=0-10, Z=-SH ,-CHO ,-COOH ,-NH ₂, and-C ₆H ₄With-C ₆H ₅Be defined as phenyl group)).

[128]

[129] the invention provides the method for amino calixarene derivative compound of the described formula 5-8 of preparation.

[130] [formula 9]

[131]

[132] tetramino cup [4] aromatic hydrocarbons of described formula 9 can carry out that reduction reaction is synthetic to be obtained behind tetranitro cup [4] aromatic hydrocarbons by with reference to the method for the reference 1 quoted cup [4] aromatic hydrocarbons being converted into.

[133]

[134] formula 5 of the present invention and 7 amino calixarene derivative compound can be by will be with tetramino cup [4] aromatic hydroxy compound of currently known methods synthetic formula 9 as starting raw materials, with the benzyl chloride among the amine functions group of formula 9 and the embodiment 13 hereinafter or the benzyl bromide derivatives reaction is synthetic obtains.Alternatively, hereinafter using reductive agent to carry out reduction reaction in the method reaction back of embodiment 16 in the functional group and the aromatic aldehyde reference of formula 9, promptly is the amino Calixarene Derivatives with benzyl chloride or benzyl bromide derivatives reaction synthesis type 5 and 7.

[135] (wherein aldehyde or mercaptan are by following-OH for formula 6 of the present invention and 8 amino calixarene derivative compound, that is, first in the pure functional group is connected with the alkyl group end with the triol functional group) can obtain with compound (wherein halogen is connected to end group with reference to the following example 17-19) reaction by compound formula 5 and 7 with functional groups such as aldehyde.

[136]

[137] in addition, the invention provides a kind of be used to prepare can be connected to the gold substrate that has various functional groups such as amine by amino Calixarene Derivatives or as the oligomer DNA chip production method of solid substrate such as glass with the oligomer DNA individual layer of the fixing various types of oligomer DNAs of high-density with described formula 6 or 8.

[138] in addition, the invention provides and a kind ofly be 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) with amido linkage and be selected from glass by imines, silicon wafer and crystalline have the self-assembled monolayer solid substrate that the solid substrate of amine functions group prepares.

[139] 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, and silicon wafer and crystalline have the self-assembled monolayer solid substrate that the solid substrate of amine functions group prepares.

[140] in addition, the invention provides and a kind ofly by irreversible molecular recognition undressed oligomer DNA with continuous base is fixed to described oligomer DNA chip base and prepares a kind of oligomer DNA individual layer, promptly a kind of method of oligomer DNA chip.

[141] Fig. 8 has shown as glass, silicon wafer, the process of preparation amino Calixarene Derivatives self-assembled monolayer of the present invention on the solid substrate such as molten silicon.

[142] concrete grammar of the amino Calixarene Derivatives self-assembled monolayer of preparation is as follows on the substrate of glass that has connected the amine functions group:

[143] substrate of glass (glass slide) of the amine functions group that uses before having connected can be with reference to the following reference of quoting 2 by (Si-OH) form of the substrate of glass that has the amine end group (amine slide or amine chip) that obtains of the chemical reaction on the glass basic surface of functional group is prepared having sufficient silanol.The substrate of glass that is connected with the amine functions group of so preparation is added the mixing solutions of chloroform and THF, and its Chinese style 6 or 8 compound are dissolved in wherein (CHCL with the concentration of 0.1-5mM ₃: THF=9: 1).After 1-5 hour, with chloroform, acetone and alcoholic acid wash in proper order, and dry, finish amino Calixarene Derivatives self-assembled monolayer as shown in Figure 8 then, 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 or glass on the market all can be used as described substrate of glass.For imine linkage used in the bonding, fracture when this key can be deposited in the water midium or long term.Yet because active materials such as oligomer DNA are dissolved in pH in the preparation usually in the buffered soln of 7-8, results verification does not have problems when forming imine linkage immediately with it after deliberation.

[144] put into and contained if should prepare A type BMT DNA chip base as shown in Figure 8 just like BH ₃-THF or 1-5%NaBH ₄Deng in the organic solvent of reductive agent 1-10 minute, this imines was reduced into stronger amine, therefore can be prolonged preservation or in different pH scopes preparation DNA chip outstanding stability is provided.

[145] Fig. 9 has shown the preparation process of amino Calixarene Derivatives self-assembled monolayer of the present invention on gold substrate.

[146] concrete grammar of the amino Calixarene Derivatives self-assembled monolayer of preparation is as follows on gold substrate:

[147] will connect the formula 6 of mercaptan or 8 compound is dissolved in as chloroform (CHCl with the concentration of 0.1-5mM ₃) wait and prepare solution in the organic solvent.After gold substrate is placed into prepared solution and places 1-5 hour, with its taking-up and with chloroform, the washing of the order of acetone and water, and dry, finish amino Calixarene Derivatives self-assembled monolayer as described in Figure 9 then.Described gold substrate can be the gold thin film of any kind; Yet, generally speaking, preferably at glass, molten silicon, silicon wafer, plastic-substrates etc. after vacuum plating is with the chromium (Cr) of 2-10nm or titanium materials such as (Ti) with the substrate of the thickness vacuum metallizing of 50-200nm.Preferably with prepared gold substrate be about to use be prepended to Piranha solution (strength sulfuric acid: 30% hydrogen peroxide=3: 1) about 1 minute, then with pure water washing and nitrogen blowing drying.The formation of described individual layer can be confirmed by infrared rays external reflection spectrum.

[148]

[149] in addition, the invention provides a kind of by polymolecular identification connect continuous guanine base and described solid substrate fixedly oligomer DNA preparing the method for oligomer DNA chip, and the oligomer DNA chip for preparing by this method.

[150] the present invention's fixing method of oligomer DNA of polymolecular identification that a kind of four continuous guanine functional groups of nitrogen-atoms molecular recognition by described amino Calixarene Derivatives more specifically are provided.

[151] fixing means of the present invention is the technology of using oligomer DNA the to prepare all kinds of biochips technology that provides the foundation, and this is that the novel method of similar result of study is not seen in a kind of whole world up to now.

[152] Figure 10 and 11 is the technology synoptic diagram for preparing high-density oligomer DNA chip by automatic molecular recognition, and the Theoretical Calculation synoptic diagram of forming the amino Calixarene Derivatives quantity of individual layer.It is with actual oligomer DNA concentration and the actual tests result that maximum oligomer DNA concentration ratio gets on the amino Calixarene Derivatives individual layer that is fixed on who obtains by Theoretical Calculation.

[153] fixedly the theoretical maximum of oligomer DNA can be passed through hereinafter Equation for Calculating.The diameter of the amino calixarene that obtains by x-ray method is 2.1-2.2nm.The quantity of desired molecule is identical with two leg-of-mutton areas that constitute by the straight line that connects three amino Calixarene Derivatives centers when it carries out self-assembly as individual layer from the teeth outwards.Therefore, the area that occupies from the teeth outwards of Calixarene Derivatives can obtain by following equation:

[154] [equation]

[155]2.1nmx2.1nmxsin60°＝3.82x10 ^-14cm ²

[156] therefore, every 1cm ²The quantity of the middle amino Calixarene Derivatives that exists is 1/3.82x10 ^-14Cm ²=2.62x10 ¹³=43.5pmol/cm ²Because 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 ²Method with reference to embodiment 24, the oligomer DNA 5 that will be used for fixing '-after GGG GGG GGG AAA TCA ACCCAC AGC TGC A-3 ' (SEQ ID NO.16) is connected to the oligomer DNA chip with c-DNA5 '-cy3-GTGCA GCT GTG GGT TGA TT-3 ' (the SEQ ID NO.19) hybridization of band fluorescence and with maximum density, be shown in the results of hybridization 2 of Figure 11 in the measuring result of the fluorescence sensitivity that records by fluorescent scanning instrument (GSI, the U.S.) after washing and the drying according to the method for embodiment 13) in.Measure the result of similar fluorescence sensitivity as using the fluorescent scanning instrument in identical c-DNA 5 '-cy3-GT GCA GCT GTG GGT TGATT-3 ' (the SEQ ID NO.19) back that is used under different concns, hybridizing of drying, apply with 3.8pmol/cm concentration and drying after can obtain the fluorescence sensitivity of similar level, this level is a half of theoretical.This results are shown in Figure 11 1) among the result of dry fluorescence oligomer DNA.This result shows that the BMT oligomer DNA chip base with reference to the present invention's exploitation can prepare the oligomer DNA chip by fixing oligomer DNA near theoretical maximum, and can obtain 50% of about maximum fluorescence after 30 minutes in hybridization, and this is the first in the world oligomer DNA chip technology of preparing of supporting ultra-high speed hybridization simultaneously and realizing the high-density technique for fixing.

[157] Figure 12 is by the concentration fixed oligomer DNA (6.75nmol/mL of the continuous oligomer DNA with identical base sequence that connects of guanine base with 5 times with reference to embodiment 22,1nL), hybridize with reference to embodiment 22, washing is also dry with after finding best base quantity continuously, the result who uses fluorescent scanning instrument (GSI, the U.S.) to analyze.In the test of Figure 12, adopt the base sequence shown in the table 4, in hybridization, adopted the fluorescence c-DNA shown in the table 4.

[158] the actual tests result is with the scan-data shown in the middle figure of Figure 12 of 6X6 display result, and this result carries out with numerical value that fluorometric analysis obtains and this analysis is shown in the histogram of Figure 12 bottom.When continuous guanine base sequence was about 1 or 4, its result was not for almost there being the surface fixing.Yet, when it has 7 continuous guanines (7G), show intense fluorescence, and when having 9 continuous guanines (9G), show the fluorescence level that doubles 7 continuous guanines (7G).When use has 12 (12G) and 15 (15G) continuously during the oligomer DNA of guanine base that is 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 show 9 continuous guanine bases be enough to by molecular recognition fixedly oligomer DNA level and when the longer guanine base of use, the oligomer DNA of 12G need be greater than the space of 9G in theory, so fluorescence sensitivity drops to 1/2 level.The oligomer DNA of 15G is compared with 9G needs about 2/3 exceptional space, and therefore the oligomer DNA that is fixed drops to about 30% level, and fluorescence sensitivity is lowered by about 1/3 level to 9G.Comprehensive these results can see that the fixing required best quantity of guanine continuously is 9.If the quantity of guanine is less than 9G continuously, then possibly can't reach abundant level by the irreversible fixedly oligomer DNA of molecular recognition.In addition, can also see the quantity that connects guanine base during, treat that fixed area (promptly fixing required area) will be greater than 9G, thereby this cause the decline of the oligomer DNA s quantity of actual fixed greater than 9G from these results.

[159] the fixedly space between the oligomer DNA of guaranteeing, the suitable space that obtains by continuous base can be by fixing oligomer DNA the time for c-DNA of Figure 13 is near chip bottom and kept the synoptic diagram of ultra-high speed near required enough spaces level.Surface area when meanwhile, it has also shown 6-7 amino Calixarene Derivatives actual fixed can be used for fixedly oligomer DNA.

[160] in Figure 14 and 15, the base sequence shown in the table 5 has been fixed different A respectively with reference to embodiment 23, C, G, T type oligomer DNA.Then, in the hybridization of carrying out for the DNA (Cy3-DNA) that detects band fluorescence and c-DNA of reference embodiment 23 to after the complementary c-DNA of bonded is hybridized with it, as described in embodiment 23, the oligomer DNA of the table 5 of this band fluorescence is used in the embodiment 23 described fluorescence connections.After this can wash with dry to carry out the fluorescence sensitivity analysis by fluorescent scanning instrument (GSI, the U.S.).Figure 14 and 15 has shown that this is a kind of even can is carrying out and can distinguish and analysis of fluorescence sensitivity in the on-off level as in about 30 minutes the short hybridization time, distinguishes the oligomer DNA chip of the ultra-high speed hybridization of SNP simultaneously in the on-off level.In addition, this result shows that the technology of preparing as the oligomer DNA chip of target of the present invention is a kind ofly to support ultra-high speed hybridization and duplicate the new technique of easy analysis list nucleosides polymorphism (SNP) with height.

[161] Figure 16 is for showing that independent guanine base and other base stationary phase are than the actual competitive reaction result who carries out the selectivity high-speed fixed.Use is to have connected the oligomer DNA (9G-1) of 9G as embodiment 25 same concentrations, it does not combine with the oligomer DNA of the table 6 of being with fluorescence, by with oligomer DNA (9G-1) 1,2,4 times concentration coating mixing 4 classes contain and is with fluorescence oligomer DNA (Cy-3-DNA) complementary base sequence and be connected 9A, 9G, 9T, the oligomer DNA mixture of 9C is with fixed order DNA shown in Figure 16.Simultaneously, if 9A, 9C, 9T, 9G is fixing faster than oligomer DNA (9G-1) with same concentrations blended oligomer DNA, then can show fluorescence by combining with band fluorescence oligomer DNA in the crossover process that reference embodiment 25 carries out, and if should to connect oligomer DNA (9G-1) of 9G fixing faster, then obviously can not show fluorescence.Can determine on the site of fixing 9G according to the actual experiment result of Figure 16, fluorescence sensitivity rises with the rising of oligomer DNA concentration fixedly the time, thereby as 9G-1: 9G=1: in the time of 1, can show to be about 50% fluorescence sensitivity, and, can show about 80% fluorescence sensitivity at 1: 4 o'clock.For 9T, it is few that 9C can determine that this fluorescence sensitivity rises, even only shown 4 times to the slight fluorescence sensitivity level of the amount of 16G when 9A.Therefore, can see and when actual fixed, having used when connecting the oligomer DNA of 9G, the base that will be used to hybridize (promptly, to be used for and c-DNA bonded base) partly do not participate in fixing, that is to say that only can prepare can be by the oligomer DNA that duplicates with maximum density and amino Calixarene Derivatives single layer of the present invention in the site of 9G selective binding.

[162] generally speaking, can be by discerning continuous guanine base irreversible fixedly oligomer DNA preparation oligomer DNA chip on the individual layer surface with the amino Calixarene Derivatives self-assembled monolayer polymolecular of exploitation thereby this result shows according to the present invention, and this is a kind of energy by supporting the fixing so that amount of the fixedly oligomer DNA that fluorometric analysis the shows basic technology near the reproducible oligomer DNA chip preparation of theoretical maximum density all the time.

[163] in addition, in the present invention, continuously guanine base be fixed on the individual layer surface and be fixed oligomer DNA shown in Figure 13 and guarantee to have firm space with described base equal length.This space can make when having the DNAs of special base sequence with diagnosis or research purpose analysis to have tens c-DNA to up to a hundred base sequences at least and can freely arrive the chip base surface with ultra-high speed like a cork, thus can as utmost point short period of time of 10 minutes-1 hour carry out ultra-high speed by the base complementrity combination that makes maximum quantity and hybridize.In addition, thus even 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 that is used to prepare the oligomer DNA chip.

[164]

[165] the present invention more specifically describes by following embodiment.Yet the present invention is not limited only to this.

[166]

Embodiment

[167] embodiment 1

[168] 5,11,17, the preparation of 23-tetramino cup [4] aromatic hydrocarbons

[169] [reaction formula 1]

[170]

[171] the light yellow solid product 5,11,17, and 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 obtains so that 75% productive rate is synthetic.Described reaction table is shown reaction formula 1.

[172]

[173] embodiment 2

[174] 5,11,17, the preparation of 23-tetrabenzyl imines-cup [4] aromatic hydrocarbons

[175] [reaction formula 2]

[176]

[177] with the TACA (1.03mmol) of 500mg and the anhydrous MgSO of 200mg ₄Put into dry round-bottomed flask, add the acetonitrile of 150ml and the phenyl aldehyde of 0.84ml (8.24mmol) simultaneously, under room temperature and nitrogen exchange, mixed two hours then.After the reaction, filter to remove solid product, then to solution decompression and remove residual solvent.Then at the CH that is dissolved in 5ml fully ₂Cl ₂After, to add the 30ml normal hexane and make 5,11,17,23-tetrabenzyl imines-cup [4] aromatic hydrocarbons (TBICA) crystallization is the lightpink solid.Filtration under diminished pressure and decompression are removed residual solvent and are obtained TBICA (822mg, productive rate 95.7%).Described reaction table is shown reaction formula 2.

[178] 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)

[179] ¹³C NMR(300MHz，CDCl ₃)：159.26，146.28，136.40，131.32，t128.86，121.90，32.47

[180]

[181] embodiment 3

[182] 5,11,17, the preparation of 23-tetrabenzyl imines alkoxyl group-cup [4] aromatic hydrocarbons

[183] [reaction formula 3]

[184]

[185] with the TBICA (0.6mmol) of 500mg, anhydrous K ₂CO ₃(830mg, 6mmol) and sodium iodide (810mg 5.4mmol) puts into dry round-bottomed flask, adds the acetonitrile of 150ml, mixes under the nitrogen exchange then.After 10 minutes, (644mg 3.6mmol), rises to 80 ℃ with the temperature of reaction in the reaction vessel then to add 6-bromine n-hexyl aldehyde.Then mixture was stirred 24 hours.After the reaction, acetonitrile is removed in decompression, and uses 150ml CH ₂Cl ₂The solubilizing reaction product.Since the KBr that obtains in the reaction process, KI, K ₂CO ₃Do not dissolve Deng material, they can be removed by filtration under diminished pressure.Solvent is removed in the filtered soln decompression also to be dissolved in the ethyl acetate of 5ml fully.If add the normal hexane of 40ml then, 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 the TBICOCA crystallization obtain light yellow solid product (570mg, productive rate 90%).Described reaction table is shown reaction formula 3.

[186] 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)

[187] 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

[188]

[189] embodiment 4

[190] 5,11,17, the preparation of 23-tetrabenzyl imines-bromine butoxy-cup [4] aromatic hydrocarbons

[191] [reaction formula 4]

[192]

[193] with TBICA (500mg, 0.6mmol), anhydrous K ₂CO ₃(830mg, 6mmol) and sodium iodide (1.08g 7.2mmol) puts into dry round-bottomed flask, adds the acetonitrile of 150ml, at room temperature mixes then 10 minutes.Add 1 to reaction vessel, (1.3g, 0.72ml 6mmol) and with the temperature in the reaction vessel rise to 80 ℃ to the 4-dibromobutane.Then with mixture reaction 24 hours.After reaction vessel was cooled to room temperature, solvent was removed in decompression, and bottom product was dissolved in the CH of 150ml ₂Cl ₂In.Since the KBr that obtains in the reaction process, KI, K ₂CO ₃Do not dissolve Deng material, they can be removed by filtration under diminished pressure, and solvent is removed in the filtered soln decompression.Behind 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 the solid recrystallization to obtain light yellow TBBCA (480mg, productive rate 72%).Described reaction table is shown reaction formula 4.

[194] 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)

[195]

[196] embodiment 5

[197] 5,11,17, the preparation of 23-tetrabenzyl imines sulfydryl butoxy cup [4] aromatic hydrocarbons

[198] [reaction formula 5]

[199]

[200] with TBBCA (500mg, 0.45mmol) and thioacetic acid potassium (114.21mg 1mmol) puts into dry round-bottomed flask, and is dissolved in the 60ml acetone, and the sound wave reaction is 90 minutes under the exchange of room temperature and nitrogen.After the reaction, solvent is removed in decompression, and is dissolved in the CH of 30ml ₂Cl ₂In.The undissolved throw out of filtration under diminished pressure then with water washing filtered soln twice, separates organic layer and with MgSO ₄Dry.The organic layer filtration under diminished pressure is obtained solid then with the filtered soln drying under reduced pressure and use ethyl acetate/normal hexane recrystallization, and filtration under diminished pressure obtains the light yellow solid crystal.The solid crystal of gained is put into round-bottomed flask, and be dissolved in CH ₂Cl ₂: in the mixing solutions of methyl alcohol=5: 1, sound wave reaction under room temperature and nitrogen exchange.After 1 minute, (1ml, 1mmol), sound wave reacted 30 minutes to add 1.0M KOH.After the reaction, solvent is removed in decompression, is dissolved in the CH of 5ml then ₂Cl ₂In and with 0.1M HCl solution washing once.Separate organic layer and with MgSO ₄After the drying, 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.

[201] 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)

[202]

[203] embodiment 6

[204] preparation of imines Calixarene Derivatives individual layer

[205] the synthetic TBICOCA that obtains among the embodiment 3 is dissolved in as CHCl ₃Deng preparation concentration in the organic solvent is the solution of 0.1-5mM.As shown in Figure 1, the slide (amine chip) that has connected the amine functions group was put into solution 1-24 hour for preparing, taken out then and with chloroform, acetone and water washing, dry then, to prepare imines calixarene individual layer of the present invention.Other imines Calixarene Derivatives individual layer is with reference to identical method preparation.

[206]

[207] embodiment 7

[208] preparation of imines Calixarene Derivatives individual layer on the gold substrate

[209] the synthetic TBIMBCA that obtains among the embodiment 5 is dissolved in as CHCl ₃Deng preparation concentration in the organic solvent is the solution of 0.1-5mM.As shown in Figure 2, gold substrate was put into solution 1-24 hour for preparing, taken out then and with chloroform, acetone and water washing, dry then, 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 various forms be used, but generally speaking, preferably at glass, molten silicon, silicon wafer, plastic-substrates etc. after the vacuum plating is with the chromium (Cr) of 5-20nm or titanium materials such as (Ti) with the substrate of the thickness vacuum metallizing of 100-300nm.Prepared gold substrate is prepended to Piranha solution (strength sulfuric acid: about 10 second-1 minute the mixing solutions of 30% hydrogen peroxide=3: 1) be about to using, with water washing and dry under the nitrogen exchange, the formation of promptly using described individual layer of existing side by side can be analyzed by infrared rays external reflection spectrum (FTIR-ERS) then.

[210]

[211] embodiment 8

[212] discern the fixedly method of oligomer DNA by polymolecular

[213] in oligomer DNA shown in Figure 3 is fixing, the microarray equipment of Genetics (Britain) and the microarray equipment of Proteogen Co. (Korea S) have been adopted.The oligomer DNA that will have 9 continuous guanine bases is dissolved in the BMT spotting solution with the preparation fixed solution with 6.75pmol/ml.Use the array contact pin to apply imines Calixarene Derivatives single-glass substrate (slide) with described fixed solution as shown in Figure 1 and use oligomer DNA is fixed in the point of diameter as 150-180 μ m with the amount of the described identical method of embodiment described 6 with 1-5nL.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, dry then.Be the fixing site of the oligomer DNA that seals other, the slide after the washing is put into the BMT lock solution of 250ml and handled 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 carry out drying.

[214]

[215] embodiment 9

[216] by the only irreversible method that fixedly has the oligomer DNA of 9 continuous guanine bases of polymolecular identification

[217] in oligomer DNA shown in Figure 4 is fixing, adopted the microarray equipment of Genetics (Britain).For determining as 6 kinds of different fixed function group 9A of table 1 hereinafter, 9G, 9C, 9T, no, the fixed efficiency of vitamin H etc. is dissolved in each oligomer DNA in the BMT spotting solution with the preparation fixed solution with 6.75pmol/ml.Use the array contact pin to apply imines Calixarene Derivatives single-glass substrate (slide) with described fixed solution as shown in Figure 1 and use oligomer DNA is fixed in the point of diameter as 150-180 μ m with the amount of the described identical method of embodiment described 6 with 1-5nL.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, dry then.Be the fixing site of the oligomer DNA that seals other, the slide after the washing is put into the BMT lock solution of 250ml and handled 30 minutes.The BMT Wa-A-1 solution washing slide of 500ml is 3 minutes then, and with twice of BMT Wa-A-2 solution washing.Remove moisture then carrying out drying, and prepare oligomer DNA chip shown in Figure 4.It is then, stable that to connect a kind of diameter be that the cell of 0.8cm is to hybridize.

[218] table 1

[219]

[220], can in the 1.5ml pipe, prepare the target DNA of 2 μ l band 2pmol/ μ l fluorescence and the mixing solutions of 58 μ lBMT hyb-mixA in order to hybridize with the target DNA of following band fluorescence.Mixing solutions was heated 3 minutes in 100 ℃ of water, and be placed on ice 3 minutes, the mixing solutions of 60 μ l is put into the slide that has connected hybridization chamber to cool off.Then, in the baking oven of fixed temperature and humidity, slide was hybridized 30 minutes down at 50 ℃.(4xSSC, 0.1%SDS) slide of solution washing through hybridizing is 2 minutes, and at room temperature washs 2 times with BMT Wa-B-2 (4xSSC) solution with BMT Wa-B-1 in 30 ℃ thermostatted.After the drying, with microarray scanner (GSI Lumonics, the U.S.) quantitative analysis fluorescence sensitivity.Actual result is presented among Fig. 5.Described result shows that the oligomer DNA with 9 guanines fixes with high-density, and that other base does not influence is fixing.

[221]

[222] embodiment 10

[223] the efficient comparative approach by the irreversible oligomer DNA that fixedly has a 1-5 continuous guanine base of polymolecular identification the time

[224] in the oligomer DNA that Fig. 6 shows is fixing, adopted the microarray equipment of Genetics (Britain).For determining as 6 kinds of different fixed function group 1G of table 2 hereinafter, 4G, 7G, 9G, 12G, the fixed efficiency of 15G etc. is dissolved in each oligomer DNA in the BMT spotting solution with the preparation fixed solution with 6.75pmol/ml.Use the array contact pin to apply imines Calixarene Derivatives single-glass substrate (slide) with described fixed solution as shown in Figure 1 and use oligomer DNA is fixed in the point of diameter as 150-180 μ m with the amount of the described identical method of embodiment described 6 with 1-5nL.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, dry then.Be the fixing site of the oligomer DNA that seals other, the slide after the washing is put into the BMT lock solution of 250ml and handled 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.Prepare oligomer DNA chip shown in Figure 4 after the drying.It is then, stable that to connect a kind of diameter be that the cell of 0.8cm is to hybridize.

[225] table 2

[226]

[227] for embodiment 9 in used identical band fluorescent DNA hybridize, can in the 1.5ml pipe, prepare the target DNA of 2 μ l band 2pmol/ μ l fluorescence and the mixing solutions of 5 μ l BMThyb-mixA.Mixing solutions was heated 3 minutes in 100 ℃ of water, and be placed on ice 3 minutes, the mixing solutions of 60 μ l is put into the slide that has connected hybridization chamber to cool off.Then, in the baking oven of fixed temperature and humidity, slide was hybridized 30 minutes down at 50 ℃.(4xSSC, 0.1%SDS) slide of solution washing through hybridizing is 2 minutes, and at room temperature washs 2 times with BMT Wa-B-2 (4xSSC) solution with BMT Wa-B-1 in 30 ℃ thermostatted.After the drying, 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 fix needs 7 continuous guanine bases at least.In addition, they show that 9 continuous guanine bases demonstrate the highest constant density.

[228]

[229] embodiment 11

[230] by verifying that with the competitive reaction of imidazoles the polymolecular identification of oligomer DNA is fixing

[231] in the oligomer DNA that Fig. 7 shows is fixing, adopted the microarray equipment of Genetics (Britain).Base sequence is 5 '-oligomer DNA of GGG GGG GGG AAA TCA ACCCAC AGC TGC A-3 ' (SEQ ID NO.1) is dissolved in the BMT spotting solution with the preparation fixed solution with 6.75pmol/ml.Use the array contact pin to apply imines Calixarene Derivatives single-glass substrate (slide) with described fixed solution as shown in Figure 1 and use oligomer DNA is fixed in the point of diameter as 150-180 μ m with the amount of the described identical method of embodiment described 6 with 1-5nL.The different concns imidazoles of forming with concentration shown in Figure 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 and dry.Be the fixing site of the oligomer DNA that seals other, the slide after the washing is put into the BMT lock solution of 250ml and handled 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.Prepare oligomer DNA chip shown in Figure 4 after removing moisture and drying.It is then, stable that to connect a kind of diameter be that the cell of 0.8cm is to hybridize.

[232]

[233] for embodiment 9 in used identical band fluorescent DNA hybridize, can in the 1.5ml pipe, prepare the target DNA of 2 μ l band 2pmol/ μ l fluorescence and the mixing solutions of 58 μ l BMThyb-mixA.Mixing solutions was heated 3 minutes in 100 ℃ of water, and be placed on ice 3 minutes, the mixing solutions of 60 μ l is put into the slide that has connected hybridization chamber to cool off.Then, in the baking oven of fixed temperature and humidity, slide was hybridized 30 minutes down at 50 ℃.(4xSSC, 0.1%SDS) slide of solution washing through hybridizing is 2 minutes, and at room temperature washs 2 times with BMT Wa-B-2 (4xSSC) solution with BMT Wa-B-1 in 30 ℃ thermostatted.After the drying, with microarray scanner (GSI Lumonics, the U.S.) analysis of fluorescence sensitivity.Actual result as shown in Figure 7.Described result shows that imidazoles has suppressed continuous guanine by polymolecular identification and fixed, and carries out the fixing of oligomer DNA when having comprised the imidazoles of 100mM at least hardly.That is to say that they have shown that imidazoles is at war with by molecular recognition, the fixed rate with oligomer DNA of 9 continuous guanine bases descends to some extent.

[234]

[235] be used for fixing in embodiment 8-11, the solution composition of purposes such as cleaning is as follows:

[236] BMT spotting solution (4xSSC, 15% glycerol, 1x PBS)

[237]BMT Wa-A-1(2xSSC，0.1％SDS)

[238]BMT Wa-A-2(0.1x SSC)

[239] BMT lock solution (5% milk casein solution)

[240] BMT hyb-mixA (4xSSC, 0.1%SDS, 50% glycerol, 1x PBS)

[241]BMT Wa-B-1(4xSSC，0.1％SDS)

[242]BMT Wa-B-2(4xSSC)

[243]

[244] embodiment 12

[245] 5,11,17,23-tetramino cup [4] aromatic hydrocarbons synthetic

[246] [reaction formula 6]

[247]

[248] the light yellow solid product 5,11,17, and 23-tetramino cup [4] aromatic hydrocarbons (TACA) can 5,11,17, and 23-tetranitro cup [4] aromatic hydrocarbons (TNCA) obtains so that 75% productive rate is synthetic with reference to following reference 3 described synthetic methods as starting raw material.

[249]

[250] embodiment 13

[251] 5,11,17, the amino cup of 23-four (2, the 4-dimethyl) dibenzyl [4] aromatic hydrocarbons synthetic

[252] [reaction formula 7]

[253]

[254] with bar magnet, TACA (1.0g, 2.05mmol) and sodium iodide (3.0g 20.5mmol) puts into the exsiccant round-bottomed flask, with the mixture drying under reduced pressure.Dry back adds the anhydrous acetonitrile of 50ml, then it is mixed under nitrogen exchange in well heater.After 5 minutes, with 2, (16.2g 82mmol) puts into reaction vessel to 4-dimethyl benzyl bromine, heats up then and stirs.(6.62ml 82mmol), made mixture reaction 6 hours to add pyridine then.After the reaction, make reaction vessel be cooled to room temperature, the solvent decompression is removed.Then, be dissolved in the CH of 150ml ₂Cl ₂After, with twice of water washing organic layer.With dried organic layer filtration under diminished pressure, and filtered soln is removed in decompression.Then with CH ₂Cl ₂/ MeOH recrystallization to be obtaining the light gray solid product 5,11,17 of 2.1g, and the amino cup of 23-four (2, the 4-dimethyl) dibenzyl [4] aromatic hydrocarbons (2,4-DMTDBACA) (productive rate 80%).

[255] 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)

[256]

[257] embodiment 14

[258] 5,11,17,23-four (2, the 4-dimethyl) benzimide-cup [4] aromatic hydrocarbons synthetic

[259] [reaction formula 8]

[260]

[261] (100mg 0.2mmol) puts into the exsiccant round-bottomed flask with magnetic stripe with TACA.Add acetonitrile and the mixing of 15ml to reaction vessel.Add 2, the 4-dimethylbenzaldehyde (322mg, 2.4mmol) after, mixing is 2 hours under nitrogen exchange and room temperature.After the reaction, with the mixture filtration under diminished pressure, and solvent is removed in decompression.Then with the CH of reactants dissolved in appropriate amount ₂Cl ₂In, the normal hexane that adds 15ml then is to obtain the light brown solid product.This product is dissolved in 3mlCH once more ₂Cl ₂In, and add the bright light brown solid product 5,11,17 of the normal hexane of 15ml with acquisition 155mg, and 23-four (2, the 4-dimethyl) benzimide-cup [4] aromatic hydrocarbons (2,4-DMICA) (productive rate 81%).

[262] 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)

[263]

[264] embodiment 15

[265] 5,11,17, the amino cup of 23-four (2, the 4-dimethyl) benzyl [4] aromatic hydrocarbons synthetic

[266] [reaction formula 9]

[267]

[268] with 2, (100mg 0.1mmol) puts into the exsiccant round-bottomed flask with magnetic stripe to 4-DMICA, and drying under reduced pressure.Add anhydrous THF and the mixing under the nitrogen exchange of 20ml then.After 10 minutes, add solution 1.0MBH ₃(0.8ml 0.8mmol), makes mixture at room temperature react 3 hours to/THF solution then.After the reaction, solvent is removed in decompression, and the product that will remain in the flask is dissolved in CH ₂Cl ₂In.Again with behind twice of the 0.1M HCl solution washing, with twice of distilled water wash.Organic layer is separated and drying, and filtered soln is removed in filtration under diminished pressure and decompression.Then with CH ₂Cl ₂/ hexane recrystallization to be obtaining the light yellow solid product 5,11,17 of 72mg, and the amino cup of 23-four (2, the 4-dimethyl) benzyl [4] aromatic hydrocarbons (2,4-TDMBACA) (productive rate 75%).

[269] 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)

[270]

[271] embodiment 16

[272] 5,11,17, the amino cup of 23-four (2,4-dimethyl tetrabenzyl) benzyl [4] aromatic hydrocarbons synthetic

[273] [reaction formula 10]

[274]

[275] with bar magnet and 2,4-TDMBACA (500mg, 0.5mmol) and sodium iodide (80mg 0.5mmol) puts into the exsiccant round-bottomed flask, and drying under reduced pressure.Dry back adds the anhydrous acetonitrile of 50ml, then it is mixed under nitrogen exchange in well heater.After 5 minutes, (1.1ml, 10mmol) also exchange mixes to add bromotoluene to reaction vessel.(0.8ml 10mmol), and reacted 6 hours to add pyridine to reaction vessel.After the reaction, make reaction vessel be cooled to room temperature, solvent is removed in decompression then.Then it is dissolved in the CH of 60ml ₂Cl ₂In and with water washing, dry then organic layer and filtration under diminished pressure.Filtered soln is removed in decompression then, and with CH ₂Cl ₂/ MeOH recrystallization is to obtain the light gray solid product 5,11,17 of 521mg, 23-four (2,4-dimethyl tetrabenzyl) benzyl amino-cup [4] aromatic hydrocarbons (TB (2,4-DMTB) ACA) (productive rate 79%).

[276] 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)

[277]

[278] embodiment 17

[279] 5,11,17, the amino cup of 23-four (2, the 4-dimethyl) dibenzyl [4]-aromatic hydrocarbons-1,3-hexanal synthetic

[280] [reaction formula 11]

[281]

[282] successively with bar magnet and 2, and 4-DMTDBACA (500mg, 0.35mmol), K ₂CO ₃(487mg, 3.5mmol), (473mg 3.15mmol) puts into the exsiccant round-bottomed flask to sodium iodide, adds the anhydrous acetonitrile of 130ml at nitrogen exchange downhill reaction container then, and mixes in well heater.Add 6-bromine hexanal (376mg, 2.1mmol) after, exchange mixed 16 hours.After the reaction, reactant is cooled to room temperature, and solvent is removed in decompression.With the CH of reactants dissolved in 130ml ₂Cl ₂After, its filtration under diminished pressure and decompression are removed filtered soln.Use MeOH/CH then ₂Cl ₂Recrystallization to be obtaining the light yellow solid product 5,11,17 of 432mg, the amino cup of 23-four (2, the 4-dimethyl) dibenzyl [4]-aromatic hydrocarbons-1, the 3-hexanal (2,4-DMTDBACAHA) (productive rate 76%).

[283] ¹H NMR(300MHz，CDCl ₃)；9.78(s，2H，CHO)，7.317.04(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)

[284]

[285] embodiment 18

[286] 5,11,17, amino bromo-butoxy cup [4] aromatic hydrocarbons of 23-four (2,4-dimethyl tetrabenzyl) benzyl synthetic

[287] [reaction formula 12]

[288]

[289] with 2, and 4-TDMBACA (500mg, 0.5mmol) and anhydrous K ₂CO ₃(619mg, 5.0mmol) and sodium iodide (674mg 4.5mmol) puts into dry round-bottomed flask.The anhydrous acetonitrile that adds 160ml to reaction vessel also at room temperature mixed 15 minutes.Add 1 to reaction vessel, (5.0mmol) also exchange mixed 20 hours the 4-dibromobutane for 1.03g, 0.6ml.After reaction vessel is cooled to room temperature, the solvent decompression is removed.Then it is dissolved in CH ₂Cl ₂In and filtration under diminished pressure.Filtered soln, recrystallization in the EA/ hexane are removed in decompression.Filtration under diminished pressure to be obtaining the light brown solid product of 610mg then, and 5,11,17, amino bromine butoxy cup [4] aromatic hydrocarbons of 23-four (2,4-dimethyl tetrabenzyl) benzyl (TB (2,4-DMTB) ABCA) (productive rate 77%).

[290] 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)

[291]

[292] embodiment 19

[293] 5,11,17,23-four (2,4-dimethyl tetrabenzyl) benzyl amino mercapto-butoxy cup [4] aromatic hydrocarbons synthetic

[294] [reaction formula 13]

[295]

[296] with TB (2,4-DMTB) ABCA (500mg, 0.31mmol) and thioacetic acid potassium (212mg 1.86mmol) puts into dry round-bottomed flask.Dissolve the anhydrous propanone of 60ml then therein and sound wave reaction 90 minutes under the exchange of room temperature and nitrogen.After the reaction, solvent is removed in decompression, and with the CH of 30ml ₂Cl ₂Dissolving.Behind the filtration under diminished pressure, filtered soln is also separated and dry organic layer for twice with water washing.With the organic layer filtration under diminished pressure and with the solid ethyl acetate/normal hexane recrystallization that obtains behind the filtered soln drying under reduced pressure, and filtration under diminished pressure obtains the light yellow solid crystal.The solid crystal of gained is put into round-bottomed flask and is dissolved in CH ₂Cl ₂: in the mixing solutions of methyl alcohol=5: 1, sound wave reaction under room temperature and nitrogen exchange.After 1 minute, (1.5ml, 1.5mmol), sound wave reacted 30 minutes to add 1.0M KOH.After the 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 ₂Behind/MeOH the recrystallization, can obtain the light yellow 5,11,17 of 320mg, 23-four (2, the 4-methoxyl group) benzyl amino mercapto butoxy cup [4] aromatic hydrocarbons (TB (2,4-DMTB) AMBCA) (productive rate 73%).

[297] ¹H NMR(300MHz，CDCl ₃)；7.84(s，2H，ArOH)，7.297.01(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)

[298]

[299] embodiment 20

[300] preparation of amino Calixarene Derivatives individual layer shown in Figure 8

[301] with among the embodiment 17 synthetic obtain 2,4-DMTDBACAHA is dissolved in as CHCl ₃Deng preparation concentration in the organic solvent is the solution of 0.1-5mM.As shown in Figure 8, the slide (amine chip) that has connected the amine functions group is put into solution 1-24 hour and the taking-up for preparing, respectively with chloroform, acetone and water washing are also dry.Prepare the amino Calixarene Derivatives individual layer (A type BMT oligomer DNA chip) of Fig. 8 then.In that being put into, above-mentioned A type contains BH ₃-THF or NaBH ₄The MeOH reductive agent in after 1-10 minute, water (deionized water) washing three times, dry amino Calixarene Derivatives individual layer (Type B BMT oligomer DNA chip) in nitrogen atmosphere with preparation embodiment 8.

[302]

[303] embodiment 21

[304] preparation of amino Calixarene Derivatives individual layer on the gold substrate shown in Figure 9

[305] (2,4-DMTB) AMBCA is dissolved in as CHCl with the synthetic TB that obtains among the embodiment 19 ₃Deng preparation concentration in the organic solvent is the solution of 0.1-5mM.As shown in Figure 9, gold substrate is put into solution 1-24 hour and the taking-up for preparing, respectively with chloroform, acetone and water washing are also dry.Prepare amino Calixarene Derivatives individual layer shown in Figure 9 then.Other amino Calixarene Derivatives individual layer is with reference to identical method preparation.Described gold substrate can various forms be used, but generally speaking, preferably at glass, molten silicon, silicon wafer, plastic-substrates etc. after the vacuum plating is with the chromium (Cr) of 2-10nm or titanium materials such as (Ti) with the substrate of the thickness vacuum metallizing of 50-200nm.With prepared gold substrate be about to use be prepended to Piranha solution (strength sulfuric acid: about 10 second-1 minute the mixing solutions of 30% hydrogen peroxide=3: 1), with water washing and dry under the nitrogen exchange, exist side by side and promptly use then.The formation of described individual layer can be analyzed by infrared rays external reflection spectrum (FTIR-ERS).

[306]

[307] embodiment 22-25 solutions employed, oligomer DNA, the oligomer DNA of band fluorescence, and c-DNA base sequence

[308] table 3

Embodiment 22-25 solutions employed

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
		The 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

[309]

[310] table 4

Best base number

[311]

[312] table 5

The SNP that is used to test

[313]

[314] table 6

Be used for competitive reaction

[315]

[316] table 7

Be used for the maximum density test

	Sequence	Remarks
				9G
	5′-GGG GGG GGG AAA TCA ACC CAC AGC TGC 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

[317]

[318] table 8

The composition (ul) that is used for 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 NH 4Cl 4	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

[319]

[320] embodiment 22

[321] measure by the irreversible best guanine base quantity that is fixed on the amino Calixarene Derivatives individual layer of Figure 12 of polymolecular identification

[322]-oligomer DNA is identified in fixing on the amino Calixarene Derivatives individual layer by polymolecular

[323] be used to measure that to be identified in the sample solution of fixed guanine quantity on the amino Calixarene Derivatives individual layer and concentration composition etc. by polymolecular as shown in table 3.At first, 10ul had the hereinafter 1G of table 4 respectively, 4G, 7G, 9G, 12G, 15G etc. continuously each oligomer DNA (100pmol/ul) of guanine base and 148ul BMT spotting solution 3 mixing solutions use microarray (Genetix Qarray2) thus with the amount of 1-5nL with solution be coated in order on the amino Calixarene Derivatives individual layer of fixing oligomer DNA with the diameter of 150-180um under the room temperature it being fixed 1 hour respectively in fixed chamber, 2 hours, 4 hours.After fixing, for taking out residual oligomer DNA, (2xSSC 0.1%SDS) at room temperature washed amino Calixarene Derivatives individual layer 1 minute with BMT Wa-A-1, at room temperature sealed 30 minutes then with BMT Wa-A-2 (0.1xSSC) washing, and with BMT lock solution (5% milk casein solution).(4xSSC, 0.1%SDS) the amino Calixarene Derivatives individual layer after the carrying out washing treatment 3 minutes at room temperature is then with the wash bottle washing of BMT Wa-A-2 (0.1XSSC) and dry with BMT Wa-B-1.

[324]

[325]-hybridize with the DNA that has fluorescence (Cy3-DNA)

[326], hybridization chamber is connected to fixed oligomer DNA and dried amino Calixarene Derivatives individual layer in order to hybridize with the Cy3-DNA that has fluorescence.Then, the BMT hyb-mixA (4xSSC, 0.002%SDS, 50% glycerol, 1x PBS) of the specified Cy3-DNA of 2ul table 4 (5nmol/ml) and 58ul was mixed in the water that is incorporated in 100 ℃ heating 3 minutes.It was placed on ice 3 minutes, apply 60ul, in 50 ℃ fixed temperature and humidity incubator, hybridized 30 minutes then with micropipet.After the hybridization, for cleaning amino Calixarene Derivatives individual layer, (4xSSC 0.1%SDS) washed 2 minutes down at 30 ℃ with BMT Wa-B-1.Then, at room temperature twice with BMT Wa-B-2 (4xSSC) washing 2 minutes and use fluorescent scanning instrument (GSI Lumonics, the U.S.) analysis of fluorescence sensitivity.Actual result is shown in Figure 12.Described result's demonstration requires 7 guanine bases at least when oligomer DNA is fixed on the amino Calixarene Derivatives individual layer by polymolecular identification, and fixes with maximum density when using 9 guanine bases.

[327]

[328] embodiment 23

[329] determine monokaryon glycosides polymorphism (SNP by the optimal spatial layout of Figure 14 and Figure 15; Single Nucleotide Polymorphism) test of Jian Bieing

[330]-definite SNP on amino Calixarene Derivatives individual layer

[331] can the mixing solutions of the BMT spotting solution 1 of the oligomer DNA s (100pmol/ul) of a table 6 that base differs from one another of the only same loci of 9.1ul and 35.9ul be coated with the thickness of 150-180um and the amount of 1-5nl by using microarray (Genetix Qarray2) earlier being used for fixing on the amino Calixarene Derivatives individual layer of oligomer DNA the test of determining SNP, at room temperature in fixed chamber, fix 3 hours then.After fixing, for taking out residual oligomer DNA, (2xSSC 0.1%SDS) at room temperature washed amino Calixarene Derivatives individual layer 1 minute with BMT Wa-A-1, at room temperature sealed 30 minutes then with BMT Wa-A-2 (0.1x SSC) washing, and with BMT lock solution (5% milk casein solution).(4xSSC, 0.1%SDS) the amino Calixarene Derivatives individual layer after the carrying out washing treatment 3 minutes at room temperature is then with the wash bottle washing of BMT Wa-A-2 (0.1XSSC) and dry with BMT Wa-B-1.

[332]

[333]-hybridize the DNA (Cy3-DNA) that has fluorescence with detection with C-DNA

[334] for hybridizing, after hybridization chamber was connected to individual layer, (50% glycerol 1xPBS) was mixed to be incorporated in 100 ℃ of water and is heated for 4xS SC, 0.002%SDS with the BMT hyb-mixA of specified each C-DNA (5nmol/ul) in the 2ul table 6 and 28ul.Placed 3 minutes and cooling on ice, use micropipet to apply 30ul then, hybridization is 30 minutes in 55 ℃ of Constant Temperature Incubators.After the hybridization, for cleaning amino Calixarene Derivatives individual layer, (4xSSC 0.1%SDS) 30 ℃ of washings 2 minutes down, at room temperature washed 2 minutes with BMTWa-B-2 (4xSSC) for twice then with BMTWa-B-1.Dry then and prepare can with the oligomer DNA individual layer of band fluorescent DNA (Cy3-DNA) hybridization.

[335]

[336]-C-DNA and the hybridization that has the Cy3-DNA of fluorescence

[337] be with the Cy3-DNA of fluorescence and the hybridization of finishing the individual layer of hybridization with C-DNA, at first, connect hybridization chamber, then with the Cy3-DNA (5nmol/ml) of 2ul table 6 and the BMT hyb-mixA (4xSSC of 28ul, 0.002%SDS, 50% glycerol, 1x PBS) mix to be incorporated in 100 ℃ of water and heated 3 minutes.Placed on ice 3 minutes and drying after, use micropipet to apply 30ul, then in hybridization 30 minutes in the constant humidity incubator under 45 ℃.After the hybridization, for cleaning amino Calixarene Derivatives individual layer, (4xSSC 0.1%SDS) 30 ℃ of washings 2 minutes down, at room temperature washed 2 minutes with BMT Wa-B-2 (4xSSC) for twice and dry then with BMT Wa-B-1.Use fluorescent scanning instrument (GSI Lumonics, the U.S.) to confirm that by fluorescence sensitivity SNP differentiates then.This results are shown in Figure 14 and 15.This result is presented at not combination in hybridization of other DNAs on the site that oligomer DNA with different bases is fixed.Therefore, this result shows that as described in the present invention technology can be by being provided with suitable space between the oligomer DNA that is fixed, thereby realizes that by the optimal spatial layout between this oligomer DNA s fast accurate SNP differentiates.

[338]

[339] embodiment 24

[340] Figure 10 and 11 high-density restraint test

[341]-oligomer DNA fixes

[342] on the amino Calixarene Derivatives individual layer fixedly the method for oligomer DNA can be used for fixing on the amino Calixarene Derivatives individual layer of oligomer DNA the mixing solutions that amount with 1-5nl applies the MBT spotting solution 1 of the oligomer DNA (100pmol/ul) of 9.1ul table 7 and 35.9ul by using microarray (Genetix Qarray2), at room temperature be fixed in fixed chamber then 3 hours.After fixing, for taking out residual oligomer DNA, (2xSSC 0.1%SDS) at room temperature washed amino Calixarene Derivatives individual layer 1 minute with BMT Wa-A-1, at room temperature sealed 30 minutes then with BMT Wa-A-2 (0.1x SSC) washing, and with BMT lock solution (5% milk casein solution).(4xSSC, 0.1%SDS) the amino Calixarene Derivatives individual layer after the carrying out washing treatment 3 minutes at room temperature is then with the wash bottle washing of BMT Wa-A-2 (0.1XSSC) and dry with BMT Wa-B-1.

[343]

[344]-hybridize with the Cy3-DNA that has fluorescence

[345] for hybridizing, at first, connect hybridization chamber with the Cy3-DNA of band fluorescence, then with the Cy3-DNA (5nmol/ml) of 2ul table 7 and the BMT hyb-mixA (4xSSC of 28ul, 0.002%SDS, 50% glycerol, 1x PBS) mix back heating and be positioned over cooled on ice in 100 ℃ of water.Then, use micropipet to apply 30ul, and in 50 ℃ of incubators, hybridized 30 minutes.After the hybridization, for cleaning amino Calixarene Derivatives individual layer, (4xSSC 0.1%SDS) 30 ℃ of washings 2 minutes down, at room temperature washed 2 minutes with BMT Wa-B-2 (4xSSC) for twice and dry then with BMT Wa-B-1.Use the fluorescent scanning instrument that fluorescence sensitivity and theoretical fluorescence sensitivity are compared and analyze then.

[346]

[347]-dry a certain amount of band fluorescence c-DNA on chip base, and itself and theoretical maximum compared and analyze

[348] by microarray (Genetix Qarray2) with the band fluorescence c-DNA of table 7 with 1-5nl, 1/2 theoretical maximum (0.675fmol/nl) uses the fluorescent scanning instrument to analyze after being coated to the inferior biological and drying of amino calixarene.The results are shown in Figure 10 and 11 of theoretical fluorescence sensitivity and the comparison of test fluorescence sensitivity, use the result of the band fluorescence Cy3-DNA of theoretical maximum half level it seems with by coming to the same thing shown in the hybridization fixed oligomer DNA.Therefore, this result shows that the oligomer DNA near theoretical maximum is fixed.

[349]

[350] embodiment 25

[351] by with the 9A of Figure 16,9T, 9G, the competitive reaction of 9C-DNA is to the only irreversible fixed validation test of 9 guanine bases

[352] being used for the oligomer DNA of the competitive reaction composition shown in can employing table 7 is prepared.At this moment, the different DNAs that are used for competitive reaction are put into table 8 and with 0 times, and 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 for preparing by method shown in the 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, (2xSSC 0.1%SDS) at room temperature washed amino Calixarene Derivatives individual layer 1 minute with BMT Wa-A-1, at room temperature sealed 30 minutes then with BMT Wa-A-2 (0.1x SSC) washing, and with BMT lock solution (5% milk casein solution).(4xSSC, 0.1%SDS) the amino Calixarene Derivatives individual layer after the carrying out washing treatment 3 minutes at room temperature is then with the wash bottle washing of BMT Wa-A-2 (0.1XSSC) and dry with BMT Wa-B-1.

[353]

[354]-hybridize with the Cy3-DNA that has fluorescence

[355] for hybridizing with the Cy3-DNA of band fluorescence, at first, connect hybridization chamber, then with the Cy3-DNA (10nmol/ml) of 10ul table 8 and the BMT hyb-mixA (4xSSC of 590ul, 0.002%SDS, 50% glycerol, 1x PBS) mix back use micropipet coating 600ul, in 50 ℃ of incubators, hybridized 30 minutes then.After the hybridization, for cleaning amino Calixarene Derivatives individual layer, (4xSSC 0.1%SDS) 30 ℃ of washings 2 minutes down, at room temperature washed 2 minutes with BMT Wa-B-2 (4xSSC) for twice and dry then with BMT Wa-B-1.Use fluorescent scanning instrument (GSI) to confirm fluorescence sensitivity then.Described the results are shown among Figure 16.This result only show 9 guanines by polymolecular identification by irreversible fixing and though whether other base continuously all can be to the fixing considerable influence that produces.This result has shown the technology of preparing that can obtain the oligomer DNA chip of reproducible results of hybridization as the actual tests result, the base that wherein is used to hybridize does not participate in fixing and only the specific base sequence participation is fixing continuously, thereby keeps the essential base quantity of hybridization all the time.

[356]

Industrial usability

[357] the present invention is different from the traditional oligomer DNA fixing means that passes through Chemical bond or physical adsorption commonly used in the world fully, it is a kind of novel method that uses continuous guanine base irreversible fixedly oligomer DNA on solid substrate by molecular recognition notion (that is polymolecular identification).

[358] the invention solves by the high price oligomer DNA be connected the traditional chemical combined techniques in chemical reaction between the aldehyde slide (aldehyde chip base) of commonly used amine functions group and many problems of producing when fixedly oligomer DNA is with preparation oligomer DNA chip on the aldehyde chip base, anyly can prepare the oligomer DNA chip easily per capita thereby make.

In fixing step, be difficult to obtain reproducibility when [359] using the oligomer DNA that has connected the high price functional group to prepare the oligomer DNA chip, so the cycle of research and development is very long, and this point has stoped many biological products to turn to commercialization from research and development.In addition, when selecting to bring the oligomer DNA of best results of hybridization, because the burden of development costs had once been abandoned the product in many exploitations by testing hundreds and thousands of kinds of oligomer DNA base sequences.

[360] as shown in Figure 3, because the continuous guanine base among the present invention is fixed on the imines Calixarene Derivatives individual layer (BMT imines chip) by polymolecular identification, the density of fixed 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 being fixed on the formed space of substrate surface, should and substrate surface bonded c-DNA can and hybridize between the oligomer DNA that is fixed by freely entering.Accordingly, it for intensive advantage of having fixed traditional oligomer DNA chip of oligomer DNA be can several times or tens times speed hybridize, thereby support quick diagnosis.Be identified in by polymolecular by molecular recognition fixed oligomer DNA and be fixed to BMT imines chip in the solution, therefore can use oligomer DNA to prepare fixedly oligomer DNA chip of high-density, even the density of this oligomer DNA is the 1/3-1/10 of conventional levels.

[361] in addition, need 2-4 hour fixedly oligomer DNA in the present invention approximately, wash then and drying.Therefore, it can make the preparation of oligomer DNA chip become easy and quick.

[362] especially, in Fig. 4 and 6, oligomer DNA is by irreversible being fixed on the imines Calixarene Derivatives self-assembled monolayer of the identification of the polymolecular by 9 continuous guanine bases, and the fluorometric analysis result of hybridization shows that the fixing constant density of oligomer DNA is 5 to 30 times of traditional method.

[363] therefore, the comparable technology by the fixing preparation of traditional oligomer DNA oligomer DNA chip of the present invention prepares the 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 process method 1/3-1/5.Correspondingly, it can be applicable to various based in the DNA chip preparation of the present invention subsequently.

[364] in addition, as another aspect of the present invention, the present invention is that a kind of variety of issue that is presented in the existing DNA chip technology of preparing that can solve (for example, can't carry out high speed hybridization according to conventional art; Can't guarantee to diagnose the technology of monokaryon glycosides polymorphism (SNP); Owing to be difficult to keep as A, the shown results of hybridization of chemical bonding that C, the amine functions group in three bases such as G carry out between rolling into a ball with the aldehyde functional group of aldehyde chip base is difficult to obtain the reproducibility of DNA chip; Being difficult to form the uniform density that the oligomer DNA homogeneous is fixed on the preparation chip fixes) new technique.

[365] the invention provides a kind of amino Calixarene Derivatives that can the continuous guanine group of irreversible molecular recognition, with the technology of the oligomer DNA chip base of its self-assembled monolayer form preparation, by oligomer DNA chip and technology of preparing thereof in the irreversible fixedly oligomer DNA preparation of the spontaneous molecular recognition on chip base surface.As described shown in embodiment and the accompanying drawing, this technique for fixing can automatically suitably between base, keep with base continuously by irreversible when being fixed on the bottom this base be arranged suitable space, occupied space.This space is to be enough to make c-DNA freely to enter the space of chip base bottom in crossover process, therefore can hybridize at a high speed.Meanwhile, this is used for can maintaining maximum value all the time in conjunction with the base quantity of base during hybridization; Therefore, this is independent base difference is confirmed in a kind of hybridization by high degree of specificity on the on-off level a new technique.

[366] in addition, the invention provides a kind of fixedly oligomer DNA individual layer that the maximum density level is kept suitable space level simultaneously that has, wherein all kinds of oligomer DNAs are connected to solid substrate (substrate of glass (amine slide) or the gold substrate (gold thin film or gold) that for example have the amine functions group) without any processing and are fixed, that is to say, imine derivative provides the chip base as individual layer production that is used for fixing oligomer DNA (that is, producing the oligomer DNA chip).The present invention thereby provide and be used to prepare the necessary basic technology of oligomer DNA chip that in all solids substrate, has identical reproducible product.

[367] in addition, according to the present invention, oligomer DNA can pass through to fix under the state of a kind of new technique (that is, polymolecular identification) no clearance spaces in the aqueous solution.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 hybridization at a high speed simultaneously with the fixing new technique of oligomer DNA s of maximum density.Therefore, thus the invention provides a kind of have the maximum sensitivity level can be by the production technology of the oligomer DNA chip diagnosed of identification c-DNA under lower concentration.Simultaneously, because oligomer DNA is fixed with maximum density all the time, it provides the oligomer DNA chip has been prepared as the necessary production technology with oligomer DNA chip of reproducibility of product.The conventional chip base of using can be by having the oligomer DNA production of high price reactor, and be difficult in the fixing stage acquisition productivity of oligomer DNA.Therefore, the cycle of these research and development is very long, and in addition, this becomes the reason that these research and development can't proceed to the production phase.Can make production identification molecular engineering of the present invention more easily by adopting, and, can select to bring the oligomer DNA of maximum results of hybridization to reduce the research and development burden in the oligomer DNA chip development by the hundreds of oligomer DNA base sequence of checking.In addition, the invention provides and a kind ofly can release the new technique of best oligomer DNA chip fast immediately by obtaining productivity.If adopt the basic technology of production chip provided by the invention, can carry out the quick production and the commercialization of range gene chip.

[368] in addition, the present invention can only be fixedly 3 to 5 times oligomer DNA s measuring of oligomer DNA s of surface by using, thereby by the fixing oligomer DNA s production high-density oligomer DNA chip of molecular recognition.Therefore, the present invention only makes and to produce oligomer DNA s with the cost of traditional oligomer DNA chip production 1/10-1/100 and become possibility, thereby has saved a large amount of production costs.

[369] in addition, the present invention is different from the traditional oligomer DNA fixing means that passes through Chemical bond or physical adsorption commonly used in the world fully, it is a kind of novel method that uses continuous guanine base irreversible fixedly oligomer DNA on solid substrate by molecular recognition definition (that is polymolecular identification).

Sequence table

＜110〉Biometrix Technology Inc.

＜120〉novel imines Calixarene Derivatives and amino Calixarene Derivatives, its preparation method by the self-assembled monolayer of this method preparation, uses this self-assembled monolayer solid

Decide the method for oligomer DNA, and by the oligomer DNA chip of this method preparation

<130>F-280-PCT

<150>KR2005-0096322

<151>2005-10-13

<150>KR2005-0105340

<151>2005-11-04

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<211>32

<212>DNA

＜213〉artificial sequence

<220>

<223>SNP-A DNA

<400>24

gggggggggt ttacatagca tatcgaggtg gg 32

<210>25

<211>32

<212>DNA

＜213〉artificial sequence

<220>

<223>SNP-T DNA

<400>25

gggggggggt ttacatagca tttcgaggtg gg 32

<210>26

<211>32

<212>DNA

＜213〉artificial sequence

<220>

<223>SNP-G DNA

<400>26

gggggggggt ttacatagca tgtcgaggtg gg 32

<210>27

<211>32

<212>DNA

＜213〉artificial sequence

<220>

<223>SNP-C DNA

<400>27

gggggggggt ttacatagca tctcgaggtg gg 32

<210>28

<211>46

<212>DNA

＜213〉artificial sequence

<220>

<223>CN-A DNA

<400>28

aatcaaccca cagctgaaaa aacccacctc gaaatgctat gtggac 46

<210>29

<211>46

<212>DNA

＜213〉artificial sequence

<220>

<223>CN-T DNA

<400>29

aatcaaccca cagctgaaaa aacccacctc gatatgctat gtggac 46

<210>30

<211>46

<212>DNA

＜213〉artificial sequence

<220>

<223>CN-G DNA

<400>30

aatcaaccca cagctgaaaa aacccacctc gagatgctat gtggac 46

<210>31

<211>46

<212>DNA

＜213〉artificial sequence

<220>

<223>CN-C DNA

<400>31

aatcaaccca cagctgaaaa aacccacctc gacatgctat gtggac 46

Claims (24)

1. imines Calixarene Derivatives that has with following formula 1:

[formula 1]

(R wherein 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).

2. imines Calixarene Derivatives that has with following formula 2:

[formula 2]

(R wherein 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, wherein R representative-CH ₃Or-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 (n=2-15 wherein, m=1-10, c=0-10, Z=-SH ,-CHO ,-COOH or-NH ₂)).

3. one kind by 5,11,17 of following formula 3, the method for the aromatic aldehyde prepared in reaction imines Calixarene Derivatives as claimed in claim 1 of 23-tetramino cup [4] aromatic hydrocarbons and following formula 4:

[formula 3]

[formula 4]

(wherein R is selected from combination-H ,-CH ₃,-C ₂H ₅,-C ₃H ₇,-OCH ₃,-Cl ,-C ₆H ₅With-COOR ', wherein R ' representative-CH ₃Or-C ₂H ₅).

4. method by imines Calixarene Derivatives as claimed in claim 1 and dual-function compound prepared in reaction imines Calixarene Derivatives as claimed in claim 2, wherein said dual-function compound are selected from 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, n=2-15 wherein, m=1-10, c=0-10, X=-Cl ,-Br ,-I or-OSO ₂C ₆H ₄CH ₃, Z=-SH ,-CHO ,-COOH or-NH ₂

5. one kind is passed through to connect the self-assembled monolayer solid substrate that the imines Calixarene Derivatives prepares, and wherein a kind of thiol group is connected to gold substrate.

6. one kind connects the imines Calixarene Derivatives and is selected from the self-assembled monolayer solid substrate that glass, silicon wafer and crystalline solid substrate prepare by imine linkage.

7. one kind by being selected from ester, and the chemical bond of ether and amido linkage is connected the imines Calixarene Derivatives and is selected from glass, silicon wafer and crystalline solid substrate and the self-assembled monolayer solid substrate for preparing.

8. oligomer DNA chip for preparing by fixing oligomer DNA, wherein at least 7 continuous guanine bases are connected to any described self-assembled monolayer solid substrate as claim 5-7 by polymolecular identification.

9. one kind prepares the method for oligomer DNA chip by fixing oligomer DNA, and wherein at least 7 continuous guanine bases are connected to any described self-assembled monolayer solid substrate as claim 5-7 by polymolecular identification.

10. as any self-assembled monolayer solid substrate described by 5,11,17 of following formula 3, that the imine derivative of 23-tetramino cup [4] aromatic hydrocarbons prepares of claim 5-7, wherein nitrogen is connected 5,11,17 of calixarene, on the 23-position:

[formula 3]

11. an oligomer DNA chip for preparing by fixing oligomer DNA, wherein at least 7 continuous guanine bases are connected to self-assembled monolayer solid substrate as claimed in claim 10 by polymolecular identification.

12. one kind prepares the method for oligomer DNA chip by fixing oligomer DNA, wherein at least 7 continuous guanine bases are connected to self-assembled monolayer solid substrate as claimed in claim 10 by polymolecular identification.

13. amino Calixarene Derivatives that has with following formula 5:

[formula 5]

(R wherein 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 representative-CH ₃Or-C ₂H ₅).

14. amino Calixarene Derivatives that has with following formula 6:

[formula 6]

(R wherein 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 representative-CH ₃Or-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 (n=2-15 wherein, m=1-10, c=0-10, Z=-SH ,-CHO ,-COOH ,-NH ₂, and-C ₆H ₄-and C ₆H ₅Be defined as phenyl group)).

15. amino Calixarene Derivatives that has with following formula 7:

[formula 7]

(R wherein 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 representative-CH ₃Or-C ₂H ₅).

16. amino Calixarene Derivatives that has with following formula 8:

[formula 8]

(R wherein 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 representative-CH ₃Or-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 ' ₈Be not simultaneously-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 (n=2-15 wherein, m=1-10, c=0-10, Z=-SH ,-CHO ,-COOH ,-NH ₂, and-C ₆H ₄-and C ₆H ₅Be defined as phenyl group)).

17. one kind by connecting the self-assembled monolayer solid substrate that amino Calixarene Derivatives prepares, and wherein is connected to gold substrate as the thiol group in claim 14 or the 16 described amino Calixarene Derivatives.

18. the amino Calixarene Derivatives by will connecting a kind of aldehyde functional group group as the end group in claim 14 or the 16 described amino Calixarene Derivatives be selected from glass, silicon wafer and crystalline have been connected the self-assembled monolayer solid substrate that the solid substrate of amine functions group is connected and prepares by imine linkage with the amine key.

19. the amino Calixarene Derivatives by will connecting a kind of aldehyde functional group group as the end group in claim 14 or the 16 described amino Calixarene Derivatives be selected from glass, silicon wafer and crystalline be by being selected from ester, and the solid substrate that the chemical bond of ether and amido linkage has been connected the amine functions group is connected and the self-assembled monolayer solid substrate for preparing.

20. an oligomer DNA chip for preparing by fixing oligomer DNA, wherein at least 7 continuous guanine bases are connected to any described self-assembled monolayer solid substrate as claim 17-19 by polymolecular identification.

21. one kind prepares the method for oligomer DNA chip by fixing oligomer DNA, wherein at least 7 continuous guanine bases are connected to any described self-assembled monolayer solid substrate as claim 17-19 by polymolecular identification.

22. as any self-assembled monolayer solid substrate described by 5,11,17 of following formula 9, that the sulfonamide derivatives of 23-tetramino cup [4] aromatic hydrocarbons prepares of claim 17-19, wherein nitrogen is connected 5,11,17 of calixarene, on the 23-position:

[formula 9]

23. an oligomer DNA chip for preparing by fixing oligomer DNA, wherein at least 7 continuous guanine bases are connected to self-assembled monolayer solid substrate as claimed in claim 22 by polymolecular identification.

24. one kind prepares the method for oligomer DNA chip by fixing oligomer DNA, wherein at least 7 continuous guanine bases are connected to self-assembled monolayer solid substrate as claimed in claim 22 by polymolecular identification.

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