CN1570140A

CN1570140A - Double probe gene chip signal amplification method

Info

Publication number: CN1570140A
Application number: CN 03141844
Authority: CN
Inventors: 宋克
Original assignee: Individual
Current assignee: Individual
Priority date: 2003-07-25
Filing date: 2003-07-25
Publication date: 2005-01-26

Abstract

The invention provides a double-probe gene chip signal amplification method. The principle is designing two or more probes which are complementary to the basic group sequence in target gene specific site, wherein the probes are the capture probe and the labelled probe. The capture probe is the nucleic acid probe fixed in the gene chip micro-array, and the labelled probe communicates with the high-charge signal carrier. The sensitivity and the signal to noise ratio can be improved by applying the high-charge signal carrier (HSC) to the gene chip signal amplification labeling and detecting process, and the high pass analysis can be completed by preparing a lot of labelled target genes and labelled probes.

Description

Two probe gene chip signal amplification method

Patent application of the present invention is the expansion of following related application technology contents and replenishes:

The Chinese invention patent application:

Gene chip combined treating system and correlation technique, application number: 01126990.1.The applying date: October 11 calendar year 2001;

Gene chip molecular probe and correlation technique, application number: 01142654.3.The applying date: December 14 calendar year 2001.

The microarray method for amplifying signal, application number: 03129124.4.The applying date: on July 23rd, 2003.

International patent application:

Gene chip molecular probe and correlation technique, international application no: PCT/CN02/00887.The applying date: on December 13rd, 2002.

Science of quoting and patent documentation

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People such as Coutlee, 1989, Anal.Biochem.181:96-105.

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People such as Stollar, Anal.Biochem.161:387-94,1987

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Huse，et?al.，1989，Science，246，1275-1281

Hames, B.D., and Higgins, people's such as S.J " nucleic acid hybridization, a kind of practical approach ", IRL Press, people such as Oxford (1985) Ellen R.Goldman, Avidin:A Natural Bridge for Quantum Dot-Antibody Conjugates.J.AM.CHEM.SOC.2002，124，6378-6382

People such as E.M.Boon, " Mutation detection by electrocatalysis at DNA-modified electrodes, " NatureBiotechnology, 18:1096-1100,2000

People such as R.M.Umek, " Electronic detection of nucleic acids:A versatile platform for moleculardiagnostics, " May 2001. for Journal of Molecular Diagnostics, 3:74-84

People such as Agne`s Labande are at " Supramolecular Gold Nanoparticles for the RedoxRecognition of Oxoanions:Syntheses; Titrations; Stereoelectronic Effects; and Selectivity " 1782-1789 VOL.124, NO.8,2,002 9 J.AM.CHEM.SOC.

People's such as Kaufman United States Patent (USP): US PAT 6,383,754.May?7，2002

People's such as Linsley U.S. Pat Pat 6,232,068, May 15,2001

Carrico United States Patent (USP) U.S.Pat.No.4,833,084, May 23,1989

Affiliated field the invention belongs to the gene chip applied technical field, particularly relates to the hybridization of gene chip, mark, wash-out, signal detection and data analysis technique.

The technology of the present invention background gene chip is to utilize parallel analysis principle not homotactic nucleic acid molecule in a large number, gene probe as target gene in the analytic sample, be solidificated in silicon chip in the microarray mode, glass, on the matrix such as nylon membrane, thereby make the gene chip on the ordinary meaning, or be called dna microarray ^(1-2)To at present, gene chip aspect the signal mark still based on fluorochrome labels such as CY3/CY5.This labelled reagent costs an arm and a leg, and sensitivity is not high, and is in use also very high to the requirement of operator's technology.In addition, Chinese patent application such as my " gene chip molecular probe and correlation technique; application number: 01142654.3 " and " microarray method for amplifying signal; application number: 03129124.4 " are carried out the labeling technique that signal amplifies though proposed high lotus signal vehicle, by hybridizing the back heteroduplex nucleic acid is carried out specific marker, but this method for from the gene of different samples at twice gene chip experiment just can compare gene content in the different samples.This relative method is particularly common in the express spectra research of gene chip.At present current technological line is with producing blue look fluorescence after the CY3[optical excitation], produce red fluorescence after the CY5[optical excitation] genome in the mark difference samples respectively, again with this not the target gene solution of isolabeling on same gene chip, hybridize.Determine the difference of the expression level of a certain gene in different samples by the fluorescence color that finally shows.

But because the not enough and expensive problem of sensitivity appears in CY3/CY5 in gene chip detects, and is not suitable for the application of gene chip aspect clinical diagnosis, so gene chip is made slow progress in the popularization and application process in clinical diagnosis field at present.

A kind of method that is used for detecting difference expression gene is to increase by PGR with specific primer from the cell, tissue or the organ that are in healthy and morbid state at first respectively to prepare target gene from sample, or the cDNA target.In preparation target gene synthetic, mixed a nucleoside monomers dNTP and a part and isotropic substance [ ³²P, ³³P, ³⁵S, ¹²⁵I] or fluorophor [as tonka bean camphor (coumarin) and derivative thereof; Cyanine dyestuff [cyaninedyes] Cy3 and Cy5] the nucleoside monomers dNTP of covalent coupling.Thereby the synthetic cDNA target gene that has various signal marks and be amplified.Select the signal tracer of different wavelength of transmitted light for use from the target gene amplification of different tissue samples.To have marker then hybridizes on the gene chip on the fixed probe fragment from the cDNA target of different cell tissue samples.Results of hybridization is with detected and relatively be used for and corresponding disease-related connection.Present fluorescent mark is time-consuming mostly and process is loaded down with trivial details, also exists the problem of the secondary structure of sensitivity deficiency and formation target-probe.

One of purpose of the present invention purpose of the present invention is to promote detection sensitivity significantly, economic and practical and signal magnifying tags technology and the test kit that can realize automatic operating for the gene chip product provides a cover.

The present invention the method that is provided among the positively effect the present invention who is produced is provided with background technology has not only greatly been improved the gene chip detection sensitivity, handle timeliness, reduce cost, also make non-specialized-technical personnel through brief training on operation or guidance simultaneously, can utilize this supermatic equipment to carry out high level and high efficiency genetic analysis.Everything is all used for the universalness of gene chip the technology impellent is provided.In addition, as general approach, no matter be applied to [micro-fluid chip under solid phase [as substrate formula gene chip etc.] or the liquid solution condition, capillary electrophoresis chip, lab-on-a-chip etc.], this signal amplification technique has the PCR[polymerase chain reaction, the gene amplification technology] technology and be used for the not available plurality of advantages of the used signal amplification technique of other gene tests.Because this technology does not rely on gene amplification, rely on simple biology, chemistry or electrochemical label, so, this technology can be accurately quantitatively, be difficult for polluting, can avoid false positive detected result, test kit with low cost, realize advantage such as automatization easily, and specimen preparation is simple and easy.Everything is that round pcr institute is inaccessible.Therefore, it can be used as general method for amplifying signal and is applied in the other forms of detection of nucleic acids.Conclusion is got up, and the beneficial effect that the present invention brought has following several aspect:

1, the technical foundation of super sensitivity detection is provided for general biochip technology product, the process instrumentation of signal amplification technique support also can produce very high signal to noise ratio thus simultaneously, satisfy the demand of practical fields such as scientific research and clinical diagnosis to the hypersensitive gene chip detecting technique, help promoting the popularization and application of biochip technology at aspects such as clinical diagnosis, industrial and commercial quarantine and environment measurings

2, signal amplification technique provided by the present invention helps realizing automatization, the integrated degree of treatment system is further improved, the system handles process is further simplified, professional and technical personnel's processing efficiency is improved, also reduced simultaneously professional technique requirement to non-specialized-technical personnel, improved gene chip use in result's repeatability and stable

3, use two probes also to help improving the specificity of the detection of gene chip among the present invention,

4, the present invention also for cost and the running cost that reduces the gene chip treatment system provides technical support, helps the popularization and application of gene chip.

Know-why the present invention of the present invention proposes the two probe magnifying tags technology of a kind of gene chip, this gene chip is made up of with the probe that is no less than two arrays arrangements that is fixed on substrate [being abbreviated as S] or coating [being abbreviated as C] surface the coating or the coating on substrate and surface thereof, is used for the biological or chemical target material of test sample.The material of substrate and/or coating or coating can by but be not limited in the following material any one or more than one differing materials combination and make:

(1) inorganic sheet or tabular material type such as glass, quartz, mica, transparent conductive material such as transparent conductive oxide class [TCO] are as tin indium oxide [indium tin oxide, ITO, or be written as In ₂O ₃: Sn], InAs, SnO ₂: coating such as F, gallium arsenide [GaAs], magnesium oxide, stannic oxide [tin oxide, SnO], ZnO, CdO, Cdln ₂O ₄, Cd ₂SnO ₄, Zn ₂SnO ₄And In ₂O ₃-ZnO etc., and carbon/pottery composite conductive ceramic, various semiconductor materials etc., and porous plate pattern, as microwell plate, droplet plate,

(2) simple substance class: platinum, gold and silver, aluminium, chromium metals such as [Au, Ag, Pt, Cu, Rh, Pd, Al, Cr] and silicon etc. are nonmetal,

(3) organism class: each organic molecular species and the self-assembled monolayer of making thus thereof or self-assembly multimolecular film,

(4) high score subclass: nylon membrane, plastics, rubber, resin, nitrocellulose filter.

The structure of substrate can be but be not limited to following kind:

(1) thin slice/flat: as sheet glass, silicon chip, nylon membrane, plastic sheet,

(2) multiaperture-type: as microwell plate, droplet is board-like etc.,

(3) electric pole type: on the sheet type gene chip, arrange the various electrod-arrays form with certain rule, as the platinum electrode in substrates such as semi-conductor, isolator, made, gold electrode etc.,

(4) micro-tubular: by kapillary or other material of kapillary tubular intracavity constructional feature is arranged, arrange various matrix form kapillary gene chips [two dimensional surface arrangement], linear kapillary gene chip [one dimension linear array] that forms and the various matrix form parallel arranged of in substrate formula structure, producing [being microfluid electrophoresis chip etc.] that are no less than a microchannel formula cavity structure by the predefined position rule of correspondence

The probe or the target that are used for detecting on the said gene chip of the present invention can be, but be not limited to certain composition of one of following kind or different types of combination or following kind, extract:

(1) Yeast Nucleic Acid [RNA], various Yeast Nucleic Acid [" ribonucleic acid " and " RNA "] comprise messenger RNA(mRNA) [being called for short mRNA], transcribe rna [being called for short tRNA] etc.,

(2) thymus nucleic acid [DNA] is as DNA, complementary DNA [cDNA] etc.,

(3) oligonucleotide [oligonucleotide] comprises dna oligo [oligodeoxynucleotide is called for short ODNs], oligomerization Yeast Nucleic Acid [oligoribonucleotide, ORNs], and polynucleotide [polynucleotide],

(4) the nucleic acid construct resemblance [DNA-DNA, RNA-RNA or RNA-DNA mimic duplexes, triplexes or quadroplexes, etc.]] as peptide nucleic acid(PNA) [peptide nucleic acids, be called for short PNA],

(5) hybrid molecule of forms such as the complementary two strands of thymus nucleic acid, Yeast Nucleic Acid and structural similitude thing thereof, three [weight] chain, four [weight] chain such as DNA-DNA, RNA-DNA, RNA-RNA, PNA-DNA, PNA-RNA, PNA-RNA-PNA, PNA-DNA-PNA] etc.

The present invention adopts high lotus signal vehicle (high molecular fluorescent microballoons or contain a large amount of fluorescence dyes in the gene chip treatment technology, the semiconductor nano quantum dot, the polymeric particles of electroluminescent molecule etc., a large amount of signaling molecules are polymerized to the various forms of huge signaling molecule of one by synthetic technology, silica optics magnetic bead [silica beads], Radioactive colloidal gold [or nm gold particles, colloidal gold nanoparticles], or pass through any other means with fluorescence dye, non-fluorescence dye, semiconductor-quantum-point, electroluminescent, chemoluminescence or noclilucence material signaling molecule etc. are fixed in the surface of particulate or are embedded in the high lotus signal vehicle of various ways such as its inside) thing that serves as a mark carries out molecular signal and amplifies.

The gene chip signal magnifying tags technology that the present invention will introduce is to be applied to the various forms of gene chip probes of mark, target or probe-target crossbred with various forms of high lotus signal vehicle or signal body.

Here said gene chip includes but not limited to following type: gene chip [comprises various DNA chips, as the glass gene chip, film chip, lab-on-a-chip, micro-fluid chip etc.], based on various biosensors of electrochemical principle or biological electronics principle etc.

The detection principle of gene chip used herein can be, but is not limited to the combination of one of following kind or different principle:

(1) carries out optical signalling [as fluorescence molecule, chemoluminescence etc.] mark by probe-target-specific affinity reaction bonded body or the crossbred that solid phase surface is positioned at different positions, then through exciting generation fluorescence, chemistry or noclilucence and detecting.With glass, silicon chip, nylon membrane etc. is that gene chip, protein chip, the immuno-chip of material taked this pattern more;

(2) by translational speed or the mobility in the liquid phase that has applied voltage detects to probe mark or unlabelled or target, be electrophoretic technique or combine with other technologies and the other technologies that produce by electrophoretic technique, as capillary electrophoresis [capillaryelectrophoresis, CE], capillary zone electrophoresis [capillary zone electrophoresis, CZE], capillary gel electrophoresis [CGE], HPCE [HPCE], capillary isoelectric focusing [capillary isoelectric focusing (CIEF)], isotachophoresis [isotachophoresis (ITP)], electrokinetic chromatography [electrokineticchromatography (EKC)], mcellar electrokinetic capillary chromatography [micellar electrokinetic capillarychromatography (MECC OR MEKC)], capillary electrochromatography method [capillaryelectrochromatography (CEC)], nonaqueous phase capillary electrophoresis [non-aqueous capillaryelectrophoresis (NACE)] etc.

(3) by probe, target or probe-target complex compound and the different avidity of their marker, carry out the chromatographic technique analysis, as affinity chromatography [affinity chromatography], SEC[size exclusion chromatography], GPC[gelpermeation chromatography], MCC[metal chelate chromatography] etc.

(4) utilization has the molecule of redox property or the part-structure of molecule [being chemical group or functional group], metal complex or (nucleic acid) intercalator be positioned and be fixed on probe on solid phase surface different positions or the electrode, target or probe-target complex compound combination is also adding under suitable mode or suitable big or small voltage (or potential difference) effect (volts DS or exchange current or pulsed electrical field or voltage), redox reaction takes place, cause from reductibility molecule or group (electron donor, electrondonor) transfer transport is to oxidisability molecule or group (electron acceptor(EA), electron acceptor), form stream of electrons, being sent to detection system by electrode measures, measured strength of current size is directly proportional with the amount of the double-strandednucleic acid of hybridizing and being labeled

(5) utilize the conductivity difference of double-strandednucleic acid and single-chain nucleic acid, or utilize by between the formed double chain acid molecule of the nucleic acid hybridization of the complete complementary pairing of sequence and double-strandednucleic acid (the nucleic acids duplexes) molecule that mispairing base pair (Mismatches) existence is arranged in conductivity difference, by various discriminatings to other aspects that nucleic acid molecule is reflected thus being carried out via the measurement of the stream of electrons size of nucleic acid molecule conduction, detect and investigate, as in transgenation, gene pleiomorphism, gene sequencing, gene expression profile research, the application of aspects such as pathogenic mechanism analysis and medical diagnosis on disease

(6) will occur on the nucleic acid molecule or and (include but not limited to by material with redox property, have the metal complex of redox property and be the polymkeric substance that monomer polymerization forms by the derivative of metal complex or metal complex, fluorescence molecule, nucleic acid intercalators etc.) electronics that is taken place or charge transfer reaction or stream of electrons convert other to and can detect the detection technique that maybe can investigate form, as by electroluminescent [Electroluminescence] principle, utilize the electroluminescent molecule that stream of electrons is converted to the detection that optical signal carries out

(7), thereby realize detection to the biological or chemical reaction that occurs in electrode surface by making marker electron-transfer reaction take place behind the mark near electrode surface directly or indirectly having oxidation-reduction quality material (as ferrocenyl) to probe or target molecule marker.ESensor Chips as Motorola Inc..

(8) pass through probe, target or probe-target complex compound and the different avidity of their marker, carry out the chromatographic technique analysis, as affinity chromatography [affinity chromatography], SEC[size exclusion chromatography], GPC[gelpermeation chromatography], MCC[metal chelate chromatography], electrokinetic chromatography [electrokinetic chromatography (EKC)], mcellar electrokinetic capillary chromatography [micellarelectrokinetic capillary chromatography (MECC OR MEKC)], capillary electrochromatography method [capillary electrochromatography (CEC)] etc.

Here the high lotus signal vehicle [HSC] of said gene chip includes but not limited to following type; the high loading optics that exists with various molecule forms; magnetic; electronics or electrochemical signals carrier; it can be quantum dot microsphere [QD-tagged Microbeads]; superpolymer fluorescent microsphere or high molecular fluorescent microballoons [polymer microspheres] or colloid microballon [latex beads]; finishing has the silica magnetic bead [silica beads] of fluorescence dye; the brilliant quantum dot [colloidal fluorescent semiconductor nanocrystalquantum dots] of colloid luminescent semiconductor nanocrystal; dendroid high polymer nano ball [dendrimers]; star dendrimer or dendrimer [star dendrimers; dendrimeric stars or dendrimers]; the semiconductor nanoparticle of protein parcel or molecule or quantum dot [as wrapping up best results with Chaperonin GroEL albumen and T.th cpn albumen among the Chaperoninmediated semiconductor nanoparticles.Chaperonin]; micella [micelles]; molecular magnet [molecular magnets]; capsule-type microballoon [encapsulated spheres]; colloidal nano gold [colloidal gold nanoparticles]; electroluminescent molecule [electroluminescent molecules]; two Metallocenyl copolymer microspheres [polymetalcene block copolymers; as ferrocene-based polymer [polyferrocene block copolymers; PFC; or polyferrocenylsilanes; PFS] microballoon; poly zirconocene [polyzirconoeene; PZC]; poly two Metallocenyls are modified or the grafted dendrimer [includes but not limited to; poly-ferrocene base grafted dendrimer (polyferrocenyl-branched dendrimers)]; two Metallocenyls or poly two Metallocenyls are modified or grafted nanometer gold colloid [includes but not limited to; the nanometer gold colloid of amido ferrocenyl functionalization (Gold colloids functionalized with amidoferrocenyl structures) etc.]; copolymer microsphere or nanometer ball; contain fullerene structure or be the superpolymer or the multipolymer of one of monomer with the soccerballene; other contains fluorescence dye or chemoluminescence [chemiluminescence]; the carrier of noclilucence [bioluminescence] material; phycobiliprotein class [phycobiliproteins] and various derivative thereof or biological or chemical modifier; and in above-mentioned each carrier different parameters [as size; the optical emitting optical wavelength; electromagnetic property; physics or chemical property such as the modification of surface biological or chemical functional group] combination of carrier, or the mutual combination between the various different carriers.Its finishing have can with the functional group of probe joint A-specific reaction in following general formula (I) compound or effect to A '-.

Here mention quantum dot microsphere [QD-tagged Microbeads, or QD-tagged Polymeric Microspheres, QD-Beads] especially.This is a kind of quantum dot to be embedded in a kind of HSC in the polymer microsphere.Because QD can be embedded in the polymer microsphere in large quantities, thereby also is the extremely strong HSC of a kind of signal.In view of one side QD can realize by the adjusting of semiconductor nano particle diameter regulating on emission wavelength easily, polymer microsphere can change the light signal strength levels of quantum dot microsphere according to the ratio difference of embedding aspect embedding QD on the other hand, therefore, quantum dot microsphere [QD-Beads] can realized diversification and stratification aspect the wavelength of fluorescent emission and the intensity very ideally.In addition, under favourable preparation condition, quantum dot microsphere can reach the homogeneous of height, aspect the electrophoresis etc. 1 very valuable application value is being arranged.

The signal amplification technique that the present invention will introduce, be a kind of pair of probe gene chip signal magnifying tags technology, it is characterized in that, two probes are made up of capture probe [Capture Probe] and label probe [Labeling Probe], capture probe is fixed in the gene chip solid phase surface, constitute gene chip micro lattice, and label probe is by high lotus signal vehicle and one or more its surface of being fixed in, with high lotus signal vehicle carry out covalent linkage or non covalent bond link coupled can with target gene on one or the sequence in the more than site specific nucleic acid sequence of carrying out complementary hybridization constitute, free being present in the label solution, the two all has the nucleotide sequence with the specific sequence complementary pairing of the different loci of target gene to be measured respectively.

This pair of probe gene chip signal amplification technique, its labeled reactant pattern can be, but be not limited to the described several sights of following general formula:

(1) target gene and the label probe in single sample source are preferentially hybridized, after heteroduplex carries out or does not carry out interchain linkage or locking, become the labels targets gene, again with gene chip on the capture probe specific hybrid, equally after capture probe and the formed heteroduplex of target gene carry out or do not carry out interchain linkage or locking, form the target gene of two probe hybridization marks

(2) target gene in two or more different samples source is preferentially hybridized with the label probe of different qualities respectively; Thereby the target gene of separate sources is defined separately by specific label probe; After heteroduplex carries out or does not carry out interchain linkage or locking; Become the labels targets gene; Again with genetic chip on the capture probe specific hybrid; Equally after capture probe and the formed heteroduplex of target gene carry out or do not carry out interchain linkage or locking; Form the target gene of two Probe Hybridization marks

(3) target gene of single source and capture probe and label probe carry out crossover process simultaneously, afterwards, carry out or do not carry out the strength of joint of the interchain locking process of heteroduplex with reinforcement mark and heteroduplex,

The structure of label probe can be used general formula (I) expression,

General formula (I): F (L-XNA) _{N '}Or F (XNA) _{N '}

Compound in order to nucleic acid double chain interchain linkage or locking is represented with X, can be respectively that one of following situation or dissimilar combination of compounds are used,

N be from zero to infinitely-great natural number: 0,1,2,3...

Wherein, X-passes through electrostatic attraction with double-strandednucleic acid, inferior bonding force is [as Van der Waals force, hydrogen bond etc.], with the major groove in the double-spiral structure or the avidity [Groove binders] of ditch, embed the avidity [intercalators] of double-stranded base pair, form the functional group that modes such as triple-helix structure or four spirane structures combine with double-spiral structure, with functional group or the molecule of nucleic acid double-spiral structure by chemical reaction formation covalent bonds, can specific recognition nucleic acid double chain structure and bonded biomolecules with it, can with nucleic acid double chain complexing or the various biological or chemical molecules of bonded, it can include but not limited to following compounds and be that the various derivatives and the biological or chemical of architecture basics or feature modified the formed compound in back with the following compounds:

The constitutional features of X-according to its with can be divided into following several types for the hybridization single-chain nucleic acid or with the nucleic acid double-spiral structure generation interactive property of complementary hybridization;

1. electrostatic attraction, inferior bonding force [as Van der Waals force, hydrogen bond etc.] are with the major groove in the nucleic acid double-spiral structure or the avidity [Groovebinders] of ditch.The X-that belongs to this situation can be, but be not limited to following several compound: (1) is the intercalator actinomycin class of the mechanism of action with duplex major groove [major grooves], as 7-amino-radiating streptozotocin D [7-amino-Actinomycin D]; (2) form the carbazoles of complex compound [as 3 with duplex ditch [minor grooves], 6-carbazole, 2,7-carbazole] and phenylbenzene furans two ammonium classes [diphenylfuran diamindine], polyamine class [polyamines] DNA intercalator, Yin quinones intercalator [6H-indoloquinolinecore], alkylamino alkyl chain [alkylaminoalkyl chain, DNA ditch intercalator]

2. can embed the avidity [intercalators] of hybrid nucleic acid duplex base pair.The X-that belongs to this situation can be: (1) can form a compounds of interchain covalent cross-linking under certain condition, as psoralene (Psoralen, relatively weak (K _d10 ^-4M)), isopsoralen and their any derivative form are as 4 '-methylol-4,5 ', 8-trimethylpsoralen (4 '-hydroxymethyl-4,5 ', 8-trioxalen, write a Chinese character in simplified form HMT), 4 '-amine methyl-4,5 ', 8-trimethylpsoralen (4 '-aminomethyl-4,5 ', 8-trioxalen, write a Chinese character in simplified form AMT), trimethylpsoralen (trimethylpsoralen writes a Chinese character in simplified form TMP), 8-methoxypsoralen (8-methoxypsoralen is abbreviated as 8-MOP), 4,7,4 '-and 4,7,5 '-trimethylammonium isopsoralen (4,7,4 '-and 4,7,5 '-trimethylallopsoralen) and psoralene itself.Optical excitation intercalator [Ru (phen) 2dppz ²⁺Phen=1,10-phenanthroline; Dppz=dipyridophenazine], Platinol [cisplatin] has the intercalator indyl quinoline positive ion salt or quinoline positive ion salt [indolo[2, the 3-b]-quinolizinium bromide of DNA light injury character concurrently]; (2) as the EDTA transition metal complex class [as methinidiumpropyl EDTA Fe (II)] of double-stranded intercalator, 2,7-diazapyrene (DAP), DAPI[4 '-6-diamidino-2-phyenylindole], N-positive ion [the N-methylated cations DAP that methylates ⁺AndDAP ²⁺], acridine compound { Acridine orange[3,6-bis (dimethylamino) acridine, HCl] }, bidentate shape acridine intercalator [bis-dentate acridine intercalators], aminoacridine or aminacrine [9-aminoacridine (9AA)], iodo or iodo acridine [2-iodine-125-iodoacridine]; (3) can cut off the intercalator adriamycin or the Zorubicin [Adriamycin of DNA chain structure, or Doxorubicin], elsamicin, Nogalamycin, Sanguinarine, Cherethyrine, anthracyclinedrug, leinamycin, Echinomycine, zhengdingmeisu or daunomycin class [Daunomycin], [Rebeccamycin]; (4) have distamycin compounds [distamycin] and T-1384 compounds [netropsin] and Hoechst 33258 and the Hoechst33342 etc. that nucleic acid base embeds function and duplex groove embedding function concurrently; (5) the following bonding force is the interactional methylene blue of accumulation base [methylene blue (MB, the MB in main and the double-spiral structure ⁺), ethidium or Ethidium Bromide [ethidiumbromide] and two ethidium bromide positive ions [diethdium cathion],

3. form the functional group that modes such as triple-helix structure or four spirane structures combine with double-spiral structure, with functional group or the molecule of nucleic acid double-spiral structure by chemical reaction formation covalent bonds, can specific recognition nucleic acid double chain structure and bonded biomolecules with it, can with nucleic acid double chain complexing or the various biological or chemical molecules of bonded, it can include but not limited to following compounds and be the various derivatives of architecture basics or feature with the following compounds: all can embed the major groove of nucleic acid duplex structure or nucleic acid double chain or the insert in the ditch (1), the poly insert, linearity or ring-type insert etc.

4. can form the paranuclein class [as Nucleolin RBD12] of various complex compounds with RNA,

5. the multiple intercalator that can a plurality of sites in double-spiral structure embeds such as the plain class of sour jujube dermatophyte [Echinomycine, the heavy intercalator of dna double] is got up by the covalent linkage coupling, or is together in series, and can form the quadruple intercalator that quadruple embeds;

6. berenil [berenil], cytotoxin class [as cytotoxin NLCQ-1], triostin [triostin], berberine class [as protoberberine-8],, oral cavity M[Gilvocarcin M], furans naphthyl pyranone [furonaphthopyrone], porphyrin or metal porphyrins [porphyrins or metalloporphyrins], carboline compounds [as beta-carbolines], proflavine [proflavin (Acr-NH ₂)], 3,7-bis (dimethylamino) phenothiazin-5-ium chloride], phenyl sulfydryl pyrans [benzothiopyranoindazole], [water-soluble] propylidene iodide [propidium iodide],

7. be that [the stacked aedamer core has two or several 1,4 for the wire multimerized dna insert of feature with the aromatic ring structure accumulation, 5,8-tetracarboxylic acid diimide (1,4,5,8-tetracarboxylic diimide) feature structure unit poly insert such as]

8. can produce the insert ferrocenyl benzene-naphthalene diimide class wire intercalator [threading ferrocenyl naphthalinediimide] of electrochemical signals;

9. anthracene class [anthryl compands such as AMAC[9-amino-6-chloro-2-methoxyl group make mute pyridine (9-amino-6-chloro-2-methoxyacridine)], anthracycline, APAC, N-Et-AMAC etc.], anthraquinone class [anthraquinones], benzene-naphthalene diimide class intercalator [naphthalene diimide intercalators], phenanthrene ion class intercalator [phenanthridinium intercalators]

10. metal species intercalator, as octahedra DNA rhodium intercalator [rhodium intercalator, bis (the chrysene quinone diimine of 2-2 ' bipyridyl), D-[Rh[(R, R)-Me 2 trien] phi] 3+, Ru (NH ₂) ₆ ³⁺], metal Ru intercalator [ruthenium intercalators, [Ru (phen) 2dppz] 2+ (phen=1,10-phenantroline of chirality complex structure are arranged; Dppz=dipyrido[3,2-a:2 ', 3 '-c] phenazine)], terpyridine platinum compound (TPH), mitoxantrone, lanthanide metal compound DNA intercalator [lanthanide], bipyridyl (quadrol) platinum salt or three pyridyl platinum salt [bipyridyl (ethylenediamine) Pt (II) ²⁺, 4-picoline-2,2 ': 6 ', 2 "-terpyridine-platinum],

11. can change benzene-naphthalene diimide compounds [two intercalators of duplex groove structure, as naphthalene diimides, N, N '-disubstituted naphthalene diimide derivatives, with the naphthalenetetracarbacidic acidic diimide pair intercalators (1 of polypeptide chain as the connecting arm that embeds the duplex ditch, 4,5,8-Naphthalene Tetracarboxylic Diimide Bis-Intercalator with theLinker (Ala) 3-Lys in the Minor Groove), with the amido alkyl DNA is embedded the functional group linearity and couple together formed pair of intercalator, three intercalators, the poly intercalator and ring two-wire shape intercalator etc. is arranged]

12. dicentrine class [dicentrine], big that mycin class [tricyclic dynemicin A] of three rings, nitro amine [nitracrine, electrophilicity DNA intercalator], endoxan class [cyclophosphamide], amsacrine, tanshinone is [as Dihydrotanshinone, I], octahydroxanthene, indazole class [indazoles, as benzothiopyranoindazole DNA intercalator], imidazoles [imidazole], N-[4-(9-acridyl amino)-3-methoxyl group-phenyl] methane-sulfonamides compound [N-[4-(9-acrydinylamino)-3-methoxy-phenyl] methane-sulphonamide, m-AMSA], macrolactams [macrocyclic lactam], quinoline class intercalator as indolo[2,3-b] and quinolines, 6H-indoloquinoline core, quinoxalines}, Amsacrine, [Xanthenes is as 1 for (folder) oxa anthracenes, 8-dioxo-9-(o-nitrophenyl)-1,2,3,4,5,6,7,8 octahydro xanthenes (1,8-dioxo-9-(o-nitrophenyl)-1,2,3,4,5,6,7,8-octahydroxanthene)], luzopeptin, camomile lopps [6a, 7-diazanaphth[3,2,1-cd] azulene and 7H-1,7-diazaindoleno[1,2e] azulene system], Ditercalinium, 4,6-diamidino-2-phenyline[DAPI], pentamidine class [pentamidine]

13.Pixantrone, roserush bases [indole-based intercalator ellipticine] based on indole structure, quinacrine class [quinacrine], diazonium compound class [4,9-diazapyrenium], estradiol class [estradiol], two phenyl acid imides [bisbenzimide], thiazoles [as thiazole orange]

14. the couplet of photolytic activity vitamin H or photobiotin [photoactivable biotin, photobiotin], photobiotin and biomolecules or chemical molecular, and the derivative of photobiotin,

15. all can carry out painted dye molecule to nucleic acid, as fluorescence dye bisbenzimide H fluorochrome[TriHCl], Toyomycin [chromomycin] A3, fluorescence intercalator [as TOTO, YOYO, SYBR Green],

16. all can form compound or biomolecules or biomolecular structure analogue and this structure and the formed complicated molecule structure of other molecule covalent couplings of triple (doubly) [triplex] spiral or quadruple (doubly) [quadruplex] spirane structure with the complementary nucleic acid double-spiral structure of hybridizing on the gene chip, as PNA[peptide nucleic acids], oligonucleotide, polypeptide is [as [N-MeCys ³, N-MeCys ⁷] TANDEM] etc.;

17. thereby,, contain the compound and the derivative thereof of benzophenone functional group in the structure as the nitrine compounds can form functional group or the molecule that covalent linkage is connected with nucleic acid double chain generation chemical reaction or photochemical reaction.Can with the various chemical cross-linking agents of nucleic acid probe or target gene generation covalent cross-linking or chemical reaction functional group as can by with target gene on hydroxyl take place the to shrink carbodiimide of reaction, or the active linking agent of photochemical reaction, as aromatic base triazo-compound [aryl azides, as N-((2-pyridyldithio) ethyl)-4-azidosalicylamide[PEAS] etc.], fluoro aromatic base triazo-compound [fluorinated arylazides], benzophenone class photoresponse reagent [benzophenone-based photoreactive reagents is as benzophenone maleimide [benzophenone maleimide]] etc.;

18. can sequence-specific ground or non-sequence-specific ground identification nucleic acid double chain structure and with it in conjunction with or be biomolecules such as the protein of substrate or enzyme with the double-spiral structure of double-strandednucleic acid, as HMGB albumen, can with the dsDNA bonded NHP6A albumen of modifying through Platinol, participate in the enzyme of dna replication dna, zymoplasm [thrombin], the enzyme mitomycin of rna transcription [Mitomycin C], can and the double-stranded two strands of nucleic acid between form the bleomycin or the bleomycin class [Bleomycin] of covalency bridge shape key, double-strandednucleic acid antibody, the specificity desmoenzyme of double-strandednucleic acid, archaeal dna polymerase or RNA polymerase

19. can carry out the complementary oligonucleotide molecule that forms complementary duplex structure, antisense nucleic acid [the anti-sense nucleic acid sequences] sequence etc. of also combining of specificity with certain or some specific sequences of probe nucleic acid molecule on target gene to be measured or the gene chip.This compounds can be applicable to the preceding mark of hybridization and two kinds of schemes of hybridization back mark simultaneously.

20. above-mentioned various intercalator or binding substances combination or the molecular structure that gets up by covalent bonds, thereby or be implemented in by chemically modified and take place to interact with nucleic acid probe, target or probe-target heteroduplex and form that covalent linkage is connected or the derivative of crosslinked above-claimed cpd, or separately and the senior intercalator or the nucleic acid binding molecule of polypeptide, linearity or chain biological or chemical synthetic macromolecule coupling formation.

21. have redox [redox] character, the give and accept nucleic acid binding substances of reaction [Electron Donor-Acceptor Reaction] or electron-transfer reaction [Electron-Transfer Reaction] and be the polymkeric substance that forms of monomer polymerization and oligopolymer, oligomer with these compounds of electronics can take place, can embed fluorescence molecule in the nucleic acid double chain as [a] has but is not limited to, methylene blue [Methylene Blue, MB ⁺] and fluorescence molecule such as Hoechst33258 etc., or [b] metal complex, [Rutheniumtris (2 as ruthenium metal complex, 2 '-bipyridine)] etc., or [c] connects by covalent linkage or the non covalent bond combination has the high lotus signal vehicle [HSC] of redox property with above-mentioned any nucleic acid intercalator generation covalent cross-linking or non covalent bond bonded, include but not limited to poly two metallocene class materials such as poly ferrocene group [polyferrocenyl], poly zirconocene group, poly ruthenocene group.

-L-can be but be not limited to following various structure or molecule: (1) solvent compatibility connecting arm, and promptly when solvent was hydrophobic systems, connecting arm also was hydrophobic connecting arm; When solvent is hydrophilic system, connecting arm also is hydrophilic connecting arm, can be PEG, synthetic polypeptide etc., length is not limit, (2) molecular wire [molecular wires, as double chain DNA molecule, the wetting ability connecting arm, pelletron type soccerballene simple linear polymer (soccerballene is imported into the pelletron type polymer of organic polymer main chain) or with soccerballene [bucky-ball, fullerene] the bucky-ball necklace that couples together with the R base etc.], (3) molecule isolator [molecular insulators is as hydrophobicity connecting arm, long chain alkane etc.], (4) be combined with the connecting arm etc. of molecular magnet, (5) under factor effects such as chemistry or optics, connecting arm that molecular chain can chain rupture etc. is as cystine linkage [SS-] etc.

F represents the high loading optics that exists with various molecule forms, magnetic, electronics or electrochemical signals carrier, it can be superpolymer or multipolymer fluorescent microsphere, finishing has the silica magnetic bead [silica beads] of fluorescence dye, the quantum dot of inorganicss such as semi-conductor [quantum dots], inorganicss such as semi-conductor nanocrystalline [nanocrystal particles], dendroid high polymer nano ball [dendrimers], the dendritic high polymer nano ball of star tree [dendrimeric stars], micella [micelles], molecular magnet [molecular magnets], capsule-type microballoon [encapsulated spheres], colloidal nano gold [colloidal gold nanoparticles], electroluminescent molecule [electroluminescentmolecules], poly two metallocene copolymer microspheres [polymetalcene block copolymers, as poly ferrocene multipolymer [polyferroceneblock copolymers, PFC] microballoon, poly zirconocene [polyzirconocene, PZC] copolymer microsphere or nanometer ball, contain fullerene structure or be the superpolymer or the multipolymer of one of monomer with the soccerballene, other contains fluorescence dye or chemoluminescence [chemiluminescence], the carrier of noclilucence [bioluminescence] material, and in above-mentioned each carrier different parameters [as size, the optical emitting optical wavelength, electromagnetic property, physics or chemical property such as the modification of surface biological or chemical functional group] combination of carrier, or the mutual combination between the various different carriers, its finishing have can with the functional group of probe molecule joint A-specific reaction in general formula (I) compound or effect to A '-.

Also at the hybridization or the probe-target-specific cohesive process of gene chip, mark has been introduced sequencing alternating electromagnetism field action comprehensively in the processes such as wash-out, be used to accelerate the efficient of said process in the present invention, shortens the process required time.

The definition of technical term involved in the present invention

[US.Pat 6 for people's such as following term of the present invention and Chenchik United States Patent (USP), 489,159] identical: peptide [peptide], oligopeptide [oligopeptide], polypeptide [polypeptide], nucleic acid [nucleic acid], Yeast Nucleic Acid [" ribonucleic acid " and " RNA "], DNA, oligonucleotide [oligonucleotide], polynucleotide [polynucleotide] etc.

High lotus signal vehicle [HSC]

The present invention as give a definition target and probe, superpolymer fluorescent microsphere or high molecular fluorescent microballoons [polymer microspheres] or colloid microballon [latex beads], quantum dot microsphere [QD-tagged Microbeads], silica magnetic bead [silica beads], the brilliant employing such as quantum dot [the colloidal fluorescent semiconductor nanocrystal quantum dots] term of etc.ing of colloid luminescent semiconductor nanocrystal by present universally recognized naming standard.

Example one present embodiment is used for gene chip carried out mark before capture probe and target gene hybridization embodiment, technical solution when X-selects psoralene [Psoralen] and derivative thereof for use [comprising gene chip hybridization chamber, HCL instrument and SPR-CCD signal detection and data handling system] can be referring to [the international patent application: gene chip molecular probe and correlation technique of patent application formerly.International application no, PCT/CN02/00887].These methods are for being suitable for sheet type gene chips such as glass, quartz, silicon, plastics and films.

Because the capture probe among the present invention is exactly little some gene probe that pin is used of some system on the gene chip substrate, belongs to very proven technique technology at present, therefore, no longer addresses the preparation of capture probe herein.The present invention will set forth the preparation of label probe emphatically.

The preparation of label probe divides three parts: with the base sequence in certain site on the target gene can specificity complementary nucleic acid probe the choosing and the two covalent linkage or non covalent bond coupling of design, high lotus signal vehicle.

(1) choosing of fluorescent microsphere: F-in the general formula (I) selects high molecular fluorescent microballoons, as the polystyrene fluorescent microsphere etc.Can purchase U.S. Molecular Probes company, the product of companies such as Duke company or Bang ' s Laboratories.About the exciting light/wavelength of transmitted light of fluorescent microsphere, consideration of the factor of the aspects such as chemical property on surface and solution thereof see also my Chinese invention patent application, and its application number is: 01142654.3.As most preferred embodiment, can select with market on gene chip scanning instrument exciting light/wavelength of transmitted light match, excitation wavelength is at the polystyrene fluorescent microsphere of 488 nanometers, 633 nanometers.

(2) covalent linkage of fluorescent microsphere and nucleic acid molecule or non covalent bond coupling: [1] selects the fluorescent microsphere of the surperficial carboxyl modified of Molecular Probes Inc company, [2] with synthetic 3 '-the terminal or amino oligonucleotide of 5 '-terminal band of dna synthesizer, [3] with water-soluble carbodiimide [EDAC] the former two is got up by the covalent linkage coupling, formation can complementary label probe of hybridizing with certain target gene.

(3), prepare and the specificity site difference complementary pairing of different target genes and the label probe of hybridization with (1) and (2) described method.

(4) hybridization: during hybridization different label probes and target gene solution are mixed, hybridize simultaneously with the capture probe on the gene chip.Crossover process is referring to Hames, B.D., and Higgins, people's such as S.J " nucleic acid hybridization, a kind of practical approach " etc.

Two other parts of example of example are identical with example one, just the F in the general formula (I) selects quantum dot [QDs], quantum dot microsphere [QD-taggedMicrobeads, or QD-tagged Polymeric Microspheres, (this is a kind of quantum dot to be embedded in a kind of HSC in the polymer microsphere to QD-Beads.Because QD can be embedded in the polymer microsphere in large quantities, thereby also is the extremely strong HSC of a kind of signal.In view of one side QD can realize by the adjusting of semiconductor nano particle diameter regulating on emission wavelength easily, polymer microsphere can change the light signal strength levels of quantum dot microsphere according to the ratio difference of embedding aspect embedding QD on the other hand, therefore, quantum dot microsphere [QD-Beads] can realized diversification and stratification aspect the wavelength of fluorescent emission and the intensity very ideally.In addition, under favourable preparation condition, quantum dot microsphere can reach the homogeneous of height, very valuable application value is arranged at aspects such as electrophoresis)], fluorescence dendrimer [dendrimers, be called for short DD], the dendritic superpolymer of star tree [dendrimeric stars, or star dendrimers, hereinafter to be referred as SD], molecular magnet [molecular magnets] electroluminescent molecule [electroluminescent molecules], poly two metallocene copolymer microspheres [polymetalcene block copolymers, as poly ferrocene multipolymer [polyferrocene block copolymers, PFC] microballoon, poly zirconocene [polyzirconocene, PZC] copolymer microsphere or nanometer ball etc.

The preparation of high lotus signal vehicle SD can be selected people's such as Ronald C Hedden method for use, makes poly-PAMAM[poly (amidoamine) earlier] SD nuclear.In this SD nuclear, grow metal [as gold, platinum, palladium etc.] or semi-conductor [nanocrystalline as CdS, as to be controlled at the 2-10 nanometer usually].On PAMAMSD nuclear surface a large amount of amidos is arranged, can connect PEG[in adding is polyoxyethylene glycol, poly (ethylene glycol), 5,000g/mol] arm the time, mix maleimide-PEG-N-hydroxyl-succinimide ester [Maleimide PEG NHS ester according to certain ratio [ratio of MAL-PEG-NHS and PEG-NHS is 1: 10], write a Chinese character in simplified form: MAL-PEG-NHS has commercially available.Apollo Scientific Ltd, Http:// www.apolloscientific.co.uk/otherProducts_Lifesciences_PE Gcross.htm], thereby make SD[SD (PEG-MAL) n that dimaleoyl imino is modified], as the F in the general formula (I).

Synthetic 3 '-end or 5 '-end are specificity site sequence complementary nucleic acid probe sulfydryl modification and a certain target gene, carry out the covalent linkage coupling with SD (PEG-MAL) n under aqueous environment.Coupling process and crossover process are referring to the relevant portion of example one and my Chinese invention patent application: microarray method for amplifying signal, application number: 03129124.4.

Example three is selected Platinol [cisplatin, i.e. (cis-diamminedichloroplatinum (II))] and derivative thereof [need not photochemical crosslinking] for use as the X-in the general formula (I), or optical excitation intercalator ruthenium complex [Ru (phen) 2dppz ²⁺Phen=1,10-phenanthroline; Dppz=dipyridophenazine], or alkylide mustargen [be dichloromethyldiethylamine [mechlorethamine (ME)], or claim nitrogenmustard, need not optical excitation crosslinked] etc.With the ruthenium complex is that X-is an example, must synthesize the embodiment of ruthenium complex succinate as general formula (I) X-L-A compound.For this reason, can adopt people's such as Dimitri Ossipov synthetic technology) the synthetic ruthenium complex succinate of solid phase synthesis technique [succinate 3 '-[Ru (tpy) is Cl (dppz)] ⁺Wherein, conduct of succsinic acid ester group and a large amount of primary amine groups [NH that modify in high lotus signal vehicle surface ₂, A '-] can carry out specificity covalent linkage labeled reactant by specific reaction centering reactive group [A-].

Other part technical solutions can be referring to patent application formerly [international patent application: gene chip molecular probe and correlation technique].

Example four substitutes the nucleic acid marking probe of the aminoterminal nucleic acid marking probe in the example one with the vitamin H end.And the polystyrene fluorescent microsphere of buying strepto-affinity element [streptavidin] pan coating replaces the polystyrene fluorescent microsphere of surperficial carboxyl modified.Other conditions are identical.

Example five present embodiments are two probe duochrome method gene chip hybridization signal magnifying tags.The purpose of duochrome method mark mainly is in order to detect the target gene from different cell or tissue sample sources simultaneously with a gene chip, and on same gene chip, compare by competitive hybridization, judge the target gene abundance or the relative values such as content or expression ratio in two kinds of sources according to final shown color of coming out.This method is especially often used in analyzing gene differential expression spectrum.At present, usually adopt to be incorporated in the PCR product the time by the base of Cy3 and Cy5 mark respectively, and be mixed together and be used for the marker gene chip sample DNA or RNA pcr amplification.The HSC signal amplification technique that is disclosed among the present invention equally can be with two probe duochrome method marks from target gene and expression ratio thereof in different sources DNA or the RNA sample, or the different expression ratio of the gene in same source under different condition, and on gene chip, detect.

Label probe synthesis step [the different target gene in source that the purpose of this step is a complex sign probe hybridization mark.Be equivalent to present gene chip commonly used be used for detecting difference expression gene the time be used for gene chip on the target gene of probe microarray hybridization.I.e. " label probe+target gene=labels targets gene "]:

(1) the synthetic earlier DNA that modifies as the terminal amino group of example one described and various target gene specific site complementary pairings, mix, under the water-soluble carbodiimide effect, carry out the covalent linkage coupling with the polystyrene microsphere of the surperficial carboxyl modified of different fluorescent emission wavelength [excitation wavelength is respectively at 488nm and 633nm] respectively with the above-mentioned dna solution of equivalent, forming label probe.

The label probe of (2) two kinds of different wavelength of transmitted light is hybridized with DNA or RNA target gene from different samples respectively, and it is covalently cross-linked with psoralene heteroduplex to be taken place, and forms two kinds of different labels targets genes respectively.Above-mentioned two solution [each 30 microlitre] of equivalent are mixed and hybridize with the capture probe on the gene chip.Hybridization buffer is: (5 * SSC, 6 * Denhardt ' s solution, 60mM Tris HCl pH7.6,0.12% sarkosyl, 48% formamide; Filter sterilised).42-47 degree centigrade 2-4 hour.

By my international patent application [gene chip molecular probe and correlation technique.International application no, PCT/CN02/00887] in the HCL instrument, crossover process is as follows:

Prehybridization:

(1) the gene chip hybridization chamber is pre-chilled to 4 degrees centigrade, the hybridization buffer of 50 microlitres is added wherein, the aperture of micro-hybridization buffer from front, gene chip hybridization chamber added, clog preventing the damping fluid volatilization with little soft rubber ball, the humidity of maintenance crossover process,

(2) bathed 2-4 hour 47 degrees centigrade of temperature,

(3) at room temperature wash gene chip 15-30 second with at least 250 microlitre elutriants,

(4) imported dry air 1-2 minute, with gene chip hybridization chamber airing.

Hybridization:

(1) two kinds of strand cDNA label probe processes of mixing are as follows: 33 μ l ss cDNA labels targets genes (488nm), 33 μ l ss cDNA labels targets genes (633nm), 4 μ l polyA DNA, human Cot1 DNA of 4 μ l and 7.0 μ l 3M sodium acetate pH5.2,218 μ l, absolute ethanol ≈ 293 μ l

(2) precipitation ss cDNAs, polyA and Cot1 DNA ,-70 ℃ 20 minutes,

(3) add the gene chip hybridization chamber, 47 ℃ of warm down baths 12-24 hour.Wash-out under the room temperature, airing,

(4), the fluorescence of above-mentioned two wavelength is detected with laser co-focusing or CCD detector.

Example six other processes are as described in the example five.Present embodiment is made label probe respectively with QD, the SD etc. of different emitting fluorescence wavelength and is used for hybridizing with DNA or RNA sample from different samples.The present embodiment nanocrystalline replacement polystyrene of semiconductor-quantum-point fluorescent microsphere.

Example seven present embodiments are that the high lotus signal vehicle [HSC] of the different sizes of different sorts are to before the gene chip hybridization or the combined mark of carrying out after the hybridization.Because being the signaling molecule that relies on the different sizes and the HSC of kind can carry different amounts, the signal amplification technique that the present invention introduced carries out the signal amplification, the carry-on signaling molecule of the HSC that size is big more is many more, can send strong more signal in signal detection, its signal amplifying power is just strong more.But, along with the increase of HSC size, they on being tagged to gene chip after occupied space will be big more.Below be the * that concerns between the size of the fluorescence polymer microballoon that provides by U.S. Molecular Probes company and the entrained signaling molecule [fluorescein equivalent].

The fluorescein equivalent of each fluorescent microsphere of fluorescent microsphere diameter [micron]

0.02 1.8×10 ²

0.04 3.5×10 ²

0.1 7.4×10 ³

0.2 1.1×10 ⁵

0.5 2.0×10 ⁶

1.0 1.3×10 ⁷

2.0 3.1×10 ⁷

10 1.1×10 ¹⁰

15 3.7×10 ¹⁰

^*Annotate: this table is selected from " Handbook of Fluorescent Probes and Research Products " the 9th edition, the 177th page.by?RichardP.Haugland。

Can calculate the gene chip spatial area that the fluorescent microsphere of every kind of size can occupy according to these data, and the diameter that calculates the gene point of fluorescent microsphere on gene chip by using this size then is 200 microns [occupied gene chip area is 10 ⁴The π micron ²] time the theoretical sensitivity that can reach detect linearity range [seeing the following form].Some diameter dimensions have also been attached in the table [generally in the 2-5 nanometer about the nanocrystalline quantum dot of colloid semi-conductor [CdSe] [QDs].Value 4 nanometers herein.According to its strength of signal of report is general fluorescent signal molecule, as 20 times of fluorescein intensity].[according to the introduction of Affymetrix company, they rely on this technology for detection lower sensitivity limit is 300,000 molecules by means of the laser co-focusing gene chip tester.When promptly only 300,000 dna moleculars being arranged in sample to be measured, adopt to detect obtain] lower sensitivity limit [but detection molecules number] that can reach.

Each fluorescent microsphere fluorescent microsphere can mark on each gene point of gene chip the scanning of maximum fluorescence laser co-focusing gene chip

The fluorescein equivalent on occupy area [micron ²] microballoon number [sensitivity linear upper limit of detection] the instrument lower sensitivity limit that can reach

1.8×10 ² 1×10 ^-4π 1×10 ⁸ 1667

3.5×10 ² 4×10 ^-4π 2.5×10 ⁷ 857

7.4×10 ³ 25×10 ^-4π 4×10 ⁶ 40

1.1×10 ⁵ 1×10 ^-2π 1×10 ⁶ 3

2.0×10 ⁶ 0.0625π 1.6×10 ⁵ 0.15

1.3×10 ⁷ 0.25π 4×10 ⁴ -

3.1×10 ⁷ π 1×10 ⁴ -

1.1×10 ¹⁰ 25π 4×10 ² -

3.7×1010 56.25π 1.7×10 ² -

The fluorescein QD of QD account on the gene chip on each gene point can mark maximum QD laser co-focusing gene chip scanning

The area of equivalent certificate [micron ²] number [sensitivity linear upper limit of detection] the instrument lower sensitivity limit that can reach

20 1.6×10 ^-5π 6.25×10 ⁸ 1.5×10 ⁴

When sensitivity improved, the linearity range order of magnitude of detection was roughly constant as can be seen from the above table, and the upper limit of linearity test begins to move down.Therefore, between the lower sensitivity limit and the upper limit, exist certain contradiction.Along with sensitivity improves, the upper limit of the sensing range of sensitivity can descend.

In order to overcome this contradiction, present embodiment adopts the HSC of unlike signal amplifying power to unite the technology of use.That is, adopt same a kind of color or the high molecular fluorescent microballoons of different colours, perhaps the fluorescence polymer microballoon of large-size and being used in combination of QD of different size [being the unlike signal amplifying power].During selection, make every effort to the upper sensitivity limit of HSC of different varieties, specification or color and the linking of lower limit as far as possible, rolling off the production line with the low linearity test of high signal amplifying power HSC with the high linearity test upper limit of the HSC that guarantees the low signal amplifying power is connected, thereby has complementary advantages.Such as, [the linear sensing range of sensitivity is 3～10 to adopt the high molecular fluorescent microballoons of 200 nanometer diameters ⁶Individual molecule]] with the linear sensing range of the semiconductor nano QD[sensitivity of 4 nanometer diameters be 1.5 * 10 ⁴～6.25 * 10 ⁸Individual molecule] unite use, can expand the linearity range of actual measurement to 3～6.25 * 10 ⁸Individual molecule.Certainly, also can select to unite use, thereby linear detection range is expanded to 3～1 * 10 with the high molecular fluorescent microballoons of 20 nanometer diameters ⁸Individual molecule.

Different-grain diameter, different emission light colors [different wavelength of fluorescence] and the fluorescence polymer microballoon that [in order to be connected different connecting arms] modified by different surfaces chemical functional group can directly be bought [Molecular Probes, Merck, Bangs Laboratories company., Brookhaven Instr Ltd. or Duke scientific company etc.].

The surface chemical modification of QD and covalent coupling technology can be with reference to people's such as Bawendi United States Patent (USP): US.Pat 6,306,610, October 23 calendar year 2001.

The quantum dot microsphere of HSC such as different emission wavelengths and strength of signal, polymer microsphere, quantum dot etc. all are extraordinary selections.

Claims

1. two probe gene chip signal magnifying tags technology, it is characterized in that, two probes are made up of capture probe [Capture Probe] and label probe [Labeling Probe], capture probe is fixed in the gene chip solid phase surface, constitute gene chip micro lattice, and label probe is by high lotus signal vehicle and one or more its surface of being fixed in, can with target gene on one or the sequence in the more than site specific nucleic acid sequence of carrying out complementary hybridization constitute, free being present in the label solution, the two all has the nucleotide sequence with the specific sequence complementary pairing of the different loci of target gene to be measured respectively.

2. gene chip according to claim 1, coating or coating by substrate and surface thereof are formed with the capture probe that is no less than two arrays arrangements that is fixed on substrate [being abbreviated as S] or coating [being abbreviated as C] surface, the gene target gene that is used for test sample, the material of substrate and/or coating or coating can by but be not limited in the following material any one or more than one differing materials combination and make:

(1) inorganic sheet material type such as glass, quartz, mica, transparent conductive material such as transparent conductive oxide class [TCO] are as tin indium oxide [indium tin oxide, ITO, or be written as In ₂O ₃: Sn], SnO ₂: coating such as F, gallium arsenide [GaAs], magnesium oxide, stannic oxide [tin oxide, SnO], ZnO, CdO, CdIn ₂O ₄, Cd ₂SnO ₄, Zn ₂SnO ₄And In ₂O ₃-ZnO etc., and carbon/pottery composite conductive ceramic, various semiconductor materials etc.,

(2) simple substance class: platinum, gold and silver, aluminium, chromium metals such as [Au, Ag, Pt, Cu, Rh, Pd, Al, Cr] and silicon are nonmetal etc.,

(4) high score subclass: nylon membrane, cellulose acetate membrane, various plastics, rubber, resin,

The structure of substrate can be but be not limited to following kind:

(1) sheet type: as sheet glass, silicon chip, nylon membrane, plastic sheet, microwell plate, droplet is board-like etc.,

(2) electric pole type: on the sheet type gene chip, arrange the various electrod-arrays form with certain rule, as the platinum electrode in substrates such as semi-conductor, isolator, made, gold electrode etc.,

(3) micro-tubular: by kapillary or other material of kapillary tubular intracavity constructional feature is arranged, arrange various matrix form kapillary gene chips [two dimensional surface arrangement], linear kapillary gene chip [one dimension linear array] that forms and the various matrix form parallel arranged of in substrate formula structure, producing [being microfluid electrophoresis chip etc.] that are no less than a microchannel formula cavity structure by the predefined position rule of correspondence

(4) particulate formula: with the molecule is solid phase carrier, biomolecules or chemical molecular are fixed on its surface, size by molecule, [luminous] color, surface potential, thereby character elements such as surface charge density carry out the particulate that sort merge produces a large amount of various combinations, the molecule of combination [specific combination of a certain size and certain emission wavelength etc.---coding] of using a certain particular group parameter respectively is as the carrier of certain biological or chemical molecule, thereby can make gene chip with a large amount of differences and specific coding molecule

3. capture probe according to claim 1, label probe or target gene can be, but be not limited to one of following kind or different types of combination:

(2) thymus nucleic acid [DNA] is as DNA, complementary DNA [cDNA] etc.,

(5) hybrid molecule of forms such as the complementary two strands of thymus nucleic acid, Yeast Nucleic Acid and structural similitude thing thereof, three [weight] chain, four [weight] chain such as DNA-DNA, RNA-DNA, RNA-RNA, PNA-DNA, PNA-RNA, PNA-RNA-PNA, PNA-DNA-PNA] etc.,

The detection principle of gene chip can be, but is not limited to the combination of one of following kind or different principle:

(1) probe by solid phase surface being positioned at different positions-target-specific hybrid reaction carrying out optical signalling [as fluorescence molecule, chemoluminescence etc.] mark produces fluorescence, chemistry or noclilucence and detects through exciting then,

(2) by translational speed or the mobility in the liquid phase that has applied voltage detects to probe mark or unlabelled or target, be electrophoretic technique or combine with other technologies and the other technologies that produce by electrophoretic technique, as capillary electrophoresis [capillary electrophoresis, CE], capillary zone electrophoresis [capillary zone electrophoresis, CZE], capillary gel electrophoresis [CGE], HPCE [HPCE], capillary isoelectric focusing [capillary isoelectric focusing (CIEF)], isotachophoresis [isotachophoresis (ITP)], electrokinetic chromatography [electrokinetic chromatography (EKC)], mcellar electrokinetic capillary chromatography [micellar electrokinetic capillary chromatography (MECC OR MEKC)], capillary electrochromatography method [capillary electrochromatography (CEC)], nonaqueous phase capillary electrophoresis [non-aqueous capillary electrophoresis (NACE)] etc.

(3) by probe, target or probe-target complex compound and the different avidity of their marker, carry out the chromatographic technique analysis, as affinity chromatography [affinity chromatography], SEC[size exclusion chromatography], GPC[gel permeationchromatography], MCC[metal chelate chromatography] etc.

(4) utilization has the molecule of redox property or the part-structure of molecule [being chemical group or functional group], metal complex or (nucleic acid) intercalator be positioned and be fixed on probe on solid phase surface different positions or the electrode, target or probe-target complex compound combination is also adding under suitable mode or suitable big or small voltage (or potential difference) effect (volts DS or exchange current or pulsed electrical field or voltage), redox reaction takes place, cause from reductibility molecule or group (electron donor, electron donor) transfer transport is to oxidisability molecule or group (electron acceptor(EA), electron acceptor), form stream of electrons, being sent to detection system by electrode measures, measured strength of current size is directly proportional with the amount of the double-strandednucleic acid of hybridizing and being labeled

(7), thereby realize detection to the biological or chemical reaction that occurs in electrode surface by making marker electron-transfer reaction take place behind the mark near electrode surface directly or indirectly having oxidation-reduction quality material (as ferrocenyl) to probe or target molecule marker.

4. according to claim 1 pair of probe gene chip signal amplification technique, its labeled reactant pattern can be, but be not limited to the described several sights of following general formula:

(1) target gene and the label probe in single sample source are preferentially hybridized, after heteroduplex carries out or does not carry out interchain linkage or locking, again with gene chip on the capture probe specific hybrid, equally after capture probe and the formed heteroduplex of target gene carry out or do not carry out interchain linkage or locking, form the target gene of two probe hybridization marks

(2) target gene in two or more different samples source is preferentially hybridized with the label probe of different qualities respectively, thereby the target gene of different sources is defined separately by specific label probe, after heteroduplex carries out or does not carry out interchain linkage or locking, again with gene chip on the capture probe specific hybrid, equally after capture probe and the formed heteroduplex of target gene carry out or do not carry out interchain linkage or locking, form the target gene of two probe hybridization marks

The structure of label probe can be used general formula (I) expression,

General formula (I): F (L ₂-XNA) _n, or F is (XNA) _n,

X-passes through electrostatic attraction with double-strandednucleic acid, inferior bonding force is [as Van der Waals force, hydrogen bond etc.], with the major groove in the double-spiral structure or the avidity [Groove binders] of ditch, embed the avidity [intercalators] of double-stranded base pair, form the functional group that modes such as triple-helix structure or four spirane structures combine with double-spiral structure, with functional group or the molecule of nucleic acid double-spiral structure by chemical reaction formation covalent bonds, can specific recognition nucleic acid double chain structure and bonded biomolecules with it, can with nucleic acid double chain complexing or the various biological or chemical molecules of bonded, it can include but not limited to following compounds and be the various derivatives of architecture basics or feature and constitutional features that biological or chemical is modified back formed compound: X-with the following compounds according to its with can be divided into following several types for the hybridization single-chain nucleic acid or with the nucleic acid double-spiral structure generation interactive property of complementary hybridization: (1) electrostatic attraction, inferior bonding force is [as Van der Waals force, hydrogen bond etc.], with the major groove in the nucleic acid double-spiral structure or the avidity [Groove binders] of ditch.Can with the various chemical cross-linking agents of nucleic acid probe or target gene generation covalent cross-linking or chemical reaction functional group as can by with target gene on hydroxyl take place the to shrink carbodiimide of reaction, or the active linking agent of photochemical reaction, as aromatic base triazo-compound [aryl azides, as N-((2-pyridyldithio) ethyl)-4-azidosalicylamide[PEAS] etc.], fluoro aromatic base triazo-compound [fluorinated aryl azides], benzophenone class photoresponse reagent [benzophenone-based photoreactive reagents is as benzophenone maleimide [benzophenonemaleimide]] etc.; (18) can sequence-specific ground or non-sequence-specific ground identification nucleic acid double chain structure and with it in conjunction with or be biomolecules such as the protein of substrate or enzyme with the double-spiral structure of double-strandednucleic acid, as HMGB albumen, can with the dsDNA bonded NHP6A albumen of modifying through Platinol, participate in the enzyme of dna replication dna, zymoplasm [thrombin], the enzyme mitomycin of rna transcription [Mitomycin C], can and the double-stranded two strands of nucleic acid between form the bleomycin or the bleomycin class [Bleomycin] of covalency bridge shape key, double-strandednucleic acid antibody, the specificity desmoenzyme of double-strandednucleic acid, archaeal dna polymerase or RNA polymerase, (19) can carry out the complementary oligonucleotide molecule that forms complementary duplex structure that also combines of specificity with certain or some specific sequences of probe nucleic acid molecule on target gene to be measured or the gene chip, antisense nucleic acid [anti-sense nucleic acidsequences] sequence etc., this compounds can be applicable to the preceding mark of hybridization and two kinds of schemes of hybridization back mark simultaneously, (20) has redox [redox] character, the give and accept nucleic acid binding substances of reaction [Electron Donor-Acceptor Reaction] or electron-transfer reaction [Electron-TransferReaction] and be polymkeric substance and the oligopolymer that monomer polymerization forms with these compounds of electronics can take place, oligomer, can embed fluorescence molecule in the nucleic acid double chain as [a] has but is not limited to, methylene blue [Methylene Blue, MB ⁺] and fluorescence molecule such as Hoechst33258 etc., or [b] metal complex, as ruthenium metal complex [Ruthenium tris (2,2 '-bipyridine)] etc., or [c] connects by covalent linkage or the non covalent bond combination has the high lotus signal vehicle [HSC] of redox property with above-mentioned any nucleic acid intercalator generation covalent cross-linking or non covalent bond bonded, include but not limited to, poly ferrocene group [polyferrocenyl], poly zirconocene group, poly two metallocene class materials such as poly ruthenocene group, (21) nucleic acid construct analogue [nucleic acid mimics], as PNA[peptide nucleic acids], can form the stable bond thing with nucleic acid

-L-can be but be not limited to following various structure or molecule: (1) solvent compatibility connecting arm, and promptly when solvent was hydrophobic systems, connecting arm also was hydrophobic connecting arm; When solvent is hydrophilic system, connecting arm also is hydrophilic connecting arm, can be PEG, synthetic polypeptide etc., length is not limit, (2) molecular wire [molecular wires, as double chain DNA molecule, the wetting ability connecting arm, pelletron type soccerballene simple linear polymer (soccerballene is imported into the pelletron type polymer of organic polymer main chain) or with soccerballene [bucky-ball, fullerene] the bucky-ball necklace that couples together with the R base etc.], [molecular insulators is as the hydrophobicity connecting arm for (3) molecule isolator, long chain alkane etc.], (4) be combined with the connecting arm of molecular magnet, (5) under factor effects such as chemistry or optics, connecting arm that molecular chain can chain rupture etc., as cystine linkage [SS-] etc.

F represents the high-load optics that exists with various molecule forms; magnetic; electronics or electrochemical signals carrier; it can be superpolymer or multipolymer fluorescent microsphere; finishing has the silica magnetic bead [silica beads] of fluorescence dye; semiconductor nano quantum dot [quantumdots; QDs]; the product quantum dot microsphere that polymer microsphere combines with quantum dot [QD-tagged nanobeads or microspheres]; semiconductor nanoparticle or the microparticle or the quantum dot [as Chaperonin mediated semiconductor nanoparticles] of protein parcel; dendrimer or dendrimer [dendrimers]; star dendrimer [dendrimeric stars]; micella [micelles]; molecular magnet [molecular magnets]; capsule-type microballoon [encapsulated spheres]; colloidal nano gold [colloidal gold nanoparticles]; electroluminescent molecule [electroluminescent molecules]; two Metallocenyl copolymer microspheres [polymetalcene block copolymers; as ferrocene-based polymer [polyferrocene block copolymers; PFC; or polyferrocenylsilanes; PFS] microballoon; poly zirconocene [polyzirconocene; PZC]; poly two Metallocenyls are modified or the grafted dendrimer [includes but not limited to; poly-ferrocene base grafted dendrimer (polyferrocenyl-branched dendrimers)]; two Metallocenyls or poly two Metallocenyls are modified or grafted nanometer gold colloid [includes but not limited to; the nanometer gold colloid of amido ferrocenyl functionalization (Gold colloids functionalized withamidoferrocenyl structures) etc.]; copolymer microsphere or nanometer ball; contain fullerene structure or be the superpolymer or the multipolymer of one of monomer with the soccerballene; other contains fluorescence dye or chemoluminescence [chemiluminescence]; the carrier of noclilucence [bioluminescence] material; phycobiliprotein class [phycobiliproteins] and various derivative thereof or biological or chemical modifier; and in above-mentioned each carrier different parameters [as size; the optical emitting optical wavelength; electromagnetic property; physics or chemical property such as the modification of surface biological or chemical functional group] combination of carrier; or the mutual combination between the various different carriers

N be from zero to infinitely-great natural number: 0,1,2,3...,

XNA represents nucleic acid and analog thereof, as double-strandednucleic acid [containing oligonucleotide] and resemblance [DNA, the RNA of strand or complementary hybridization, PNA, DNA-DNA, RNA-RNA, RNA-DNA or RNA-DNA mimic duplexes, triplexes or quadroplexes, PNA-DNA, PNA-RNA, PNA-RNA-PNA, PNA-DNA-PNA etc.]

5. two probe signals marks of making according to claim 1 of gene chip and amplify test kit, promptly the BAT test kit has comprised gene chip and has handled the needed whole biochemical reagents of each process, contains but is not limited to the represented core component of following general formula:

General formula (I) compound.