CN1435493A - Solid phase nucleic acid detection probe and preparing method thereof - Google Patents
Solid phase nucleic acid detection probe and preparing method thereof Download PDFInfo
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- CN1435493A CN1435493A CN 03112923 CN03112923A CN1435493A CN 1435493 A CN1435493 A CN 1435493A CN 03112923 CN03112923 CN 03112923 CN 03112923 A CN03112923 A CN 03112923A CN 1435493 A CN1435493 A CN 1435493A
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Abstract
固相化核酸检测探针及其制备方法是一种固定在固体基片上寡核苷酸探针及其用此法制作的微阵列芯片，是一种检测核酸序列信息的非标记的寡核苷酸探针，该探针在固体基片1上通过手臂分子2固定有荧光淬灭材料3，在荧光淬灭材料3表面上制备有由荧光基团5、寡核苷酸探针分子的茎杆部6、寡核苷酸探针的环部7组成的寡核苷酸探针，寡核苷酸探针的一端固定在荧光淬灭材料3表面上，寡核苷酸探针的另一端附近的碱基标记有荧光基团5，寡核苷酸探针两端附近的序列分别有3至15个碱基为互补序列，可使该寡核苷酸探针两端附近的序列能够形成杂交，寡核苷酸探针中间部分的碱基序列为被检测的核酸序列的互补序列。
The solid-phase nucleic acid detection probe and its preparation method are an oligonucleotide probe fixed on a solid substrate and a microarray chip made by this method, and a non-labeled oligonucleotide for detecting nucleic acid sequence information An acid probe, the probe is fixed with a fluorescence quenching material 3 through an arm molecule 2 on a solid substrate 1, and a stem composed of a fluorescent group 5 and an oligonucleotide probe molecule is prepared on the surface of the fluorescence quenching material 3. An oligonucleotide probe composed of a rod portion 6 and a ring portion 7 of the oligonucleotide probe, one end of the oligonucleotide probe is fixed on the surface of the fluorescence quenching material 3, and the other end of the oligonucleotide probe The nearby bases are marked with a fluorescent group 5, and the sequences near both ends of the oligonucleotide probe have 3 to 15 bases as complementary sequences, which enable the sequences near the two ends of the oligonucleotide probe to form In hybridization, the base sequence in the middle part of the oligonucleotide probe is the complementary sequence of the detected nucleic acid sequence.
Description
One, technical field
The present invention relates to a kind of micro-array chip that is fixed on oligonucleotide probe on the solid substrate and makes of this method, is a kind of cold oligonucleotide probe that detects nucleic acid sequence information.
Two, background technology
Along with going deep into of genome research, from the difference of gene level understanding life, disease takes place, the rule of development, and the interaction of medicine and life entity will become possibility.The high throughput testing of nucleic acid sequence information and analytical technology will become one of core technology of life sciences such as medical science.People need be developed the detection method of high-throughput, accurate, low-cost gene information.Recently, biochip technology more and more is subject to people's attention.But biochip technology remains in some problems at present, influences its application in biology and clinical medicine.1) testing process complexity.The sample DNA that extracts need increase, and mixes fluorescent marker.In this process,, influence the reliability that detects because of the labeling effciency problem.2) though the non-marked that has some technology can carry out gene order at present detect, all not really ripe.Therefore, can to carry out the biochip technology cheaply that non-marked detects to detected gene order be the key that realizes biochip a large amount of practical applications in fields such as medical science and life science in development.3) the single base mismatch detection still is a difficult problem at present, and main method is still measured the sequence of nucleic acid, measures not accurate enough convenience for the transgenation of heterozygosity.(4) detection method of primer specificity still can not be used for the detection of heterozygosity transgenation.
Three, summary of the invention
1, goal of the invention:
The purpose of this invention is to provide immobilization detection of nucleic acids probe non-a kind of mark, that hang down use cost and high reliability ground detection nucleotide sequence gene chip and preparation method thereof.
2, technical scheme:
The present invention is a kind of immobilization detection of nucleic acids probe, on solid substrate, be fixed with the fluorescent quenching material by arm molecule, preparing on the fluorescent quenching material surface has by fluorophor, the stipe part of oligonucleotide probe molecule, the oligonucleotide probe that the ring portion of oligonucleotide probe is formed, one end of oligonucleotide probe is fixed on the fluorescent quenching material surface, near the other end of oligonucleotide probe base is marked with fluorophor, it is complementary sequence that near the oligonucleotide probe two ends sequence has 3 to 15 bases respectively, can make near the sequence in these oligonucleotide probe two ends can form hybridization, the base sequence of oligonucleotide probe middle portion is the complementary sequence of detected nucleotide sequence, oligonucleotide probe is the oligonucleotide probe array that multiple probe is formed, it is gene chip, the immobilized oligonucleotide probe is a thymus nucleic acid, or Yeast Nucleic Acid, peptide nucleic acid(PNA) or their combination, fluorescent quenching material on the solid substrate of immobilized oligonucleotide probe can be a nano particle, comprises metal nanoparticle, metal oxide nanoparticles, the metal-salt nano particle; Also can be macromolecular material and the composite high-molecular material that includes the fluorescent quenching group, fluorescent quenching material on the solid substrate of immobilized oligonucleotide probe can be direct covalently bound fluorescent quenching group on solid substrate, be fixed with the solid substrate of nano particle, its material is a glass, silicon, pottery, plastics, cellulose nitrate, a kind of in nylon or the rubber, method is: a, with difunctional active agent by chemical reaction active group on the solid substrate surface bond, it is arm molecule, b, has on the solid substrate of active group fixedly fluorescent quenching material, c, surface at the fluorescent quenching material is modified with the agent of dual-functional group group chemistry degree, form arm molecule, d, one of the oligonucleotide probe of the specific nucleic acid sequence that includes at least one fluorescence chromophoric group that solid state chemistry is synthetic good terminates on the fluorescent quenching material, make the preparation of fluorescent quenching material surface go up oligonucleotide probe, the method for preparing oligonucleotide probe on the fluorescent quenching material surface is an in-situ synthesis, promptly directly at fluorescent quenching material surface chemical synthesising DNA probe, 3 ' end of oligonucleotide probe is fixed on the fluorescent quenching material, the method that on the fluorescent quenching material surface, prepares oligonucleotide probe be with chemical process will be in advance synthetic oligonucleotide probe overall fixed on the fluorescent quenching material, oligonucleotide probe can be whole original position synthetic, also can be that the part original position is synthetic, connect by chemical group then and form complete oligonucleotide probe, the method that on solid substrate, prepares the nm gold particles film be to use vapour deposition method with material evaporations such as metals on the solid substrate surface, form compact arranged nanometer particle film.
The present invention is by the method for chemistry or physics; nano-metal particle is fixed on the solid substrate; the perhaps metallic film of evaporation one deck nanometer grade thickness on solid substrate; modify chemical group at nano level metallic particles or film surface, synthetic or other original position synthetic method synthesizes nucleic acid at the solid phase substrate surface by the molecular seal original position then.When synthetic last several base, a synthetic amino or fluorescently-labeled nucleic acid monomer directly synthesized on nucleic acid chains on base therein selectively.Because it is complementary that the probe of the detection usefulness of design has base sequence at its two ends; after synthetic the finishing; chip can be formed secondary structure in damping fluid; at this moment; nano-metal particle or film can make the fluorophor emitted fluorescence cancellation of modifying on the nucleic acid effectively, and probe can not detect fluorescence.But after the identification of detected target gene and probe, make fluorophor away from nano-metal particle behind target gene and the probe hybridization, thereby fluorophor can be excited and detect the fluorescence that it sends.
If this kind stationary probe method is used for the probe of the tested nucleotide sequence of difference is constituted microarray, promptly constitutes a kind of original position synthetic non-marked gene chip micro-array chip.The preparation method of this novel immobilization nucleic acid probe is as follows: nano-metal particle is fixed on the material of solid phase by arm molecule, two or more mark fluorescent oligonucleotide probe of chemosynthesis is connected on the solid phase carrier by covalent bonds or physical adsorption.This probe comprises one or more fluorescence molecule groups, because transmission ofenergy or the effect of electronic cloud eclipsed, the fluorescent signal of fluorescence molecule group is by the cancellation of nano-metal particle institute; The fixed label probe can be that strand or oneself form various ways such as secondary structure, can constitute micro-array chip.
Its principle is to introduce nano-metal particle and fluorescence chromophoric group in nucleic acid probe, by under tested nucleic acid and interaction probe, realizes the non-marked of tested nucleotide sequence (target sequence) in the biological sample is measured.The non-marked that this novel nucleic acid probe and micro-array chip thereof can be used for bio-science and medical field amplifying nucleic acid sequence detects.
3, technique effect:
Immobilization nucleic acid probe that the present invention proposes and preparation method thereof has following characteristics: need not be carried out fluorescent mark or isotopic labeling by cls gene; Can improve the mispairing recognition capability of single base greatly by by cls gene and the hybridization of being at war with property of fixed probe; Probe can directly be fixed on the solid carrier by multi-form, form the micro-array chip of higher density, utilize the space to offer an explanation to distinguish different detection site, realize that high-flux parallel detects, avoided the selection of fluorescence dye in the existing quantitative PCR instrument to be subject to the problem of excitation wavelength range; For not with the probe of target molecule hybridization, the fluorescence of its fluorescence molecule group can be by the efficient cancellation of nano-metal particle, so the fluorescence background of entire chip is very low, detection highly sensitive; Can carry out the detection by quantitative of target gene; By making up micro-fluid chip, the automatization that realizes whole testing process can be arranged.
Compare with prior art, this method has following advantage: utilize the nucleic acid hybridization principle of dynamics, detected nucleic acid competitiveness and the fluorophor labeling nucleic acid probe hybridization that is fixed on the nano-metal particle, have good signal-to-noise, successfully solved the technical barrier that single base mismatch detects.Simultaneously, this detection method non-marked that can realize detecting sample detects and (can realize that sample high-throughput, cold automatization detect.) significantly reduced and detected the required time, and have the potentiality that realize micro-total analysis etc.Not only shortened Diagnostic Time greatly, and improved the accuracy that single base mismatch detects greatly, reduced the detection cost, the detection of especially can tumour relevant single base mismatch polymorphism.
The present invention proposes immobilization nucleic acid probe and micro-array chip thereof, and genome times afterwards comprehensively non-marked, real-time, high-throughout nucleotide sequence are detected, and particularly single base polymorphisms detects, and oncogene polymorphism detection etc. all has significant application value.
Four, description of drawings
Fig. 1 is the structural representation that oligonucleotide probe is synthesized or fixed on the nm gold particles surface on the solid substrate 1.
Fig. 2 is on the solid substrate 1 behind fluorescent quenching material 3 surperficial synthetic or fixed oligonucleotide probe and the detected nucleic acid hybridization, and the fluorophor 5 on the oligonucleotide probe sends the synoptic diagram of fluorescence away from fluorescent quenching material 3 surfaces after the irradiation of Stimulated Light.
Have among the above figure: solid substrate 1, arm molecule 2,4, fluorescent quenching material 3, fluorophor 5, the stipe part 6 of oligonucleotide probe molecule, the ring portion 7 of oligonucleotide probe, tested nucleic acid molecule 8.
Five, embodiment
The present invention is a kind of immobilization detection of nucleic acids probe, on solid substrate 1, be fixed with fluorescent quenching material 3 by arm molecule 2, preparing on fluorescent quenching material 3 surfaces has by fluorophor 5, the stipe part 6 of oligonucleotide probe molecule, the oligonucleotide probe that the ring portion 7 of oligonucleotide probe is formed, one end of oligonucleotide probe is fixed on fluorescent quenching material 3 surfaces, near the other end of oligonucleotide probe base is marked with fluorophor 5, it is complementary sequence that near the oligonucleotide probe two ends sequence has 3 to 15 bases respectively, can make near the sequence these oligonucleotide probe two ends can form hybridization, the base sequence of oligonucleotide probe middle portion is the complementary sequence of detected nucleotide sequence.By after a plurality of these type of probe combinations in same device, promptly constitute a kind of non-marked gene chip.
On solid substrate 1, be fixed with fluorescent quenching material 3 by arm molecule 2, preparing on fluorescent quenching material 3 surfaces has by fluorophor 5, the stipe part 6 of oligonucleotide probe molecule, the oligonucleotide probe that the ring portion 7 of oligonucleotide probe is formed, one end of oligonucleotide probe is fixed on fluorescent quenching material 3 surfaces, near the other end of oligonucleotide probe base is marked with fluorophor 5, it is complementary sequence that near the oligonucleotide probe two ends sequence has 3 to 15 bases respectively, can make near the sequence these oligonucleotide probe two ends can form hybridization, the base sequence of oligonucleotide probe middle portion is the mutual row of detected nucleotide sequence.
1. the preparation of solid substrate: the material requirements surface that is used for fixing nano particle has can be modified chemical active radical, good optical character and have certain stability.With sheet glass (glass slides), silicon chip (silicon chip), polystyrene (polystyrene), polypropylene (polypropylene) or polycarbonate (polycarbonate) etc. is common used material;
2. the activation of solid substrate is with synthetic: with difunctional active agent being arranged by chemical reaction active group on the surface bond of carrier, so that with corresponding aglucon covalent attachment, formation has the affiliation carrier of different biologic specificities, is used for fixing different nucleic acid probes.
3. fluorescent quenching material: the fluorescent quenching material with the immobilization nucleic acid probe of the present invention that fixes can be a nano-metal particle, as nm gold particles, and nano-Ag particles, its surface is modified with the bifunctional group chemical reagent; Also can be organic molecule or the composite high-molecular material that includes the fluorescent quenching group.
4. the preparation of immobilization nucleic acid probe: the oligonucleotide probe that adopts the synthetic fluorescence chromophoric group specific nucleic acid sequence that includes at least one that designs of commercialization solid state chemistry synthetic method; The method for preparing nucleic acid probe also can be to adopt in-situ synthesis directly to carry out the synthetic and fluorescent mark of nucleic acid on the fluorescent quenching material.
5. probe is fixing: the synthetic good probe of solid state chemistry is transferred to the solid substrate surface by modes such as machines, is connected with solid substrate under proper condition.Each point includes two kinds at least and uses different fluorescently-labeled probes respectively.
6. hybridization and detection: add suitable ion and damping fluid etc. in tested systems, target gene and immobilization probe of the present invention carry out hybridization, endonuclease reaction or amplified reaction.Corresponding reaction system is carried out the detection of fluorescent signal, analyze its result, obtain detected gene information by corresponding software.
Embodiment one, original position synthetic non-marked gene chip
1. slide cleans: soak slide with washing lotion and spend the night, wash down, used the alkali alcohol solution dipping again two hours, after distilled water washed down, nitrogen dried up standby.
2. slide is modified: get the edulcoration slide, soaked 5 minutes in the acetone soln of triethoxy aminosilane, clean, baking is 40 minutes under 100 degree, and glutaraldehyde solution soaked after 2 hours, and clean nitrogen dries up.
3. nanometer gold is fixed: the nano-Au solution immersion slide of modifying with mercaptoethylamine spends the night, and clean nitrogen dries up.
4. oligonucleotide probe is synthetic: carry out nucleotide sequence with above-mentioned slide in the anhydrous glove box of anaerobic and synthesize, carry out multiple sequence with the molecular seal method and synthesize, make chip, wherein last base is to have fluorescein-labeled nucleic acid monomer, therefore, the chip of making mark fluorescein.
Embodiment two, the fixedly making non-marked of synthetic oligonucleotide probe gene chip
1. slide cleans: soak slide with washing lotion and spend the night, wash down, used the alkali alcohol solution dipping again two hours, after distilled water washed down, nitrogen dried up standby.
2. slide is modified: get the edulcoration slide, soaked 5 minutes in the acetone soln of triethoxy aminosilane, clean, baking is 40 minutes under 100 degree, and glutaraldehyde solution soaked after 2 hours, and clean nitrogen dries up.
3. nanometer gold is fixed: the nano-Au solution immersion slide of modifying with mercaptoethylamine spends the night, and clean nitrogen dries up.
4. oligonucleotide probe is synthetic: synthetic with ordinary method, at a terminal modified amino of oligonucleotide probe, modified fluorescein at the other end.
5. chip manufacturing: be fixed on the slide of having fixed nanometer gold with point sample method synthetic oligonucleotide probe.
Embodiment three nano-Au films non-marked gene chips
1. slide cleans: soak slide with washing lotion and spend the night, wash down, used the alkali alcohol solution dipping again two hours, after distilled water washed down, nitrogen dried up standby.
2. the making of nano-Au films: be equipped with the gold nano film with chemical solid precipitation legal system.
3. oligonucleotide probe is synthetic: carry out nucleotide sequence with above-mentioned slide in the anhydrous glove box of anaerobic and synthesize, carry out multiple sequence with the molecular seal method and synthesize, make chip, wherein last base is to have fluorescein-labeled nucleic acid monomer, therefore, the chip of making mark fluorescein.
Claims (10)
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| CN 03112923 CN1206368C (en) | 2003-03-05 | 2003-03-05 | Solid phase nucleic acid detection probe and preparing method thereof |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101666805A (en) * | 2009-07-15 | 2010-03-10 | 苏州纳米技术与纳米仿生研究所 | Method for preparing specific protein detection chip |
| CN102072955A (en) * | 2010-11-05 | 2011-05-25 | 苏州大学 | Preparation method of modified porous plate |
| CN102575988A (en) * | 2009-07-07 | 2012-07-11 | 拓克西密特有限公司 | Fluorescent polymers and solid-phase extraction methods |
| CN101942516B (en) * | 2006-06-28 | 2012-09-26 | 邓兴旺 | Method for detecting specific nucleotide sequence using visual film sensor chip |
| CN104109712A (en) * | 2004-02-18 | 2014-10-22 | 克罗莫塞尔公司 | Methods and materials using signaling probes |
| CN106119344A (en) * | 2016-06-20 | 2016-11-16 | 清华大学 | A kind of combined with fluorescent intensity and the nano-probe of fluorescence polarization detection DNA |
| CN110726840A (en) * | 2019-11-29 | 2020-01-24 | 中山大学 | DNA-two-dimensional material array sensor, microbial detection kit, microbial detection method and antibacterial drug classification method |
| CN114481334A (en) * | 2020-11-13 | 2022-05-13 | 北京华牛世纪生物技术研究院 | Method for in-situ synthesis of gene chip probe by using DNA polymerase |
-
2003
- 2003-03-05 CN CN 03112923 patent/CN1206368C/en not_active Expired - Fee Related
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104109712A (en) * | 2004-02-18 | 2014-10-22 | 克罗莫塞尔公司 | Methods and materials using signaling probes |
| CN101942516B (en) * | 2006-06-28 | 2012-09-26 | 邓兴旺 | Method for detecting specific nucleotide sequence using visual film sensor chip |
| CN102575988A (en) * | 2009-07-07 | 2012-07-11 | 拓克西密特有限公司 | Fluorescent polymers and solid-phase extraction methods |
| CN101666805A (en) * | 2009-07-15 | 2010-03-10 | 苏州纳米技术与纳米仿生研究所 | Method for preparing specific protein detection chip |
| CN102072955A (en) * | 2010-11-05 | 2011-05-25 | 苏州大学 | Preparation method of modified porous plate |
| CN102072955B (en) * | 2010-11-05 | 2013-08-07 | 苏州大学 | Preparation method of modified porous plate |
| CN106119344A (en) * | 2016-06-20 | 2016-11-16 | 清华大学 | A kind of combined with fluorescent intensity and the nano-probe of fluorescence polarization detection DNA |
| CN106119344B (en) * | 2016-06-20 | 2020-06-09 | 清华大学 | A nanoprobe that combines fluorescence intensity and fluorescence polarization to detect DNA |
| CN110726840A (en) * | 2019-11-29 | 2020-01-24 | 中山大学 | DNA-two-dimensional material array sensor, microbial detection kit, microbial detection method and antibacterial drug classification method |
| CN114481334A (en) * | 2020-11-13 | 2022-05-13 | 北京华牛世纪生物技术研究院 | Method for in-situ synthesis of gene chip probe by using DNA polymerase |
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| CN1206368C (en) | 2005-06-15 |
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