CN2597478Y - Oligonucleotides brobe and chip fixed on solid substrate - Google Patents
Oligonucleotides brobe and chip fixed on solid substrate Download PDFInfo
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- CN2597478Y CN2597478Y CNU032202083U CN03220208U CN2597478Y CN 2597478 Y CN2597478 Y CN 2597478Y CN U032202083 U CNU032202083 U CN U032202083U CN 03220208 U CN03220208 U CN 03220208U CN 2597478 Y CN2597478 Y CN 2597478Y
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- oligonucleotide probe
- solid substrate
- fixed
- chip
- probe
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Abstract
The utility model relates to a scant nucleotide copolymer detector and core which is fixed on the solid base sheet. The utility model is a nucleotide copolymer detector without mark and can detect the serial information of the nucleotide copolymer; the detector is fixed on a solid base (1) which is fixed with a fluorescence putting out material (3) though an arm molecule(2) ; the surface of the fluorescence putting out material (3) is provided with a scant nucleotide copolymer detector which consists of a fluorescence base (5), a culm part (6) of the scant nucleotide copolymer and a ring part (7) of the scant nucleotide copolymer; one end of the scant nucleotide copolymer is fixed on the surface of the fluorescence putting out material (3); the base next to the other end of the scant nucleotide copolymer is marked with the fluorescence base (5); the serial next to the two ends of the scant nucleotide copolymer are three to fifteen mutually complemented to make the serial neat to the two ends of the scant nucleotide copolymer crossbreed; the middle part base serial is the complementary serial of the detected nucleotide serial of the scant nucleotide copolymer.
Description
One, technical field
The utility model 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 utility model provides, and use cost non-a kind of mark, low and high reliability ground detect the oligonucleotide probe and the chip on the solid substrate of being fixed on of nucleotide sequence gene chip.
2, technical scheme:
The utility model is a kind of be fixed on oligonucleotide probe and chip on the solid substrate, 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, by after a plurality of these type of probe combinations in same device, promptly constitute a kind of non-marked gene chip, 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 kind of in glass, silicon, pottery, plastics, cellulose nitrate, nylon or the rubber.
The utility model 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:
The immobilization nucleic acid probe that the utility model proposes 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 probe 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 utility model proposes immobilization nucleic acid probe and micro-array chip thereof, genome times afterwards comprehensively non-marked, real-time, high-throughout nucleotide sequence are detected, 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 utility model is a kind of be fixed on oligonucleotide probe and chip on the solid substrate, 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.
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 in same device, promptly constituting a kind of non-marked gene sheet after a plurality of these type of probe combinations.
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 (nano-metal particle): with the nano-metal particle of the immobilization nucleic acid probe of the present invention that fixes, as nm gold particles, nano-Ag particles.The surface is modified with the bifunctional group chemical reagent;
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.
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 (6)
1, a kind ofly be fixed on oligonucleotide probe and chip on the solid substrate, it is characterized in that upward being fixed with fluorescent quenching material (3) by arm molecule (2) at solid substrate (1), preparing on fluorescent quenching material (3) surface has by fluorophor (5), the stipe part of oligonucleotide probe molecule (6), the oligonucleotide probe that the ring portion of oligonucleotide probe (7) is formed, one end of oligonucleotide probe is fixed on fluorescent quenching material (3) surface, 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.
2, according to claim 1ly be fixed on oligonucleotide probe and chip on the solid substrate, it is characterized in that by after a plurality of these type of probe combinations in same device, promptly constitute a kind of non-marked gene chip.
3, according to claim 1ly be fixed on oligonucleotide probe and chip on the solid substrate, it is characterized in that 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.
4, according to claim 1 and 2ly be fixed on oligonucleotide probe and chip on the solid substrate, it is characterized in that the fluorescent quenching material (3) on the solid substrate (1) of immobilized oligonucleotide probe can be a nano particle, comprise metal nanoparticle, metal oxide nanoparticles, metal-salt nano particle; Also can be organic molecule or the composite high-molecular material that includes the fluorescent quenching group.
5, according to claim 1 and 2ly be fixed on oligonucleotide probe and chip on the solid substrate, it is characterized in that: the fluorescent quenching material (3) on the solid substrate of immobilized oligonucleotide probe (1) can be direct covalently bound fluorescent quenching group on solid substrate (1).
6, describedly be fixed on oligonucleotide probe and chip on the solid substrate according to claim 1 and 2, it is characterized in that being fixed with the solid substrate (1) of nano particle, its material is a kind of in glass, silicon, pottery, plastics, cellulose nitrate, nylon or the rubber.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNU032202083U CN2597478Y (en) | 2003-03-05 | 2003-03-05 | Oligonucleotides brobe and chip fixed on solid substrate |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNU032202083U CN2597478Y (en) | 2003-03-05 | 2003-03-05 | Oligonucleotides brobe and chip fixed on solid substrate |
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| CN2597478Y true CN2597478Y (en) | 2004-01-07 |
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| CNU032202083U Expired - Lifetime CN2597478Y (en) | 2003-03-05 | 2003-03-05 | Oligonucleotides brobe and chip fixed on solid substrate |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111394432A (en) * | 2020-03-27 | 2020-07-10 | 深圳闪量科技有限公司 | Multiple quantitative PCR detection system based on universal probe chip |
-
2003
- 2003-03-05 CN CNU032202083U patent/CN2597478Y/en not_active Expired - Lifetime
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111394432A (en) * | 2020-03-27 | 2020-07-10 | 深圳闪量科技有限公司 | Multiple quantitative PCR detection system based on universal probe chip |
| CN111394432B (en) * | 2020-03-27 | 2023-03-24 | 深圳闪量科技有限公司 | Multiple quantitative PCR detection system based on universal probe chip |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| AV01 | Patent right actively abandoned |
Effective date of abandoning: 20050622 |
|
| C25 | Abandonment of patent right or utility model to avoid double patenting |