CN1850988A - Fluorescent quantum dot marking DNA bioprobe, and its preparing method - Google Patents
Fluorescent quantum dot marking DNA bioprobe, and its preparing method Download PDFInfo
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- CN1850988A CN1850988A CN 200610018426 CN200610018426A CN1850988A CN 1850988 A CN1850988 A CN 1850988A CN 200610018426 CN200610018426 CN 200610018426 CN 200610018426 A CN200610018426 A CN 200610018426A CN 1850988 A CN1850988 A CN 1850988A
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Abstract
The invention discloses the DNA biologic probe with the fluorescent quanta sign and the producing process, the DNA probe with the fluorescent quanta sign connects the fluorescent nanometer quanta point on the end of the mercapto DNA, The surface of the fluorescent quanta point can be connected with the one or the several DNA segments. The producing process can be showed in the below steps: firstly, the quanta point decorated by the mercapto acetic acid is produced; secondly, the mercapto DNA probe substitutes the mercapto acetic acid molecule on the surface of the quanta point using the competing and substituting method, the fluorescent quanta point is connected with the mercapto DNA probe. The double functional nanometer material are gained, the material has the good fluorescence characteristic and has the merits such as the easy handling, the low cost, finished easy in the common chemic lab. So it can be applied in the domains of the biology and the medicinal testing and analyzing.
Description
Technical field
The present invention relates to DNA bioprobe of a kind of fluorescence quantum point mark and preparation method thereof, belong to analytical chemistry, nanomaterial science and bioanalysis chemical field.
Background technology
Semiconductor nano crystal grain one quantum dot, shell/caryogram quantum dot particularly, owing to its quantum size effect has peculiar character, comprise that the fluorescent emission wavelength is controlled, the exciting light spectrum width and continuously, fluorescence strong, good light stability, fast light bleaching, special performance such as multi-color marking when can realize the polynary emission of an elementary excitation, can remedy the deficiency of existing organic fluorescent dye, even alternative in some cases organic fluorescent dye.The special performance of quantum dot will help its application on medical experiment and clinical detection, and comparing conventional fluorescent reagent has huge advantage aspect bioanalysis.
At present, have on core/shell type quantum dot shell according to relevant report and to connect Thiovanic acid, link to each other the method for the biological functional of realization quantum dot then by amido bond with target protein matter.
The detailed process that its preparation Thiovanic acid is modified fluorescence quantum is as follows:
1, preparation fluorescence quantum;
2, the oil soluble fluorescence quantum is dissolved in the chloroform, and with the Thiovanic acid of about 1mL reaction two hours;
3, the 0.1mol/L PBS that adds pH7.4 with 1: 1 ratio extracts as solvent;
4, after the fierce concussion, centrifugal.Removal contains the chloroformic solution layer of unreacted oil soluble quantum dot;
5, repeat said process, remove unnecessary Thiovanic acid.
But also do not occur similar we fluorescence quantum and sulfhydrylation DNA are built into the method for bifunctional material with biological function.The dna fragmentation of sulfhydrylation can make the fluorescence quantum point mark dna probe can be used for cell DNA at different target DNA designs, comprises the research work of various pathogenic bacteria DNA.
Summary of the invention
For further expanding fluorescence quantum in the application aspect the bioanalysis, the invention provides a kind of dna probe and preparation method thereof of biologically active of fluorescence quantum point mark, this method is connected to quantum dot on the sulfhydrylation dna probe effectively, obtained existing excellent fluorescent characteristic biologically active again, can be applicable to the difunctional nano material of the research field relevant with DNA.
Fluorescence quantum point mark dna probe of the present invention is to be connected with the fluorescence nano quantum dot at the DNA of sulfhydrylation end, and the fluorescence quantum surface can connect one or more dna fragmentations.This number can be adjusted by the ratio of control sulfhydrylation DNA and quantum dot.
Fluorescence quantum wherein is at least one of CdTe, CdSe, InP, InAs, CdSe/CdS, CdSe/ZnS, CdSe/ZnSe, CdTe/ZnS.Fluorescence quantum is water miscible, and used dna fragmentation can be the dna fragmentation of different sequences, can be strand or double-stranded DNA.
Preparation method's concrete steps of fluorescence quantum point mark dna probe provided by the invention are as follows:
1., preparation fluorescence nano quantum dot;
2., as required get the hexane solution of an amount of fluorescence quantum, elder generation to remove its surperficial organic molecule, dries up into solid with nitrogen with the anhydrous methanol repetitive scrubbing then;
3., in solid, add and N, N '-dimethyl formamide (DMF) is scattered in quantum dot wherein under ultrasound condition fully equably, adds excessive Thiovanic acid again, after the fierce concussion reaction with reactant centrifugal and disgorging;
4., take out supernatant liquid, add tetrahydrofuran (THF) and tetramethyl ammonium hydroxide solution or sodium hydroxide solution therein, mixing the centrifugal precipitation that obtains in back is the fluorescence quantum that Thiovanic acid is modified.
5., with resolution of precipitate in the phosphoric acid buffer (PBS) or the aqueous solution, form the quantum dot that water miscible Thiovanic acid is modified;
6., the dna probe that in the PBS solution of the Thiovanic acid modification quantum dot that takes out, adds the sulfhydrylation of required amount of substance, the molar ratio of quantum dot and DNA is determined as required, to form a quantum dot surface product of one or one above dna fragmentation is arranged, wherein dna fragmentation can be strand or two strands;
7., solution is at room temperature slowly shaken fully reaction;
8., centrifugal to remove the precipitation that may form, the supernatant of gained is quantum dot-labeled DNA active organism probe.
Wherein used fluorescence quantum is water miscible, and the dna fragmentation of used biologically active can be the Different Alkali basic sequence as required, can be strand or two strands.
The present invention is under the inspiration of the quantum dot method that existing preparation Thiovanic acid is modified, successfully fluorescent nano material and sulfhydrylation DNA are built into the bifunctional material with biological function first, the existing excellent fluorescent characteristic of this material is biologically active again, and good dispersity, high specificity are the difunctional nano materials that can be applicable to the DNA Related Research Domain.Its preparation method is to make the quantum dot that Thiovanic acid is modified earlier, the water-soluble quantum dot that is about to obtain by the competition alternative method joins in the dna probe powder or PBS solution of sulfhydrylation again, blow even gently, room temperature reaction spends the night, make the Thiovanic acid small molecules on the dna probe lieu of quantum dots surface of sulfhydrylation, fluorescence quantum is connected on the sulfhydrylation dna probe, this preparation method is simple, repeatability is high, cost is lower, all can finish at general chemical laboratory, but wide popularization and application detects and analysis field in biology and medical science.
Description of drawings
Fig. 1 is the atomic force microscope photo of fluorescence quantum point mark dna probe.
Fig. 2 is the fluorescence inverted microscope photo of fluorescence quantum point mark dna probe.
Fig. 3 is the dot hybridization photo of fluorescence quantum point mark dna probe.
The intestinal bacteria fluorescence in situ hybridization photo of Fig. 4 fluorescence quantum point mark dna probe
Embodiment
Below by specific embodiment the present invention is described in further detail, but content of the present invention be not limited to for embodiment.
The dna probe of fluorescence quantum point mark is that to be connected with particle diameter at the DNA of sulfhydrylation end be the water miscible fluorescence nano quantum dot of 4nm-8nm, and particle diameter is that the fluorescence quantum surface of 4nm-8nm can connect one or more dna fragmentations.
Wherein the fluorescence nano quantum dot is at least one of CdTe, CdSe, InP, InAs, CdSe/CdS, CdSe/ZnS, CdSe/ZnSe, CdTe/ZnS.Dna fragmentation can be the dna fragmentation of different sequences, can be strand or double-stranded DNA.
Preparation method embodiment:
Fluorescence quantum adopts No. 02139152.1 described method of patent of invention, makes feedstock production with cadmium acetate or zinc acetate.
Handle fluorescence quantum as follows: get the hexane solution of an amount of fluorescence quantum, wash repeatedly for several times with anhydrous methanol earlier, remove the organic molecule on surface, dry up into solid with nitrogen then.In solid precipitation, add the isopyknic N of hexane solution with fluorescence quantum, N '-dimethyl formamide (DMF), under ultrasound condition, quantum dot is scattered in wherein fully equably, add excessive Thiovanic acid again, fierce concussion is reacted after 30 minutes reactant centrifugal and disgorging under the 12000rpm condition.Take out supernatant liquid, according to following operation sequential: adding the 0.3mL mass percent concentration at the 1mL supernatant liquor is 10% tetramethyl ammonium hydroxide solution (or sodium hydroxide solution of 1mol/L) and 0.7mL tetrahydrofuran (THF), mixes the back is the Thiovanic acid modification in the centrifugal precipitation that obtains of 12000rpm fluorescence quantum.
To be dissolved in the fluorescence quantum that aforesaid method obtains in the 0.1mol/L phosphoric acid buffer (PBS) of pH7.5, form the quantum dot that water miscible Thiovanic acid is modified;
Take out an amount of Thiovanic acid and modify the dna probe that adds the sulfhydrylation of required amount of substance in the PBS solution of quantum dot, the molar ratio of quantum dot and DNA is determined as required, to form a quantum dot surface product of one or one above dna fragmentation is arranged, wherein dna fragmentation can be strand or two strands; Solution is at room temperature slowly shaken, reacted 12 hours; At 5000rpm centrifugal 5 minutes at last, to remove the precipitation that may form, the supernatant of gained was quantum dot-labeled DNA active organism probe.
The DNA bioprobe of the fluorescence quantum point mark that makes is characterized (as Fig. 1) by atomic force microscope, can see intuitively that fluorescence quantum has good monodispersity.Characterize (as Fig. 2) by inverted fluorescence microscope, the fluorescent label DNA probe is rendered as good monodispersity and fluorescence radiation characteristic.Easy dot hybridization (as Fig. 3) can see that the dna probe of fluorescence quantum point mark presents excellent specificity.
This routine fluorescent label DNA probe is specially adapted to biological DNA activity and distributional analysis.
Adopt the DNA active organism probe of the inventive method synthetic fluorescence quantum point mark to have the following advantages: the fluorescent characteristic that (1) is excellent.Quantum dot, shell/caryogram quantum dot particularly, the incomparable advantage of traditional organic fluorescent dye is arranged, very wide as excitation spectrum, can realize that the fluorescent emission wavelength is controlled, the exciting light spectrum width and continuously, fluorescence strong, good light stability, fast light bleaching, multi-color marking etc. when can realize the polynary emission of an elementary excitation, so it has the great potential that replaces conventional fluorescent reagent to be used for long-time online bioanalysis in real time; (2) dna fragmentation of sulfhydrylation can design primer as required and increases, perhaps directly synthetic corresponding probe, thus observation is studied in activity and the distribution etc. in cell to the paired target DNA; (3) when the fluorescent label DNA probe, not only simple to operate, and can realize the controllable devices of a plurality of dna fragmentations of quantum dot, thereby realize different types of receptor biological molecule is studied by introduce different dna fragmentations or identical dna fragmentation on the surface.Whole making method is simple, and cost is lower, all can finish at general chemical laboratory, for wide popularization and application is laid a good foundation.Our study group has utilized the dna probe of fluorescence quantum point mark that the distribution of pUC18 in the intestinal bacteria is studied, and illustrates that by intestinal bacteria fluorescence in situ hybridization experiment (as Fig. 4) dna probe of this modifying method formation fluorescence quantum point mark has excellent specificity and biological activity.More than these character fluorescence quantum point mark dna probe of making us invent can be used for the research of DNA in the cell.
Claims (6)
1, a kind of dna probe of fluorescence quantum point mark is characterized in that: it is that the fluorescence quantum surface can connect one or more dna fragmentations at the terminal fluorescence quantum that connects of the DNA of sulfhydrylation.
2, the dna probe of fluorescence quantum point mark according to claim 1 is characterized in that: fluorescence quantum is at least one of CdTe, CdSe, InP, InAs, CdSe/CdS, CdSe/ZnS, CdSe/ZnSe, CdTe/ZnS.
3, the dna probe of fluorescence quantum point mark according to claim 1 and 2 is characterized in that: dna fragmentation can be the dna fragmentation of different sequences, can be strand or double-stranded DNA.
4, the dna probe of fluorescence quantum point mark according to claim 1 and 2 is characterized in that: fluorescence quantum is water miscible.
5, a kind of preparation method of dna probe of fluorescence quantum point mark is characterized in that adopting following concrete steps:
1., preparation fluorescence nano quantum dot;
2., as required get the hexane solution of an amount of fluorescence quantum, elder generation to remove its surperficial organic molecule, dries up into solid with nitrogen with the anhydrous methanol repetitive scrubbing then;
3., in solid, add capacity N, N '-dimethyl formamide (DMF) is scattered in quantum dot wherein under ultrasound condition fully equably, adds excessive Thiovanic acid again, after the fierce concussion reaction with the centrifugal disgorging of reactant;
4., get supernatant liquid, add an amount of tetrahydrofuran (THF) and tetramethyl ammonium hydroxide solution or sodium hydroxide solution therein, mixing the precipitation that the back obtains at high speed centrifugation is the fluorescence quantum that Thiovanic acid is modified;
5., with resolution of precipitate in the phosphoric acid buffer (PBS) or the aqueous solution, form the quantum dot that water miscible Thiovanic acid is modified;
6., the dna probe that in the PBS solution of the Thiovanic acid modification quantum dot that takes out, adds the sulfhydrylation of required amount of substance, the molar ratio of quantum dot and DNA is determined as required, to form a quantum dot surface product of one or one above dna fragmentation is arranged, wherein dna fragmentation can be the Different Alkali basic sequence as required, can be strand or two strands;
7., solution is at room temperature slowly shaken fully reaction;
8., centrifugal to remove the precipitation that may form, the supernatant of gained is quantum dot-labeled DNA active organism probe.
6, according to the preparation method of the dna probe of the described fluorescence quantum point mark of claim 5, it is characterized in that: fluorescence quantum is water miscible quantum dot, is reflected in the aqueous solution and carries out.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101289446B (en) * | 2007-04-16 | 2010-05-19 | 中国科学院化学研究所 | Polarity sensitive fluorescent probe suitable for marking sulfhydryl, preparation method thereof and applications |
| CN102181441A (en) * | 2011-04-15 | 2011-09-14 | 武汉大学 | Method for coupling single long-chain DNA (deoxyribonucleic acid) molecules through single quantum dots |
| CN102191325A (en) * | 2011-04-15 | 2011-09-21 | 武汉大学 | Detection method of vegetal single copy genes |
| CN102942935A (en) * | 2012-09-28 | 2013-02-27 | 武汉大学 | One-step method for synthesizing DNA functionalized Zn doped CdTe quantum dot |
| CN101525668B (en) * | 2009-03-11 | 2013-11-06 | 中国人民解放军第三军医大学第一附属医院 | Nuclear acid probe marked with quantum dots and preparation method and application thereof |
| CN103881707A (en) * | 2013-12-30 | 2014-06-25 | 安徽师范大学 | Phosphorescent energy transfer system, synthesis method, application of system and detection method of single-stranded deoxyribonucleotide |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102121030B (en) * | 2010-12-21 | 2013-01-02 | 中南林业科技大学 | Fluorescent quantum dot marked chitosan DNA nano composite vector and preparation and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1148457C (en) * | 2001-09-30 | 2004-05-05 | 武汉大学 | Nanometer particle mark gene probe and its preparing method and use |
| CN1174080C (en) * | 2002-10-10 | 2004-11-03 | 武汉大学 | Prepn of CdSe/CdS or CdSe/ZnS core-shell quantum dot |
| US20050123974A1 (en) * | 2003-11-17 | 2005-06-09 | U.S. Genomics, Inc. | Methods and compositions relating to single reactive center reagents |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101289446B (en) * | 2007-04-16 | 2010-05-19 | 中国科学院化学研究所 | Polarity sensitive fluorescent probe suitable for marking sulfhydryl, preparation method thereof and applications |
| CN101525668B (en) * | 2009-03-11 | 2013-11-06 | 中国人民解放军第三军医大学第一附属医院 | Nuclear acid probe marked with quantum dots and preparation method and application thereof |
| CN102181441A (en) * | 2011-04-15 | 2011-09-14 | 武汉大学 | Method for coupling single long-chain DNA (deoxyribonucleic acid) molecules through single quantum dots |
| CN102191325A (en) * | 2011-04-15 | 2011-09-21 | 武汉大学 | Detection method of vegetal single copy genes |
| CN102191325B (en) * | 2011-04-15 | 2013-01-23 | 武汉大学 | Detection method of vegetal single copy genes |
| CN102942935A (en) * | 2012-09-28 | 2013-02-27 | 武汉大学 | One-step method for synthesizing DNA functionalized Zn doped CdTe quantum dot |
| CN103881707A (en) * | 2013-12-30 | 2014-06-25 | 安徽师范大学 | Phosphorescent energy transfer system, synthesis method, application of system and detection method of single-stranded deoxyribonucleotide |
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