CN101525668A - Nuclear acid probe marked with quantum dots and preparation method and application thereof - Google Patents
Nuclear acid probe marked with quantum dots and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a nuclear acid probe marked with quantum dots and a preparation method and application thereof. Probes with different sequences are marked with quantum dots with different emission spectrum characteristics; the preparation method of the probe has the following steps: synthesizing numerous probes with different sequences and marking the probes with X; synthesizing numerous quantum dots with different emission spectrum characteristics and marking the dots with Y; integrating probes marked with X and quantum dots marked with Y through specificity of X and Y and carrying out coupling on the probes and the quantum dots; facilitating probes with different sequences to be marked with quantum dots with different emission spectrum characteristics; the probes are used for multicolor fluorescence in situ hybridization, spectral karyotype analysis and chromosome karyotype analysis, can improve sensitivity, repetitiveness and specificity of detection, simplify detection device, lower detection cost, increase detection speed and prevent false positive result from occurring.
Description
Technical field
The present invention relates to molecular biology and cytogenetics field, particularly nuclear acid probe marked with quantum dots and its production and application.
Background technology
The karyomit(e) of different plant species all has specific separately morphological structure feature (comprising chromosomal length, position, kinetochore, arm ratio, satellite size etc.), and this morphological structure feature is metastable.Therefore, chromosome karyotype analysis is the important content of genetic research, and generation, development, metastasis research sick for human chromosomal and malignant tumour also have important scientific meaning.
The traditional method of chromosome karyotype analysis is mainly Chromosome G and shows the band method, has advantage simple to operate, that expense is cheap, can accurately distinguish out each number karyomit(e) and bigger segmental disappearance, repetition and inversion, but can't determine the source of marker chromosomes, can't detect small or complicated chromosome translocation.(fluorescence in situ hybridization FISH) is a kind of new hybridization in situ technique that forms with fluorescent mark replacement isotopic labeling phase late 1980s to fluorescence in situ hybridization on the basis of radioactive in situ hybridization technology.Because of have economy, safety, fast, characteristics such as stablize, be widely used in numerous areas such as the structural research of animal-plant gene group, the analysis of variance of karyomit(e) fine structure, virus infection analysis, human antenatal diagnosis, cancer genetics and genome evolution research at present.Its ultimate principle is to be probe with known mark single-chain nucleic acid, according to the base complementrity principle, carries out specificity with single-chain nucleic acid unknown in the material to be checked and combines the heteroduplex nucleic acid that formation can be detected.Because dna molecular is to be linear array along the karyomit(e) longitudinal axis on karyomit(e), thereby thereby can with probe directly and karyomit(e) hybridize special genes located on karyomit(e).Multi-color fluorescence in situ hybridization (M-FISH) and spectral karyotyping (SKY) on the basis of FISH, have been set up in recent years again, use 5 kinds of fluorescence dyes by different combinations and 24 kinds of probes of ratio mark, hybridize back 24 karyomit(e)s and present special fluorescence separately, thereby realized the drafting of human full genome karyotype collection of illustrative plates, for providing, the researchist enriches detailed cytogenetics information more, comprise the source of determining marker chromosomes, detect small or complicated chromosome translocation etc., especially provide brand-new for the tumour cell chromosome analysis of highly resetting, high-efficiency method.
The inventor uses the M-FISH technology to carry out multinomial early-stage Study: the variation of cervical exfoliated cell human chromosomal terminal enzyme (hTERC) gene copy number has been carried out detecting to judge that itself and human papillomavirus (HPV) infect, the dependency of somatotype; Transitional cell carcinoma of bladder patient urine cast-off cells have been carried out detecting and confirmed that the clinicopathologic stage of 3,7 and No. 17 numerical chromosome aberrations and bladder cancer has significant correlation.But early-stage Study found that there are the following problems for existing M-FISH technology: the fluorescence intensity of (1) fluorescence dye is weak and background noise is high, and detection sensitivity is lower; (2) serious interference influences the result and judges between the spectrum of different fluorescence dyes, detection specificity a little less than; (3) the photobleaching phenomenon of fluorescence dye is obvious, and it is relatively poor to detect repeatability; (4) exciting of different fluorescence dyes needs with different filter groups, and is easy to generate false positive results during switching between the filter group.Therefore, sensitivity, specificity and repeatability, the minimizing false positive results that how to improve detection is that the M-FISH technology is applied to the key issue that urgent clinical needs solve.
(quantum dots, appearing as QDs) overcomes above problem and brought hope quantum dot.Quantum dot be a kind of diameter between 1～10nm, can accept the semiconductor nanoparticle of excitation light generation fluorescence, elementary composition by II-VI family in the periodictable or III-V family.It has many advantages with respect to fluorescence dye: (1) emission wavelength tunable is humorous, by the size and the composition of control quantum dot, the color that can regulate its emitting fluorescence; (2) excitation wavelength range is wide, from the ultraviolet region to the near-infrared region, and can be with the quantum dot of the optical excitation different emission of same wavelength; (3) emission peak is narrow and symmetrical, and peak width at half height is generally 25～45nm; (4) fluorescence intensity is high and stable, and photobleaching is had the intensive resistancing action.Because above-mentioned good spectral response curve, quantum dot has demonstrated great potential as the novel fluorescence marker in fields such as tumour living imaging and targeted therapy, molecular diagnosis in recent years.But do not see up to now, the research report of nuclear acid probe marked with quantum dots and its production and application as yet.
Summary of the invention
In view of this, one of purpose of the present invention is to provide a kind of nuclear acid probe marked with quantum dots, can overcome the fluorescence intensity that existing fluorochrome label nucleic acid probe exists weak, easily photobleaching, different fluorochrome label nucleic acid probe need different wave length to excite and spectrum between disturb defectives such as bigger.
For reaching this purpose, the invention provides a kind of nuclear acid probe marked with quantum dots, have not homotactic probe mark the quantum dot with different emission spectrum characteristics is arranged.
Further, described quantum dot is selected from MgS, MgSe, MgTe, CaS, CaSe, CaTe, ZnO, ZnS, ZnSe, ZnTe, SrS, SrSe, SeTe, CdS, CdSe, CdTe, BaS, BaSe, BaTe, HgS, HgSe, HgTe, PbSe, CaAs, InP, InAs, InCaAs, ZnS/CdS, ZnS/CdS/ZnS, ZnS/HgS/ZnS/CdS, CdS/ZnS, CdS/Ag
2S, CdS/HgS, CdS/HgS/CdS, CdS/PbS, CdS/Cd (OH)
2, among CdSe/CuSe, CdSe/ZnS, CdSe/ZnSe, CdSe/CdS, CdSe/HgSe, CdSe/HgSe/CdSe, CdSe/HgTe, CdTe/HgS, CdTe/HgTe, InAs/ZnSe, InAs/CdSe, InAs/InP, ZnS:Mn, ZnS:Cu, CdS:Mn and the CdS:Cu any, and be that nuclear, silicon-dioxide are the core-shell type quantum point of shell with above-mentioned any;
Further, described quantum dot is selected from any among CdS, CdSe, CdTe, PbSe, ZnS/CdS, CdS/ZnS, CdSe/ZnS, the CdSe/CdS, and is that nuclear, silicon-dioxide are the core-shell type quantum point of shell with above-mentioned any;
Further, carry out coupling by following specificity coupling system between described nucleic acid probe and the quantum dot: biotin-avidin system, Ag-Ab system or ligand-receptor system.
Two of purpose of the present invention is to provide the preparation method of described nuclear acid probe marked with quantum dots.
For reaching this purpose, the invention provides the preparation method of described nuclear acid probe marked with quantum dots, may further comprise the steps:
A, synthetic multiplely have not homotactic probe and carry out mark with X;
B, synthetic multiplely have the quantum dot of different emission spectrum characteristics and carry out mark with Y;
C, the X label probe of step a gained combined with the specificity of Y by X with the Y mark quantum dot of step b gained carry out coupling, make to have the quantum dot that has different emission spectrum characteristics on the not homotactic probe mark, promptly get nuclear acid probe marked with quantum dots.
Further, described quantum dot is selected from MgS, MgSe, MgTe, CaS, CaSe, CaTe, ZnO, ZnS, ZnSe, ZnTe, SrS, SrSe, SeTe, CdS, CdSe, CdTe, BaS, BaSe, BaTe, HgS, HgSe, HgTe, PbSe, CaAs, InP, InAs, InCaAs, ZnS/CdS, ZnS/CdS/ZnS, ZnS/HgS/ZnS/CdS, CdS/ZnS, CdS/Ag
2S, CdS/HgS, CdS/HgS/CdS, CdS/PbS, CdS/Cd (OH)
2, among CdSe/CuSe, CdSe/ZnS, CdSe/ZnSe, CdSe/CdS, CdSe/HgSe, CdSe/HgSe/CdSe, CdSe/HgTe, CdTe/HgS, CdTe/HgTe, InAs/ZnSe, InAs/CdSe, InAs/InP, ZnS:Mn, ZnS:Cu, CdS:Mn and the CdS:Cu any, and be that nuclear, silicon-dioxide are the core-shell type quantum point of shell with above-mentioned any;
Further, described quantum dot is selected from any among CdS, CdSe, CdTe, PbSe, ZnS/CdS, CdS/ZnS, CdSe/ZnS, the CdSe/CdS, and is that nuclear, silicon-dioxide are the core-shell type quantum point of shell with above-mentioned any;
Further, be selected to described X and Y pairing following specificity coupling system: biotin-avidin system, Ag-Ab system or ligand-receptor system;
Further, be selected to described X and Y pairing biotin-avidin system.
Three of purpose of the present invention is to provide the application of described nuclear acid probe marked with quantum dots.
For reaching this purpose, the invention provides the application of described nuclear acid probe marked with quantum dots in multi-color fluorescence in situ hybridization, spectral karyotyping and chromosome karyotype analysis.
Beneficial effect of the present invention is: the present invention adopts the quantum dot-labeled not homotactic nucleic acid probe with different emission spectrum characteristics, can easily realize the nucleic acid marking of tens kinds of colors, and have fluorescence intensity height, fluorescent stability strong, be difficult for photobleaching, have different emission spectrum characteristics quantum dot can with single wavelength excite and spectrum between disturb advantages such as little; The preparation method of probe of the present invention is easy, and the gained probe is stable; Probe of the present invention can be used for M-FISH, SKY and chromosome karyotype analysis, not only can improve detection sensitivity, repeatability and specificity, and can simplify test set, reduce and detect cost, improve detection speed, can also prevent the generation of false positive results when the filter group is switched; The present invention has opened up a new direction for the application of quantum dot in life science, for cytogenetic research provides a kind of efficient, easy method, solid basis has been established in the research and the clinical diagnosis of the generation of and malignant tumour sick for human chromosomal, development, metastasis, has good, wide application prospect.
Description of drawings
In order to make the purpose, technical solutions and advantages of the present invention clearer, the present invention is described in further detail below in conjunction with accompanying drawing, wherein:
Fig. 1 is the preparation of nuclear acid probe marked with quantum dots and uses synoptic diagram;
The M-FISH image of Fig. 2 for gathering;
Fig. 3 is the human full genome karyotype collection of illustrative plates of 24 looks that obtain with software processes.
Embodiment
The quantum dot that the present invention uses mainly comprises the quantum dot (as CdS/ZnO, CdSe/ZnS, CdTe/CdS etc.) of monokaryon quantum dot (as CdS, CdSe, CdTe etc.) and nucleocapsid structure.The quantum dot of nucleocapsid structure is to coat the quantum dot that another kind of material (as CdS, ZnS etc.) with big band gap is constituted in the outside of a kind of material (as CdSe etc.), its optical characteristics is determined that by kernel shell has tangible raising quantum yield and strengthens the effect of photochemical stability.In addition, also comprise the quantum dot (as CdS:Mn, CdS:Cu etc.) of containing transition metal element and silicon dioxide coated quantum dot etc., by mixing the spectral quality that transition metals such as a certain proportion of Mn, Cu can effectively improve quantum dot, and the structure of silica shell is except improving the fluorescent stability of quantum dot, the function of surface of being convenient to quantum dot is outside the pale of civilization, also helps reduction and decomposes the cytotoxicity problem that is caused because of quantum dot light.
The synthetic method of quantum dot mainly contains two kinds: a kind of is synthetic in organic phase, and another kind is synthetic at aqueous phase.Adopt the colloid chemistry method in organic phase, to synthesize and to make the quantum dot that particle size distribution range is less, fluorescence quantum yield is higher, but it is water insoluble, when being applied to biomolecular labelings such as protein, nucleic acid, must modify its surface earlier with certain double-functional group, make its possess the water miscible while again can with biomolecules generation coupling.The at present existing multiple method for preparing water-soluble quantum dot: (1) ligand exchange method, substitute trioctyl-phosphine oxide/tri-n-octyl phosphine (TOPO/TOP) part on former quantum dot surface with hydrophilic difunctionalization part, this difunctionalization part mainly is made of two portions, a part can be fixed on quantum dot the surface (as-SH), another part be hydrophilic radical (as-OH ,-COOH); (2) method of packing into, as by polymeric silicon shell quantum dot being packed into, the internal layer of polymerization silicon shell is-SH that skin is a hydrophilic radical; (3) new part preparation method keeps the TOPO/TOP part on former quantum dot surface, uses the variant of the amphiphile, amphiphilic molecule of diblock or triblock polymer to insert by hydrophobic interaction and intersect mutually and obtains wetting ability.Compare with the organic synthesis method, aqueous phase synthesis method is by adding stablizers such as mercaptoethanol, mercaptan carboxylic acid, cysteamine in the aqueous solution, can directly make finishing hydroxyl, carboxyl or amino water-soluble quantum dot are arranged, have advantages such as simple to operate, that cost is low, toxicity is little, stability is high in the aqueous solution, bio-compatibility is good, but the particle size distribution range of quantum dot is relatively large, and fluorescence quantum yield is relatively low.
The quantum dot-labeled of nucleic acid probe carries out coupling by specificity coupling systems such as biotin-avidin (comprising avidin and Streptavidin), Ag-Ab or ligand-receptors, as with vitamin H, antigen or part on the probe mark, avidin, antibody or acceptor on the quantum dot pairing ground mark, the specificity of then utilizing biotin-avidin, Ag-Ab or ligand-receptor is in conjunction with realizing the quantum dot-labeled of probe.
Nuclear acid probe marked with quantum dots of the present invention can be used for many M-FISH, SKY and chromosome karyotype analysis.
Hereinafter with reference to accompanying drawing, the preferred embodiments of the present invention are described in detail.
One, the preparation of nuclear acid probe marked with quantum dots
Fig. 1 is the preparation of nuclear acid probe marked with quantum dots and uses synoptic diagram that as shown in the figure, the preparation method of the nuclear acid probe marked with quantum dots of present embodiment may further comprise the steps:
A, 4 kinds of whole chromosomes of Synthetic 2 are smeared probe and are carried out mark with vitamin H
Smear probe according to a whole set of 24 kinds of whole chromosomes of the full genome chromosomal DNA sequence design of the mankind, the whole chromosome of design is smeared probe and is prepared according to ordinary method.Certainly, also can directly adopt and commercially do not smear probe with the whole chromosome of fluorochrome label.The biotin labeling that whole chromosome is smeared probe can adopt ordinary methods such as nick-translation, PCR method.
B, Synthetic 2 have the quantum dot of different emission spectrum characteristics and carry out mark with Streptavidin for 4 kinds
With Cadmium chloride fine powder (CdCl
22.5H
2O) and the mixing solutions concentration of Thiovanic acid (MAA) be that the NaOH solution of 1mol/L is regulated pH to 10.2, logical nitrogen is after 30 minutes, adds sodium hydrogen telluride (NaHTe) solution (with sodium borohydride reacted make by the tellurium powder) of new system under agitation condition, Cd
2+/ Te
2-The mol ratio of/MAA is 1: 0.5: 2.4, continues to stir 20 minutes, makes orange CdTe quantum dot precursor solution; With this forerunner's liquid solution back flow reaction different time in 100 ℃ of water-baths, make 24 kinds and have the CdTe quantum dot solution (maximum emission wavelength is respectively 440nm, 450nm, 460nm, 475nm, 490nm, 510nm, 520nm, 530nm, 540nm, 550nm, 560nm, 575nm, 580nm, 590nm, 605nm, 610nm, 620nm, 630nm, 640nm, 650nm, 660nm, 670nm, 680nm and 690nm) that different emission spectrum characteristics and finishing have carboxyl; In above-mentioned CdTe quantum dot solution, add an amount of Virahol respectively to reduce the solubleness of CdTe quantum dot, centrifugal 30 minutes again in 10000r/min, abandon supernatant, promptly get the CdTe quantum dot of purifying.
In the optimization experiment of CdTe quantum dot synthesis condition, proportioning raw materials, solution acid alkalinity, reflux time have been investigated to synthesizing the influence of CdTe quantum dot.Found that: work as Cd
2+/ Te
2-The mol ratio of/MAA is 1: 0.5: 2.4 o'clock, and gained CdTe quantum dot solution is the most stable; When the pH of CdTe quantum dot precursor solution is 10.2, the fluorescence efficiency height of the fast growth of nanocrystal and gained CdTe quantum dot; The fluorescence emission spectrum of back flow reaction different time gained CdTe quantum dot all has stable, the symmetric hyperfluorescence peak that transmits, and along with the prolongation of reflux time, the fluorescent emission signals peak red shift gradually of quantum dot; The visible absorption spectra of back flow reaction different time gained CdTe quantum dot shows that the excitation wavelength range of quantum dot is very wide and size distribution is more even; CdTe quantum dot almost spherical and the size distribution that can observe back flow reaction different time gained under transmission electron microscope are more even, and its corresponding electron-diffraction diagram is a diffraction ring, show that the CdTe quantum dot of generation is cube microlitic structure.
In concentration is that 0.1mol/L, pH are in 8.5 the phosphate buffered saline buffer (PBS), add finishing the CdTe quantum dot 0.6mg of carboxyl and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) 250 μ L that concentration is 8.7g/L (the EDC consumption is 50～100 times of quantum dot) are arranged, mixing, add Streptavidin 0.12mg again, stirring reaction is after 3 hours under the room temperature, last Sephadex G100 chromatography column, with concentration is that 0.1mol/L, pH are 7.4 PBS wash-out, gets the CdTe quantum dot solution of the marked by streptavidin of purifying; According to the method described above, have different emission spectrum characteristics and finishing has the CdTe quantum dot of carboxyl to carry out marked by streptavidin respectively to 24 kinds, make 24 kinds of CdTe quantum dots with different emission spectrum characteristics and marked by streptavidin.
C, biotin labeled whole chromosome is smeared probe combine with the specificity of Streptavidin by vitamin H with the quantum dot of marked by streptavidin and carry out coupling, make to have not homotactic whole chromosome and smear the quantum dot that has different emission spectrum characteristics on the probe mark
With the CdTe quantum dot of marked by streptavidin is that 7.4 bovine serum albumin solution seals non-specific site with pH earlier, smearing probe with biotin labeled whole chromosome again is room temperature lucifuge reaction 24 hours among 8.0 the PBS at pH, use the chromatography column separation and purification at last, promptly get the quantum dot-labeled whole chromosome of CdTe and smear probe; According to the method described above, 24 kinds of CdTe quantum dots with different emission spectrum characteristics and marked by streptavidin and 24 kinds of biotin labeled whole chromosomes are smeared probe to be combined with the specificity of Streptavidin by vitamin H and carries out coupling, make and have not homotactic whole chromosome and smear the quantum dot that has different emission spectrum characteristics on the probe mark, the quantum dot-labeled whole chromosome that promptly gets 24 kinds of emission different colours fluorescence is smeared probe, puts-20 ℃ and keeps in Dark Place.
Two, the application of nuclear acid probe marked with quantum dots
Fig. 1 is the preparation of nuclear acid probe marked with quantum dots and uses synoptic diagram, as shown in the figure, uses the above-mentioned quantum dot-labeled whole chromosome that makes and smears the method that probe carries out chromosome karyotype analysis, may further comprise the steps:
A, preparation Metaphase Chromosome sample
Get the multiple myeloma patients bone marrow smear, with concentration is that the Omaine of 0.4 μ g/mL was handled 1.5 hours, 37 ℃ of trysinizations 6 minutes, adding concentration is the Klorvess Liquid 10mL of 0.075mol/L, piping and druming is evenly gently, 37 ℃ of water-baths 30 minutes, stationary liquid (the methyl alcohol: Glacial acetic acid=3: 1) fix, again cell suspension is dripped to slide glass dried overnight that adds precooling, promptly get the Metaphase Chromosome sample, put-20 ℃ of preservations.
B, nuclear acid probe marked with quantum dots and Metaphase Chromosome sample are carried out in situ hybridization
Hybridization comprises prehybridization, hybridization and rinsing totally three steps operation, and wherein, the purpose of prehybridization is with the non-specific site sealing in the determined nucleic acid molecule, to avoid the non-specific binding of these sites and nucleic acid probe; The purpose of rinsing be the flush away non-specific hybridization and not hybridization label probe, to avoid interference the detection of specific hybrid signal.
The prehybridization of Metaphase Chromosome sample: get the Metaphase Chromosome sample, drip concentration and be the RNA enzyme solution [be the RNA enzyme solution of 10mg/ml dilute make] of 100 μ g/mL with 2 * SSC damping fluid (is that the sodium-chlor of 0.3mol/L and trisodium citrate that concentration is 0.03mol/L are formed by concentration) with concentration, covered, hatched 30 minutes for 37 ℃, float cover glass and washed 5 minutes with 2 * SSC damping fluid, drip mass percentage concentration again and be 0.05% pepsin solution (is that 10% pepsin solution concentration is that the HCl solution dilution of 1mol/L makes with mass percentage concentration), covered, hatched 10 minutes for 37 ℃, float cover glass and washed 5 minutes with 2 * SSC damping fluid, put concentration expressed in percentage by volume again and be in 1% the formaldehyde solution, fix 10 minutes for 37 ℃, with PBS washing 5 minutes, the concentration expressed in percentage by volume of putting precooling more immediately successively is 70%, 90%, dewater in 100% the ethanol series solution, each 5 minutes, seasoning.
The in situ hybridization of nuclear acid probe marked with quantum dots and Metaphase Chromosome sample: will put concentration expressed in percentage by volume through the Metaphase Chromosome sample that prehybridization is handled is 70% the middle sex change of formamide soln (with the preparation of 2 * SSC damping fluid) 3 minutes, the concentration expressed in percentage by volume of putting precooling more immediately successively is to dewater in 70%, 90%, 100% the ethanol series solution, each 5 minutes, seasoning; With 24 kinds of nuclear acid probe marked with quantum dots incubation 5 minutes in 75 ℃ of waters bath with thermostatic control, left standstill 5～10 minutes at 0 ℃ immediately afterwards, make the double chain DNA probe sex change; 24 kinds of nuclear acid probe marked with quantum dots of sex change are dripped on the Metaphase Chromosome sample of sex change, and covered is used the plasticine mounting, transfers to (control hybridization pH is 6～7) in the moist magazine, hybridizes 24 hours for 37 ℃.
The rinsing of Metaphase Chromosome sample: after hybridization finishes, take out the Metaphase Chromosome sample, gently cover glass is taken off with blade, be that 0.3% Nonidet P40 (NP-40) solution (with the preparation of 0.4 * SSC damping fluid) washed 2 minutes earlier with the concentration expressed in percentage by volume that is preheated to 73 ℃, be 0.3% NP-40 solution (with the preparation of 0.4 * SSC damping fluid) washing 1 minute at room temperature again with concentration expressed in percentage by volume, PBS washing 2 minutes, putting concentration expressed in percentage by volume again is 70%, 90%, dewater in 100% the ethanol series solution, each 5 minutes, seasoning, use 4 again, 6-diamino-2-phenylindone (DAPI) is redyed, the DAPI that the PBS washing is unnecessary ,-20 ℃ of preservations are put in seasoning.
C, hybridization signal is carried out fluoroscopic examination
Metaphase Chromosome sample after the hybridization is put under the fluorescent microscope that cold CCD camera is installed, as seen 24 karyomit(e)s present special fluorescence separately in the same nucleus, carry out IMAQ and the data processing of M-FISH with commercialization image capture software and self-editing data analysis software, and draw 24 looks human full genome karyotype collection of illustrative plates.For avoiding interaction between different fluorescence to cause wrong classification, the pixel displacement in the time of when detecting, need thinking over about an image to another image and the order of pickup image.Carry out testing conditions optimization by regulating excitation light source wavelength, sampling time, sampling interval etc.The M-FISH image of gathering as shown in Figure 2, the 24 looks human full genome karyotype collection of illustrative plates that obtains with software processes as shown in Figure 3, arrow indication place is the karyomit(e) of undergoing mutation.
Explanation is at last, above embodiment is only unrestricted in order to technical scheme of the present invention to be described, although by invention has been described with reference to the preferred embodiments of the present invention, but those of ordinary skill in the art is to be understood that, can make various changes to it in the form and details, and the spirit and scope of the present invention that do not depart from appended claims and limited.
Claims (10)
1, nuclear acid probe marked with quantum dots is characterized in that: not homotactic labeled nucleic acid probe has the quantum dot with different emission spectrum characteristics.
2, nuclear acid probe marked with quantum dots according to claim 1 is characterized in that: described quantum dot is selected from MgS, MgSe, MgTe, CaS, CaSe, CaTe, ZnO, ZnS, ZnSe, ZnTe, SrS, SrSe, SeTe, CdS, CdSe, CdTe, BaS, BaSe, BaTe, HgS, HgSe, HgTe, PbSe, CaAs, InP, InAs, InCaAs, ZnS/CdS, ZnS/CdS/ZnS, ZnS/HgS/ZnS/CdS, CdS/ZnS, CdS/Ag
2S, CdS/HgS, CdS/HgS/CdS, CdS/PbS, CdS/Cd (OH)
2, CdSe/CuSe, CdSe/ZnS, CdSe/ZnSe, CdSe/CdS, CdSe/HgSe, CdSe/HgSe/CdSe, CdSe/HgTe, CdTe/HgS, CdTe/HgTe, InAs/ZnSe, InAs/CdSe, InAs/InP, ZnS:Mn, ZnS:Cu, CdS; Among Mn and the CdS:Cu any, and be that nuclear, silicon-dioxide are the core-shell type quantum point of shell with above-mentioned any.
3, nuclear acid probe marked with quantum dots according to claim 2, it is characterized in that: described quantum dot is selected from any among CdS, CdSe, CdTe, ZnS/CdS, CdS/ZnS, CdSe/ZnS, the CdSe/CdS, and is that nuclear, silicon-dioxide are the core-shell type quantum point of shell with above-mentioned any.
4, nuclear acid probe marked with quantum dots according to claim 3 is characterized in that: carry out coupling by following specificity coupling system between described nucleic acid probe and the quantum dot: biotin-avidin system, Ag-Ab system or ligand-receptor system.
5, the preparation method of the described nuclear acid probe marked with quantum dots of claim 1 is characterized in that: may further comprise the steps:
A, synthetic multiplely have not homotactic probe and carry out mark with X;
B, synthetic multiplely have the quantum dot of different emission spectrum characteristics and carry out mark with Y;
C, the X label probe of step a gained combined with the specificity of Y by X with the Y mark quantum dot of step b gained carry out coupling, make to have the quantum dot that has different emission spectrum characteristics on the not homotactic probe mark, promptly get nuclear acid probe marked with quantum dots.
6, the preparation method of nuclear acid probe marked with quantum dots according to claim 5 is characterized in that: described quantum dot is selected from MgS, MgSe, MgTe, CaS, CaSe, CaTe, ZnO, ZnS, ZnSe, ZnTe, SrS, SrSe, SeTe, CdS, CdSe, CdTe, BaS, BaSe, BaTe, HgS, HgSe, HgTe, PbSe, CaAs, InP, InAs, InCaAs, ZnS/CdS, ZnS/CdS/ZnS, ZnS/HgS/ZnS/CdS, CdS/ZnS, CdS/Ag
2S, CdS/HgS, CdS/HgS/CdS, CdS/PbS, CdS/Cd (OH)
2, among CdSe/CuSe, CdSe/ZnS, CdSe/ZnSe, CdSe/CdS, CdSe/HgSe, CdSe/HgSe/CdSe, CdSe/HgTe, CdTe/HgS, CdTe/HgTe, InAs/ZnSe, InAs/CdSe, InAs/InP, ZnS:Mn, ZnS:Cu, CdS:Mn and the CdS:Cu any, and be that nuclear, silicon-dioxide are the core-shell type quantum point of shell with above-mentioned any.
7, the preparation method of nuclear acid probe marked with quantum dots according to claim 6, it is characterized in that: described quantum dot is selected from any among CdS, CdSe, CdTe, ZnS/CdS, CdS/ZnS, CdSe/ZnS, the CdSe/CdS, and is that nuclear, silicon-dioxide are the core-shell type quantum point of shell with above-mentioned any.
8, the preparation method of nuclear acid probe marked with quantum dots according to claim 5 is characterized in that: be selected to described X and Y pairing following specificity coupling system: biotin-avidin system, Ag-Ab system or ligand-receptor system.
9, the preparation method of nuclear acid probe marked with quantum dots according to claim 8 is characterized in that: be selected to described X and Y pairing biotin-avidin system.
10, the application of the described nuclear acid probe marked with quantum dots of claim 1 in multi-color fluorescence in situ hybridization, spectral karyotyping and chromosome karyotype analysis.
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| CN101974626A (en) * | 2010-10-12 | 2011-02-16 | 上海交通大学 | Detection method of symbiotic bacteria in sponge cell based on quantum dot fluorescence in-situ hybridization |
| CN102538683A (en) * | 2012-01-11 | 2012-07-04 | 徐州师范大学 | Optical imaging method for measuring distance smaller than diffraction limit distance |
| CN102590160A (en) * | 2011-01-13 | 2012-07-18 | 索尼公司 | Fluorescent quantum dot/nano-metal particle conjugate and preparation and application thereof |
| CN103033463A (en) * | 2012-12-26 | 2013-04-10 | 江南大学 | Method for simultaneously detecting two pathogenic bacteria by employing quantum dot marked aptamer recognition and flow cytometry |
| CN103048298A (en) * | 2012-12-21 | 2013-04-17 | 中国人民解放军第三军医大学第一附属医院 | Method of using glycine modified quantum dot probes to mark living cell |
| CN104297323A (en) * | 2014-11-01 | 2015-01-21 | 济南大学 | Preparation and application of ZnO@CdTe-carboxylation C3N4 photoelectric DNA sensor |
| CN105603109A (en) * | 2016-03-25 | 2016-05-25 | 苏州达麦迪生物医学科技有限公司 | Probe labeling method for detecting BCR/ABL gene fusion under single channel |
| CN106867540A (en) * | 2017-03-27 | 2017-06-20 | 天津工业大学 | The quick preparation of fluorescence CdTe quantum dot high |
| CN107002137A (en) * | 2014-09-22 | 2017-08-01 | 加利福尼亚大学董事会 | Unimolecule RNA is detected |
| CN107101981A (en) * | 2017-03-03 | 2017-08-29 | 南京农业大学 | A kind of method that utilization utilizing total internal reflection fluorescence microscope detects single biological marker |
| CN108872169A (en) * | 2018-05-07 | 2018-11-23 | 广西大学 | The method for quantitative determining mixed component CdS/ZnS quantum dot in plant roots epidermal tissue |
| CN109957607A (en) * | 2017-12-14 | 2019-07-02 | 深圳先进技术研究院 | A kind of detection probe and its application |
| CN110117639A (en) * | 2019-05-22 | 2019-08-13 | 扬州大学 | The method of CdSe/CdS quanta point resonance light scattering determining DNA content |
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| CN100400677C (en) * | 2006-02-28 | 2008-07-09 | 武汉大学 | Fluorescent quantum dot marking DNA bioprobe, and its preparing method |
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Cited By (19)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101935704B (en) * | 2010-08-27 | 2012-12-19 | 中国科学院广州生物医药与健康研究院 | Liquid-phase chip detection system based on biological bar code |
| CN101935704A (en) * | 2010-08-27 | 2011-01-05 | 中国科学院广州生物医药与健康研究院 | Liquid-phase chip detection system based on biological bar code |
| CN101974626A (en) * | 2010-10-12 | 2011-02-16 | 上海交通大学 | Detection method of symbiotic bacteria in sponge cell based on quantum dot fluorescence in-situ hybridization |
| CN101974626B (en) * | 2010-10-12 | 2012-08-29 | 上海交通大学 | Detection method of symbiotic bacteria in sponge cell based on quantum dot fluorescence in-situ hybridization |
| CN102590160A (en) * | 2011-01-13 | 2012-07-18 | 索尼公司 | Fluorescent quantum dot/nano-metal particle conjugate and preparation and application thereof |
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| CN102538683A (en) * | 2012-01-11 | 2012-07-04 | 徐州师范大学 | Optical imaging method for measuring distance smaller than diffraction limit distance |
| CN103048298A (en) * | 2012-12-21 | 2013-04-17 | 中国人民解放军第三军医大学第一附属医院 | Method of using glycine modified quantum dot probes to mark living cell |
| CN103033463A (en) * | 2012-12-26 | 2013-04-10 | 江南大学 | Method for simultaneously detecting two pathogenic bacteria by employing quantum dot marked aptamer recognition and flow cytometry |
| CN107002137A (en) * | 2014-09-22 | 2017-08-01 | 加利福尼亚大学董事会 | Unimolecule RNA is detected |
| CN104297323A (en) * | 2014-11-01 | 2015-01-21 | 济南大学 | Preparation and application of ZnO@CdTe-carboxylation C3N4 photoelectric DNA sensor |
| CN104297323B (en) * | 2014-11-01 | 2016-01-20 | 济南大学 | The carboxylated C of a kind of ZnO CdTe- 3n 4the preparation of photoelectricity DNA sensor and application thereof |
| CN105603109A (en) * | 2016-03-25 | 2016-05-25 | 苏州达麦迪生物医学科技有限公司 | Probe labeling method for detecting BCR/ABL gene fusion under single channel |
| CN107101981A (en) * | 2017-03-03 | 2017-08-29 | 南京农业大学 | A kind of method that utilization utilizing total internal reflection fluorescence microscope detects single biological marker |
| CN106867540A (en) * | 2017-03-27 | 2017-06-20 | 天津工业大学 | The quick preparation of fluorescence CdTe quantum dot high |
| CN109957607A (en) * | 2017-12-14 | 2019-07-02 | 深圳先进技术研究院 | A kind of detection probe and its application |
| CN108872169A (en) * | 2018-05-07 | 2018-11-23 | 广西大学 | The method for quantitative determining mixed component CdS/ZnS quantum dot in plant roots epidermal tissue |
| CN108872169B (en) * | 2018-05-07 | 2021-02-05 | 广西大学 | Method for quantitatively determining mixed component CdS/ZnS quantum dots in plant root epidermal tissue |
| CN110117639A (en) * | 2019-05-22 | 2019-08-13 | 扬州大学 | The method of CdSe/CdS quanta point resonance light scattering determining DNA content |
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