CN109439318A - A kind of Ratio-type viscosity solar flare fluorescence probe and its preparation method and application - Google Patents
A kind of Ratio-type viscosity solar flare fluorescence probe and its preparation method and application Download PDFInfo
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Abstract
The present invention discloses a kind of Ratio-type viscosity solar flare fluorescence probe and its preparation method and application.Probe using gold nano grain as carrier, its surface connect identification Telomerase the DNA double chain with different fluorophors: FL-R flare long-chain and with the short chain of SH-C-FL strand of long-chain moiety complementary series.FL-R flare long-chain from 5 ' ends be followed successively by fluorescent dye, auxiliary sequencel, Telomerase extension increasing sequence complementation sequence and Telomerase primer sequence, no matter Telomerase presence or absence, FL-R flare long-chain is all luminous, can be used as intrinsic fluorescence signal reference.One terminal modified sulfydryl of the short chain of SH-C-FL strand, another terminal modified another fluorescent dye, in the presence of Telomerase, short chain is extended, and is formed hairpin structure, is split away off from nanogold, fluorescence is opened, for telomerase activity in cells in situ detection, in conjunction with long-chain fluorescence signal, it can be achieved that effective differentiation of quantitative analysis and tumour cell and normal cell to tumour cell Telomerase Activity.This probe, without cleaning, can be directly used for laser confocal imaging during cell experiment.
Description
Technical field
The present invention relates to analysis detections and area of medical diagnostics, and in particular to telomerase activity in cells in situ detection
Ratio-type viscosity solar flare fluorescence probe and its preparation method and application.
Background technique
Telomere is to be located at end of chromosome, chromosome degradation, rearrangement and terminal fusion is prevented, to maintain genome complete
The special construction of property.In recent years the study found that telomere length in fission process constantly shorten lead to body cell
The reason of " replicative senescence ", and in tumour cell, the Telomerase of excessive activation can prolong the telomere reparation constantly shortened
It is long, telomere will not be lost because of cell division, so that fissional number increases.Telomerase is in normal somatic cell
In inactivated state, but it is in excessive activation state in almost all of tumour cell, maintains the table of tumour cell immortality
Type or infinite multiplication state.Therefore, telomerase activation becomes the generation, development and evaluation tumor prognosis of detection malignant tumour
The important symbol object of effect.
The detection method of telomerase activation is mainly based upon the telomeric repeat amplification method of polymerase chain reaction at present
(TRAP), the detection method and on this basis extended, such as electrochemical method, surface-enhanced Raman method.These methods
Although having very high sensitivity and detection limit, process is complicated, takes a long time, and needs expensive instrument, it is often more important that,
These methods carry out telomerase activation detection using cell pyrolysis liquid, can not provide the direct letter of living cells telomerase activity
Breath.Fluorescent method has many advantages, such as strong signal, real-time detection, using simple and visualization, mentions for the detection of telomerase activation
Powerful is supplied.Although more and more seminars start with fluorescent method to detect the activity of cell telomerase,
It is that there are still some problems.For example, most of fluorescence detection is all a kind of DNA probe of luminophore modification, holding
Telomerase activity catalysis extends the process of DNA probe, and fluorophor is opened or shut, to reach the mesh of detection telomerase activation
's.These methods are difficult to distinguish different tumour cells with naked eye, and are easy to produce false positive results.Furthermore these DNA
The luminophore of probe is normally at telomerase catalytic and extends on the complementary strand of chain, and in the presence of Telomerase, luminophore is detached from
Telomerase catalytic extends chain, is unable to reach the in situ detection truly of cell telomerase activity.
Summary of the invention
In order to solve technical problem present in existing Telomerase activity means, the present invention designs and has synthesized a series of tools
Have that fluorescence background is low, specific high, membrane penetrating is good and can be with the Ratio-type viscosity solar flare of quantitative detection telomerase activation
(sticky-flare) fluorescence probe and its preparation method and application.
The present invention solves technical problem and adopts the following technical scheme that a kind of Ratio-type sticky-flare fluorescence probe,
Ratio-type sticky-flare fluorescence probe for the quantitative detection telomerase activation detected for telomerase activation；The ratio
Type sticky-flare fluorescence probe is connected using the gold nano grain that diameter is 13 ± 5nm as carrier on the surface of the carrier
Identify the DNA double chain with different fluorophors of Telomerase；
It is FL-R flare long-chain that a DNA in the DNA double chain is single-stranded, the FL-R flare long-chain from 5 ' ends according to
Secondary is fluorescent dye F1, auxiliary sequencel R1, Telomerase extension increasing sequence complementation sequence (CCCTAA)mAnd Telomerase primer sequence TP
(AATCCGTCGAGCAGAGTT), m is positive integer；
The single-stranded SH-C-FL for the FL-R flare long-chain complementary series of another DNA in the DNA double chain
The short chain of strand, and a terminal modified sulfydryl of the short chain of SH-C-FL strand, another terminal modified another fluorescent dye F2；
Total length >=30 base of the short chain of SH-C-FL strand, and base wherein complementary with the FL-R flare long-chain
Range is 12-18.
As a further improvement of the foregoing solution, the base sequence of the FL-R flare long-chain are as follows: 5 '-F1-R1-
(CCCTAA)m- TP-3 ', the auxiliary sequencel R1Base number range be 5~10, and its sequence be thymine alkali bases T, gland
Any combination of purine bases A, guanine base G and cytosine base C.
As a further improvement of the foregoing solution, the base sequence of the short chain of SH-C-FL strand are as follows: 3 '-SH-R2-
R3-R4-F2- 5 ', in which: R2Base number be 5~10 A, T, G, any combination base sequence；R3It is and R1-(CCCTAA)m
The base sequence of partial complementarity；R4For 9~15 A, T, G, any combination base sequence, but this section of sequence cannot with it is described
The base sequence complementary of FL-R flare long-chain.
As a further improvement of the foregoing solution, the numberical range of m is 3~5.
As a further improvement of the foregoing solution, the fluorescent dye F1With the fluorescent dye be selected from Cy3, Cy5,
Cy5.5, FAM, FITC, TAMRA or Texas Red, the fluorescent dye F1It cannot be selected with the fluorescent dye same glimmering
Photoinitiator dye, and fluorescence resonance transfer cannot occur between both fluorescent dyes.
The present invention also provides a kind of preparation methods of Ratio-type viscosity solar flare fluorescence probe, are used to prepare above-mentioned any ratio
Rate type viscosity solar flare fluorescence probe, the preparation method comprises the following steps:
The FL-R flare long-chain is mixed with the short chain of SH-C-FL strand by mol/L ratio 1:1,70-95
It is incubated for 5 minutes in DEG C water-bath, then natural cooling is down to 16-30 DEG C and hybridizes it sufficiently under the conditions of being protected from light, and obtains sulfydryl
Double fluorophor heteroduplex DNA of modification；
After double fluorophor heteroduplex DNA reduction treatments, with the molar ratio of 300:1/liter be added to gold nano
In grain solution, at least 16h is vibrated at room temperature, and PBS buffer solution then is added in three times and sodium chloride, final concentration are respectively 0.01M
And 0.2M；
Centrifugation 20 minutes, centrifuging temperature is 4 DEG C, revolving speed 13,000rpm, and is 0.1M/L's with pH=7.4, concentration
PBS buffer solution is cleaned twice, and not connected heteroduplex DNA is removed, and is finally resuspended with 1mlPBS buffer, and it is viscous to obtain Ratio-type
Property solar flare fluorescence probe, saves backup under the conditions of 4 DEG C.
As a further improvement of the foregoing solution, the gold nano grain solution preparation method the following steps are included:
Gold nano grain, the HAuCl for being first 0.01% by concentration are synthesized using the method for reduction of sodium citrate gold chloride4
Solution is heated with stirring to boiling, and the sodium citrate solution that concentration is 1%, HAuCl is then added4Solution and sodium citrate solution
Volume ratio is 50mL: 1.75mL, when mixed solution color becomes claret from yellowish discoloration, continues to heat 10min, then stop
Only heating, which continues stirring, makes mixed solution be naturally cooling to room temperature, and after obtained nanogold particle filtering, it is standby to be placed on 4 DEG C of preservations
With.
Further, the nanogold particle obtained passes through 0.45 μm of Millipore film filtering.
As a further improvement of the foregoing solution, double fluorophor heteroduplex DNA are managed through three microcosmic salt hydrochlorate original places；
Preferably, the three microcosmic salts hydrochlorate is three (2- carbonylethyl) microcosmic salt hydrochlorates (TCEPHCl).
The present invention also provides a kind of above-mentioned arbitrary ratio type viscosity solar flare fluorescence probes to detect telomere in living cells in situ
As the application of detection reagent when enzymatic activity, realized by the quantitative analysis to telomerase activation to tumour cell and normal cell
Differentiation and different tumour cell between differentiation.
Probe of the invention without that can be directly entered cell in any transfection reagent there is very high cell to take the photograph
Take rate and transfection efficiency.Two fluorophors are connected on probe of the invention, a fluorophor exists in Telomerase and do not deposit
It all shines under the conditions, can be used as internal reference.Another nucleic acid chains with fluorophor, which belong to, rings Telomerase specificity
Answer fluorescence " on/off " structure.In the presence of Telomerase, this chain is extended, and hairpin structure is formed, under falling off from nanogold
Come, fluorescence is opened, and telomerase activity in cells in situ detection is used for.In conjunction with the design of internal reference chain, realize in tumour cell
The quantitative analysis of Telomerase and effective differentiation of tumour cell.This probe, can be with without cleaning during cell experiment
It is directly used in laser confocal imaging.
Compared with existing activity test method of telomerase, the present invention has following advantages:
1, Ratio-type sticky-flare fluorescence probe preparation method of the present invention is easy, easily operated, Er Qiexi
Cellular toxicity is low, and background fluorescence is weak, and after cell culture, imaging of directly taking pictures realizes cell telomerase activity without washing
Fluorescence imaging and detection；
2, probe of the present invention is using AuNPs as carrier, the higher DNA chain of surface Connection Density, is easy by cell
Intake, the booster action without transfection reagents such as liposomes；
3, probe of the present invention has fluorescence instruction base group modification in one end containing Telomerase primer strand, in telomere
Extend and formed hairpin structure under the action of enzyme, fluorescence is opened, and realizes telomerase activation in situ detection truly；
4, fluorophor is modified in another DNA chain of probe of the present invention, is constantly in fluorescence opening state, it can be with
As inherent object of reference, false positive results are excluded, realize the quantitative detection to telomerase activation；
5, the fluorescence probe that the present invention synthesizes realizes the in situ detection to tumour cell Telomerase Activity；
6, the fluorescence probe that the present invention synthesizes can carry out quantification detection and area to different cell line telomerase othernesses
Point, realize tumour cell and effective differentiation between normal cell and different tumour cells.
Detailed description of the invention
Fig. 1 is the schematic illustration of fluorescence probe of the invention.
Fig. 2 is the fluorescence response and selective figure of fluorescence probe of the invention to telomerase activation.
Fig. 3 is fluorescence probe in situ detection telomerase activation figure of the invention.
Fig. 4 is that fluorescence probe of the invention distinguishes tumour cell and normal cell figure.
Fig. 5 is to be directed to cervical cancer cell (HeLa), lung carcinoma cell (A549), liver cell (QSG-7701) three using the present invention
The fluorescence signal figure of kind cell collection.
Fig. 6 is fluorescence probe of the invention to different tumour cell telomerase activity otherness quantitative analysis figures.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to the accompanying drawings and embodiments, right
The present invention is further elaborated.It should be appreciated that described herein, specific examples are only used to explain the present invention, not
For limiting the present invention.
Fluorescence probe of the invention is a kind of Ratio-type of quantitative detection telomerase activation for telomerase activation detection
Sticky-flare fluorescence probe is with the gold nano grain (AuNPs) of diameter about 13nm (about 13 ± 5nm) for carrier, at it
Surface connects the DNA double chain with different fluorophors of highdensity specific recognition Telomerase.
It is FL-R flare long-chain that a DNA in DNA double chain is single-stranded, the FL-R flare long-chain from 5 ' ends successively
For fluorescent dye (F1), auxiliary sequencel (R1), the sequence (CCCTAA) of Telomerase extension increasing sequence complementationmAnd Telomerase primer sequence
TP(AATCCGTCGAGCAGAGTT).Another DNA in the heteroduplex DNA single-stranded is complementary with FL-R flare long-chain
The short chain of SH-C-FL strand of sequence, a terminal modified sulfydryl, a terminal modified another fluorescent dye (F2)。SH-C-FL
Total length >=30 base of the short chain of strand, wherein the base range complementary with FL-R flare long-chain is 12-18,
(CCCTAA)mThe numberical range of middle m is 3~5.
The base sequence of the FL-R flare long-chain are as follows: 5 '-F1-R1-(CCCTAA)m- TP-3 ', the auxiliary sequencel R1
Base number range be 5~10, and its sequence be thymine alkali bases T, adenine base A, guanine base G and cytimidine
Any combination of base C.
The base sequence of the short chain of SH-C-FL strand are as follows: 3 '-SH-R2-R3-R4-F2- 5 ', in which: R2Base number
For 5~10 A, T, G, any combination base sequence；R3It is and R1-(CCCTAA)mThe base sequence of partial complementarity；R4For 9~
15 A, T, G, any combination base sequence, but this section of sequence cannot be mutual with the base sequence of the FL-R flare long-chain
It mends.
Fluorescent dye F1And F2It can choose Cy3, Cy5, Cy5.5, FAM, FITC, TAMRA or Texas Red etc., but F1
And F2Same fluorescent dye cannot be selected, and fluorescence resonance transfer cannot occur between both fluorescent dyes.
Ratio-type sticky-flare fluorescence probe has without that can be directly entered cell in any transfection reagent
There are very high cellular uptake rate and transfection efficiency.Two fluorophors are connected on the probe, a fluorophor is deposited in Telomerase
And all shine under conditions of be not present, can be used as internal reference.Another nucleic acid chains with fluorophor belong to Telomerase
Specificly-response fluorescence " on/off " structure.In the presence of Telomerase, this chain is extended, and hairpin structure is formed, from nanogold
It splits away off, fluorescence is opened, and telomerase activity in cells in situ detection is used for.In conjunction with the design of internal reference chain, realize thin to tumour
The quantitative analysis of Telomerase in born of the same parents and effective differentiation of tumour cell.This probe is during cell experiment, without clear
It washes, is used directly for laser confocal imaging.
Ratio-type sticky-flare fluorescence probe substantially may include two steps in the preparation:
One, the preparation of gold nano grain；
Two, the preparation of Ratio-type sticky-flare fluorescence probe.
When preparing gold nano grain, gold nano grain is synthesized using the method for reduction of sodium citrate gold chloride in the present invention.
The HAuCl for being first 0.01% by concentration4Solution is heated with stirring to boiling, and the sodium citrate solution that concentration is 1% is then added,
HAuCl4The volume ratio of solution and sodium citrate solution is 50mL: 1.75mL, when mixed solution color becomes wine from yellowish discoloration
When red, continue to heat 10min, then stopping heating continuing to stir makes mixed solution be naturally cooling to room temperature, obtained nanometer
After gold particle filtering, it is placed on 4 DEG C and saves backup.
In the present embodiment, by the 0.01%HAuCl of 50mL4It is vigorously stirred and is heated to boiling, 1.75mL lemon is then added
Lemon acid sodium (1%).When solution colour becomes claret from yellowish discoloration, continue to heat 10min, then stops heating and continue to stir
Mixing makes solution slow cooling to room temperature.After obtained nanogold particle is by 0.45 μm of Millipore film filtering, with projection electricity
Sub- microscope (TEM) and ultraviolet specrophotometer characterize gold nano grain.The gold nano grain average-size of preparation
About 13 ± 2nm is placed on 4 DEG C and saves backup.
When preparing Ratio-type sticky-flare fluorescence probe, by the FL-R flare long-chain and the SH-C-
The short chain of FLstrand is mixed by mol/L ratio 1:1, is incubated for 5 minutes in 70-95 DEG C of (preferably 75 DEG C) water-bath, is then being protected from light
Under the conditions of natually cooled to room temperature (generally 16-30 DEG C) hybridize it sufficiently, it is miscellaneous to obtain mercapto-modified double fluorophors
Hand over double-stranded DNA；After double fluorophor heteroduplex DNA reduction treatments, with the molar ratio of 300:1/liter be added to gold nano
In particle solution, at least 16h is vibrated at room temperature, and PBS buffer solution then is added in three times and sodium chloride, final concentration are respectively
0.01M and 0.2M；Centrifugation 20 minutes, centrifuging temperature is 4 DEG C, revolving speed 13,000rpm, and with pH=7.4, concentration 0.1M/L
PBS buffer solution cleaning twice, remove not connected heteroduplex DNA, finally with 1ml/ rise PBS buffer solution be resuspended, compared
Rate type sticky-flare fluorescence probe, saves backup under the conditions of 4 DEG C.
In the present embodiment, by FL-R flare long-chain, 1:1 is mixed in molar ratio with the short chain of SH-C-FL strand, and 75 DEG C
It is incubated for 5 minutes in water-bath, then natually cooled to room temperature hybridizes it sufficiently under the conditions of being protected from light, and obtains mercapto-modified
Double fluorophor heteroduplex DNA；Double fluorophor heteroduplex DNA are through three (2- carbonylethyl) microcosmic salt hydrochlorates
(TCEPHCl) it after reduction treatment, is added to the molar ratio of 300:1 in the gold nano grain solution of step 1 preparation, at room temperature
At least 16h is vibrated, is then slowly added to PBS buffer solution and sodium chloride in three times, final concentration is respectively 0.01M and 0.2M.4 DEG C from
The heart (13,000rpm), 20 minutes, and with pH=7.4, the PBS buffer solution cleaning that concentration is 0.1M twice, removed not connected miscellaneous
Double-stranded DNA is handed over, is finally resuspended with 1ml PBS buffer solution, Ratio-type sticky-flare fluorescence probe is obtained, under the conditions of 4 DEG C
It saves backup.
Purposes of the present invention for the Ratio-type sticky-flare fluorescence probe of telomerase activation detection, is to examine in situ
Application when surveying living cells Telomerase Activity as detection reagent is realized by the quantitative analysis to telomerase activation to tumour
Differentiation between the differentiation of cell and normal cell and different tumour cells.
The working principle of Ratio-type sticky-flare fluorescence probe of the present invention is as follows:
Ratio-type sticky-flare fluorescence probe of the present invention is largely meticulous in AuNPs area load using AuNPs as kernel
Design can form a kind of spherical shape of densification with the band of specific recognition Telomerase there are two the heteroduplex DNA of fluorophor
Nucleic acid structure.FL-R flare long-chain contains four functional areas interconnected:
(1) 3 ' end is fluorophor, as detection fluorescence signal indicative function；
(2) sequence matched with SH-C-FL strand short chain moieties；
(3) several repetitive sequences complementary with telomere amplification sequence (CCCTAA)m；
(4) Telomerase primer sequence can extend the end TTAGGG with specific recognition Telomerase and under the action of Telomerase
Grain repetitive sequence.
The short chain of SH-C-FL strand is the DNA chain of one with FL-R flare long-chain moiety complementary pairing, and 5 ' ends are repaired
Mercapto groups are adornd, are used to connect with AuNPs, 3 ' terminal modified fluorescence radiation groups, as inherent object of reference.The double-strand of hybridization
DNA arrives the surface of AuNPs by the interaction modification of sulfydryl and gold, and the fluorescent dye of the short chain of SH-C-FL strand is arrived in modification
Distance AuNPs is greater than certain distance, and fluorescence will not be quenched, and fluorescence is in an open state always, can be used as inherent object of reference；
Close to AuNPs, fluorescence is quenched the fluorescent dye of FL-R flare long-chain, and fluorescent state at this time is to close.Exist in Telomerase
In the case where, FL-Rflare long-chain is extended by catalysis, and after extending to certain length, FL-R flare long-chain will form one
A more stable hairpin structure is separated from the short chain of SH-C-FL strand and AuNPs, and fluorescence is restored, by glimmering
Optical signal realizes detection and bio-imaging to telomerase activation.The present invention may be implemented to telomerase activity in cells original position
Detection and the differentiation between tumour cell and normal cell and different tumour cells.
Fluorescence probe when in use, first does the preparation of cell pyrolysis liquid, then does the inspection of cell pyrolysis liquid Telomerase Activity
It surveys, tumour cell and normal cell is then distinguished by the detection of telomerase activation, finally to different cell line telomerases
The fluorescence imaging of the poor activity opposite sex.
One, the preparation of cell pyrolysis liquid.
Specific steps are as follows: with 100mm culture dish culture cell, it is (probably raw in cell index when cell covers with
The long stage) collect cell.It is first cleaned with PBS, pancreatin digestion collects cell into 1.5mL centrifuge tube, and supernatant is abandoned in centrifugation, uses
PBS (pH 7.4) washs CHAPS cell lysis buffer solution (the 10mM Tris-HCl, pH for being then resuspended in 1ml refrigeration three times
7.5,1mM MgCl2, 0.1mM PMSF, 1mM EGTA, 0.1%RNA enzyme inhibitor, 0.5%CHAPS, and 10%
Glycerol cracking in).Centrifuge tube is placed on to be placed on shaking table on ice and shakes 30min, with 12000rpm in 4 DEG C of centrifuges from
Heart 20min collects supernatant, and the cell concentration of cell pyrolysis liquid is about 107A/mL.Finally cell pyrolysis liquid is dispensed into small
In EP pipe, liquid nitrogen is stored in spare in -80 DEG C after instantaneously freezing.The cell pyrolysis liquid of preparation is added in 90-100 DEG C of water-bath
5-10 minutes hot, with the activity of this killed cells telomerase, this lysate is the cell pyrolysis liquid of inactivation, is stored in -80 DEG C
In it is spare.
In conjunction with Fig. 2, the cell pyrolysis liquid containing different amounts of Telomerase is added to the reaction system containing fluorescence probe
In (50ul (6nM)) probe, in 20 μ L10 × TRAP buffers and 4 μ L 10mM dNTPs mixed liquors, the final concentration of reaction system
For 200 μ L, deficiency DEPC water polishing.), 2h is incubated in 37 DEG C of water-baths.Under telomere enzyme effect, FL-R flare long-chain
It is extended, is split away off from nanogold particle, fluorescence restores.Fluoremetry is carried out with sepectrophotofluorometer, fluorescence is strong
Degree and telomere enzyme concentration are in a linear relationship in low concentration, and may eventually reach a platform.
Two, the in situ detection of living cells Telomerase Activity.
In conjunction with Fig. 3, inspection in situ is carried out to living cells telomerase activity using Ratio-type sticky-flare fluorescence probe
Survey and fluorescence imaging, for the fluorescence picture that the A549 cell after probe changes over time is added.It, will by taking A549 cell as an example
Probe is added in culture dish by co-focusing special culture dish of the A549 cell kind at glass dish bottom after culture for 24 hours, with
Cell is incubated for jointly.Fluorescence imaging is carried out to cell by laser confocal microscope (LSM710, Zeiss), when collecting different
Between section fluorescence signal.Excitation wavelength is 633nm, and collection wave-length coverage is 640nm-750nm.
Three, tumour cell and normal cell are distinguished by the detection of telomerase activation.
In conjunction with Fig. 4, different cell telomerase activities are carried out using Ratio-type sticky-flare fluorescence probe in situ
Detection and fluorescence imaging.Choose human cervical carcinoma cell (HeLa), human lung carcinoma cell (A549), three kinds of human liver cell (QSG-7701)
Cell, wherein QSG-7701 is normal human liver cell, and probe is incubated for 2h with these cells respectively jointly, is copolymerized using laser
Focusing microscope carries out fluorescence signal acquisition to cell, as shown in figure 5, the fluorescence signal of tumour cell is significantly stronger than normal cell,
Normal cell is nearly no detectable fluorescence signal.
Four, to the fluorescence imaging of different cell line telomerase activity othernesses.
In conjunction with Fig. 6, different tumour cell telomerase activities are carried out using Ratio-type sticky-flare fluorescence probe
Detection and quantitative analysis.The tumour cell chosen in this experiment is respectively as follows: that choose human cervical carcinoma cell (HeLa), human embryo kidney (HEK) thin
Born of the same parents (293T), human lung carcinoma cell (A549), human breast cancer cell (MCF-7), human oral cavity epithelial cancer cell (KB), human bladder cancer are thin
Born of the same parents (5637), gastric carcinoma cells (N87), human pancreatic cancer cell (BXPC-3), human ovarian cancer (NIH:OVCAR-3) and human liver cancer are thin
Born of the same parents (Hep G2).Quantitative analysis is carried out to cell telomerase activity by fluorescence signal intensity, distinguishes different tumour cells.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.
Claims (10)
1. a kind of Ratio-type viscosity solar flare fluorescence probe, which is characterized in that it is the quantitative detection detected for telomerase activation
The Ratio-type sticky-flare fluorescence probe of telomerase activation；The Ratio-type sticky-flare fluorescence probe is with diameter
The gold nano grain of 13 ± 5nm is carrier, in the surface of carrier connection identification Telomerase with different fluorophors
DNA double chain；
A DNA in the DNA double chain single-stranded is FL-R flare long-chain, and the FL-R flare long-chain is followed successively by from 5 ' ends
Fluorescent dye F1, auxiliary sequencel R1, Telomerase extension increasing sequence complementation sequence (CCCTAA)mAnd Telomerase primer sequence TP
(AATCCGTCGAGCAGAGTT), m is positive integer；
The single-stranded SH-C-FL for the FL-R flare long-chain complementary series of another DNA in the DNA double chain
The short chain of strand, and a terminal modified sulfydryl of the short chain of SH-C-FL strand, another terminal modified another fluorescent dye F2；
Total length >=30 base of the short chain of SH-C-FL strand, and base wherein complementary with the FL-R flare long-chain
Range is 12-18.
2. Ratio-type viscosity solar flare fluorescence probe as described in claim 1, it is characterised in that: the FL-R flare long-chain
Base sequence are as follows: 5 '-F1-R1-(CCCTAA)m- TP-3 ', the auxiliary sequencel R1Base number range be 5~10, and its sequence
It is classified as any combination of thymine alkali bases T, adenine base A, guanine base G and cytosine base C.
3. Ratio-type viscosity solar flare fluorescence probe as described in claim 1, it is characterised in that: the SH-C-FL strand is short
The base sequence of chain are as follows: 3 '-SH-R2-R3-R4-F2- 5 ', in which: R2Base number be 5~10 A, T, G, any combination alkali
Basic sequence；R3It is and R1-(CCCTAA)mThe base sequence of partial complementarity；R4For 9~15 A, T, G, any combination base sequence
Column, but this section of sequence cannot be with the base sequence complementary of the FL-R flare long-chain.
4. Ratio-type viscosity solar flare fluorescence probe as described in claim 1, it is characterised in that: the numberical range of m is 3~5.
5. Ratio-type viscosity solar flare fluorescence probe as described in claim 1, it is characterised in that: the fluorescent dye F1With it is described
Fluorescent dye is selected from Cy3, Cy5, Cy5.5, FAM, FITC, TAMRA or Texas Red, the fluorescent dye F1With it is described glimmering
Photoinitiator dye cannot select same fluorescent dye, and fluorescence resonance transfer cannot occur between both fluorescent dyes.
6. a kind of preparation method of Ratio-type viscosity solar flare fluorescence probe, it is characterised in that: its be used to prepare as claim 1 to
Ratio-type viscosity solar flare fluorescence probe described in any one of 5, the preparation method comprises the following steps:
The FL-R flare long-chain is mixed with the short chain of SH-C-FL strand by mol/L ratio 1:1,70-95 DEG C of water
It is incubated for 5 minutes in bath, then natural cooling is down to 16-30 DEG C and hybridizes it sufficiently under the conditions of being protected from light, and obtains sulfydryl modification
Double fluorophor heteroduplex DNA；
It is molten with the molar ratio of 300:1/liter be added to gold nano grain after double fluorophor heteroduplex DNA reduction treatments
In liquid, vibrate at least 16h at room temperature, be then added PBS buffer solution and sodium chloride in three times, final concentration be respectively 0.01M and
0.2M；
Centrifugation 20 minutes, centrifuging temperature is 4 DEG C, revolving speed 13,000rpm, and with pH=7.4, concentration be 0.1M/L PBS it is slow
Fliud flushing is cleaned twice, and not connected heteroduplex DNA is removed, and is finally resuspended with 1ml PBS buffer solution, and the credit of Ratio-type viscosity is obtained
Spot fluorescence probe saves backup under the conditions of 4 DEG C.
7. the preparation method of Ratio-type viscosity solar flare fluorescence probe as claimed in claim 6, it is characterised in that: the gold nano
The preparation method of particle solution the following steps are included:
Gold nano grain, the HAuCl for being first 0.01% by concentration are synthesized using the method for reduction of sodium citrate gold chloride4Solution
It is heated with stirring to boiling, the sodium citrate solution that concentration is 1%, HAuCl is then added4The volume of solution and sodium citrate solution
Than being 50mL: 1.75mL, when mixed solution color becomes claret from yellowish discoloration, continues to heat 10min, then stop adding
Heat, which continues stirring, makes mixed solution be naturally cooling to room temperature, after obtained nanogold particle filtering, is placed on 4 DEG C and saves backup.
8. the preparation method of Ratio-type viscosity solar flare fluorescence probe as claimed in claim 7, it is characterised in that: obtained nanometer
Gold particle passes through 0.45 μm of Millipore film filtering.
9. the preparation method of Ratio-type viscosity solar flare fluorescence probe as claimed in claim 6, it is characterised in that: double fluorescence
Group heteroduplex DNA is managed through three microcosmic salt hydrochlorate original places；Preferably, the three microcosmic salts hydrochlorate is three (2- carbonylethyl) microcosmic salts acid
Salt (TCEPHCl).
10. a kind of Ratio-type viscosity solar flare fluorescence probe as described in any one of claim 1 to 5 detects living thin in situ
As the application of detection reagent when born of the same parents' Telomerase Activity, it is characterised in that: realized by the quantitative analysis to telomerase activation
The differentiation between differentiation and different tumour cells to tumour cell and normal cell.
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