CN107576418A - A kind of fluorescence nano thermometer based on DNA nanostructure and preparation method thereof - Google Patents
A kind of fluorescence nano thermometer based on DNA nanostructure and preparation method thereof Download PDFInfo
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- CN107576418A CN107576418A CN201710810329.8A CN201710810329A CN107576418A CN 107576418 A CN107576418 A CN 107576418A CN 201710810329 A CN201710810329 A CN 201710810329A CN 107576418 A CN107576418 A CN 107576418A
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Abstract
The present invention discloses a kind of fluorescence nano thermometer based on DNA nanostructure, and described fluorescence nano thermometer includes silver nanoclusters, Thermo-sensitive hairpin structure DNA sequence dna and rich guanine DNA sequence dna；Wherein, the silver nanoclusters and rich guanine DNA sequence dna are connected to the both ends of Thermo-sensitive hairpin structure DNA sequence dna.The present invention further discloses the preparation method of above-mentioned fluorescence nano thermometer.The shortcomings of fluorescence nano thermometer of the present invention avoids the hairpin structure thermometer cytotoxicity of conventional temperature meter fluorescence molecule modification, easily bleaching, difficult preparation and sensitivity are limited by energy transfer efficiency, the Thermo-sensitive of silver nanoclusters is enhanced simultaneously, and there is good development prospect.
Description
Technical field
The present invention relates to nanothermometer field.Received more particularly, to a kind of fluorescence based on DNA nanostructure
Rice thermometer and preparation method thereof.
Background technology
Temperature affects chemical reaction equilibrium therein as a highly important thermodynamic parameter in cellular environment
And the physical state of nucleic acid and protein.So the temperature of the small system of accurate measurement, to recognizing life mistake from cellular level
Journey is significant.
DNA hairpin structures have Thermo-sensitive.In lower temperature, DNA hairpin structures fold, and at high temperature, folding
DNA hairpin structures can be opened.Using this temperature sensitive property of DNA hairpin structures, and further distinguish at the both ends of hairpin structure
Fluorescence molecule and quencher molecule are connected, is formed the fluorescence nano thermometer based on energy transfer.Its sensitive section is (molten with Tm
Change temperature) it is relevant.However, some inevitable shortcomings be present in this hairpin structure thermometer, such as：Cytotoxicity, easily bleaching,
Hardly possible preparation etc..In addition, its sensitivity is limited by energy transfer efficiency.
Accordingly, it is desirable to provide a kind of fluorescence nano thermometer, to overcome disadvantage mentioned above.
The content of the invention
It is an object of the present invention to provide a kind of fluorescence nano thermometer based on DNA nanostructure, can use
In the detection of small system temperature.
It is another object of the present invention to provide a kind of preparation method of above-mentioned fluorescence nano thermometer.
To reach above-mentioned purpose, the present invention uses following technical proposals：
The invention provides a kind of fluorescence nano thermometer based on DNA nanostructure, described fluorescence nano temperature
Degree meter includes silver nanoclusters, Thermo-sensitive hairpin structure DNA sequence dna and rich guanine DNA sequence dna；Wherein, the silver nanoclusters and richness
Guanine DNA sequence dna is connected to the both ends of Thermo-sensitive hairpin structure DNA sequence dna.
Preferably, the silver nanoclusters are to be synthesized with CCCTTAATCCCC (as shown in SEQ ID No.1) for DNA profiling.
Preferably, the Thermo-sensitive hairpin structure DNA sequence dna is designed according to required response range, such as can be with
For ATCATAATTATTGTTTTTTTTTTTTTTTACTATTTTTTTGAT (such as SEQ ID No.2 show),
ATCTAATCATTATTGTTTTTTTTTTTTTTTACTATTATGTTTAGAT (such as SEQ ID No.3 show), or
ATATACATTTGTTTTTTTTTTTTACATATGTATAT (such as SEQ ID No.4).
Preferably, the rich guanine DNA sequence dna is GGGTGGGGTGGGGTGGGG (such as SEQ ID No.5 show).
Present invention also offers a kind of preparation method of the fluorescence nano thermometer based on DNA nanostructure, including
Following steps：
1) according to Range of measuring temp Demand Design and specific dna sequence is synthesized：
The interlude of the specific dna sequence is Thermo-sensitive hairpin structure DNA sequence dna, and its both ends is respectively to be used to synthesize silver
The DNA profiling sequence of nano-cluster and rich guanine DNA sequence dna；
2) silver nanoclusters are synthesized in specific dna sequence：Specific dna sequence and silver nitrate are added in buffer solution, shaken,
Ice bath 20min-30min, sodium borohydride is added afterwards, shake, ice bath 1-1.5h, produce.
Preferably, the amount ratio of the material of the specific dna sequence and silver nitrate is 1:6-7.
Preferably, the amount ratio of the material of the silver nitrate and sodium borohydride is 1:1-1.2.
Preferably, the buffer solution is 10mM, pH 7.0 phosphate buffer.
Further, fluorescence nano thermometer of the present invention based on DNA nanostructure is tested on fluorescence spectrum,
Excitation wavelength is 580nm, launch wavelength 636nm.
Fluorescence nano thermometer of the present invention based on DNA nanostructure is utilized under certain temperature, hair clip in DNA sequence dna
Structure, which is unwind, changes silver nanoclusters and the distance of rich guanine fragment, regulates and controls the interaction between them, utilizes fluorescence intensity
Change realize detection of the DNA nanostructure to temperature.
Beneficial effects of the present invention are as follows：
Fluorescence nano thermometer of the present invention based on DNA nanostructure utilize using DNA as template silver nanoclusters with
The principle design synthesis that red fluorescence is remarkably reinforced when rich guanine sequence is close, suitable for the measurement in various temperature section.
Fluorescence nano thermometer of the present invention avoids the hairpin structure thermometer cytotoxicity of conventional temperature meter fluorescence molecule modification, easily drift
In vain, the shortcomings of difficult preparation and sensitivity are limited by energy transfer efficiency, while the Thermo-sensitive of silver nanoclusters is enhanced, have
Good development prospect.
Brief description of the drawings
The embodiment of the present invention is described in further detail below in conjunction with the accompanying drawings.
Fig. 1 shows the structure and operation principle of fluorescence nano thermometers of the embodiment 1-3 based on DNA nanostructure
Schematic diagram.
Fig. 2 shows embodiment 1-3DNA nanostructured transmission electron microscope pictures, and a is the transmission electron microscope picture under low power, and b is high-resolution
Transmission electron microscope picture, d=0.23 nanometers are the interstitial void in silver-colored (111) face.
Fig. 3 shows change of fluorescence nano thermometer fluorescence spectrum of the embodiment 1 based on DNA nanostructure with temperature
Change, excursion is 0 DEG C -50 DEG C, at intervals of 5 DEG C.
Fig. 4 shows change of fluorescence nano thermometer fluorescence intensity of the embodiment 1 based on DNA nanostructure with temperature
Change, excursion is 0 DEG C -50 DEG C, at intervals of 5 DEG C.
Fig. 5 shows change of fluorescence nano thermometer fluorescence intensities of the embodiment 1-3 based on DNA nanostructure with temperature
Change, excursion is 0 DEG C -50 DEG C, at intervals of 5 DEG C.
Fig. 6 shows change of fluorescence nano thermometer fluorescence intensity of the embodiment 3 based on DNA nanostructure with temperature
Change, excursion is 30 DEG C -50 DEG C, at intervals of 5 DEG C.
Embodiment
In order to illustrate more clearly of the present invention, the present invention is done further with reference to preferred embodiments and drawings
It is bright.Similar part is indicated with identical reference in accompanying drawing.It will be appreciated by those skilled in the art that institute is specific below
The content of description is illustrative and be not restrictive, and should not be limited the scope of the invention with this.
A kind of fluorescence nano thermometer based on DNA nanostructure of embodiment 1
A kind of fluorescence nano thermometer based on DNA nanostructure, comprise the following steps：
1) specific dna sequence (T is synthesizedmFor 22.4 DEG C)：
5‘-ATCATAATTATTGTTTTTTTTTTTTTTTACTATTTTTTTGATGGGTG GGGTGGGGTGGGG- 3 ' (as shown in SEQ ID No.6, thickened portion is the DNA profiling sequence for synthesizing silver nanoclusters, in
Between section be hairpin structure DNA sequence dna, underscore part is rich guanine DNA sequence dna).
2) specific dna sequence synthesis silver nanoclusters structure nanothermometer：
It is 1 by the amount ratio of material:6 specific dna sequence and silver nitrate is added to 10mM, pH 7.0 phosphate buffer
In, 20s is shaken, then ice bath reacts 20min in the dark；The sodium borohydride newly configured, silver nitrate and hydroboration are added afterwards
The amount ratio of the material of sodium is 1:1,20s is shaken, continues to keep 1h in ice-water bath, produces fluorescence nano thermometer, structure such as Fig. 1
Shown, transmission electron microscope picture is as shown in Figure 2.
The above-mentioned obtained fluorescence nano thermometer with DNA nanostructure is placed on XRF and tested,
Excitation wavelength is respectively 460nm and 580nm, and launch wavelength is respectively 543nm and 636nm, and test scope is 0-50 DEG C, between temperature
It is divided into 5 DEG C.It is can be seen that with reference to Fig. 3 and Fig. 4 in 543nm, fluorescence intensity uniformly declines with temperature, but sensitivity is poor.
636nm, between 15 DEG C -30 DEG C, fluorescence intensity change is most obvious, illustrates that sensitivity is high in this section of temperature range, with hair clip knot
The Tm of structure is relevant, and by contrast, illustrating the fluorescence nano thermometer of design has strengthened the Thermo-sensitive of silver nanoclusters.
A kind of fluorescence nano thermometer based on DNA nanostructure of embodiment 2
A kind of fluorescence nano thermometer based on DNA nanostructure, comprise the following steps：
1) synthesize specific dna sequence (Tm is 38.7 DEG C)
5‘-ATCTAATCATTATTGTTTTTTTTTTTTTTTACTATTATGTTTAGATG GGTGGGGTGGGGTGGGG- 3 ' (as shown in SEQ ID No.7, thickened portion is the DNA profiling sequence for synthesizing silver nanoclusters
Row, interlude is hairpin structure DNA sequence dna, and underscore part is rich guanine DNA sequence dna).
2) specific dna sequence synthesis silver nanoclusters structure nanothermometer
It is 1 by the amount ratio of material：7 specific dna sequence and silver nitrate is added in buffer solution, 30s is shaken, then black
Ice bath reaction 30min in the dark；Add the sodium borohydride newly configured afterwards, the amount ratio of the material of silver nitrate and sodium borohydride is 1：
1.2,30s is shaken, continues to keep 1.5h in ice-water bath, produces fluorescence nano thermometer, structure is as shown in figure 1, transmission electron microscope
Figure is as shown in Figure 2.
The above-mentioned obtained fluorescence nano thermometer with DNA nanostructure is placed on XRF and tested,
Excitation wavelength is 580nm, launch wavelength 636nm.Test scope is 0-50 DEG C, and temperature interval is 5 DEG C.As a result as shown in figure 5,
It can be seen that between 25 DEG C -40 DEG C, fluorescence intensity change is most obvious, illustrates that sensitivity is high in this section of temperature range,
It is relevant with the Tm of hairpin structure.
A kind of fluorescence nano thermometer based on DNA nanostructure of embodiment 3
A kind of fluorescence nano thermometer based on DNA nanostructure, comprise the following steps：
1) synthesize specific dna sequence (Tm is 48.7 DEG C)
5‘-ATATACATTTGTTTTTTTTTTTTACATATGTATATGGGTGGGGTGG GGTGGGG- 3 ' (as shown in SEQ ID No.8, thickened portion is the DNA profiling sequence for synthesizing silver nanoclusters, and interlude is
Hairpin structure DNA sequence dna, underscore part are rich guanine DNA sequence dna)
2) specific dna sequence synthesis silver nanoclusters structure nanothermometer
It is 1 by the amount ratio of material：6 specific dna sequence and silver nitrate is added in buffer solution, 25s is shaken, then black
Ice bath reaction 25min in the dark；Add the sodium borohydride newly configured afterwards, the amount ratio of the material of silver nitrate and sodium borohydride is 1：
1.1,25s is shaken, continues to keep 1.2h in ice-water bath, produces fluorescence nano thermometer, structure is as shown in figure 1, transmission electron microscope
Figure is as shown in Figure 2.
The above-mentioned obtained fluorescence nano thermometer with DNA nanostructure is placed on XRF and tested,
Excitation wavelength is 580nm, launch wavelength 636nm.Test scope is 0-50 DEG C, and temperature interval is 5 DEG C.As a result as shown in figure 5,
It can be seen that between 30 DEG C -50 DEG C, fluorescence intensity change is most obvious, illustrates that sensitivity is high in this section of temperature range.
Further, tested only for 30-50 DEG C of test scope on XRF, as shown in Figure 6, it can be seen that temperature
There is good linear relationship with fluorescence intensity.
1,2 and 3 (Fig. 5) in conjunction with the embodiments, it can be seen that different hairpin structures based on DNA nanostructure
The sensitive volume of fluorescence nano thermometer is also different, shows that fluorescence nano thermometer of the present invention can be set by rational structure
Count to change temperature sensitive scope, with suitable for the requirement of the temperature test of different systems.
Obviously, the above embodiment of the present invention is only intended to clearly illustrate example of the present invention, and is not pair
The restriction of embodiments of the present invention, for those of ordinary skill in the field, may be used also on the basis of the above description
To make other changes in different forms, all embodiments can not be exhaustive here, it is every to belong to this hair
Row of the obvious changes or variations that bright technical scheme is extended out still in protection scope of the present invention.
SEQUENCE LISTING
<110>Physical Chemistry Technology Inst., Chinese Academy of Sciences
<120>A kind of fluorescence nano thermometer based on DNA nanostructure and preparation method thereof
<130> JLC17I0528E
<160> 8
<170> PatentIn version 3.3
<210> 1
<211> 12
<212> DNA
<213>It is artificial synthesized to be used to synthesize silver nanoclusters sequence
<400> 1
cccttaatcc cc 12
<210> 2
<211> 42
<212> DNA
<213>Artificial synthesized hairpin structure DNA sequence dna
<400> 2
atcataatta ttgttttttt ttttttttac tatttttttg at 42
<210> 3
<211> 46
<212> DNA
<213>Artificial synthesized hairpin structure DNA sequence dna
<400> 3
atctaatcat tattgttttt tttttttttt actattatgt ttagat 46
<210> 4
<211> 35
<212> DNA
<213>Artificial synthesized hairpin structure DNA sequence dna
<400> 4
atatacattt gttttttttt tttacatatg tatat 35
<210> 5
<211> 18
<212> DNA
<213>Artificial synthesized rich guanine DNA sequence dna
<400> 5
gggtggggtg gggtgggg 18
<210> 6
<211> 72
<212> DNA
<213>The specific dna sequence of artificial synthesized embodiment 1
<400> 6
cccttaatcc ccatcataat tattgttttt tttttttttt actatttttt tgatgggtgg 60
ggtggggtgg gg 72
<210> 7
<211> 76
<212> DNA
<213>The artificial synthesized specific dna sequence of embodiment 2
<400> 7
cccttaatcc ccatctaatc attattgttt tttttttttt ttactattat gtttagatgg 60
gtggggtggg gtgggg 76
<210> 8
<211> 65
<212> DNA
<213>The artificial synthesized specific dna sequence of embodiment 3
<400> 8
cccttaatcc ccatatacat ttgttttttt tttttacata tgtatatggg tggggtgggg 60
tgggg 65
Claims (8)
- A kind of 1. fluorescence nano thermometer based on DNA nanostructure, it is characterised in that described fluorescence nano thermometer Including silver nanoclusters, Thermo-sensitive hairpin structure DNA sequence dna and rich guanine DNA sequence dna；Wherein, the silver nanoclusters and rich bird are fast Purine DNA sequence dna is connected to the both ends of Thermo-sensitive hairpin structure DNA sequence dna.
- 2. fluorescence nano thermometer according to claim 1, it is characterised in that the silver nanoclusters are with sequence such as SEQ DNA profiling synthesis shown in ID No.1.
- 3. fluorescence nano thermometer according to claim 1, it is characterised in that the Thermo-sensitive hairpin structure DNA sequence dna As shown in SEQ ID No.2, as shown in SEQ ID No.3 or as shown in SEQ ID No.4.
- 4. fluorescence nano thermometer according to claim 1, it is characterised in that the rich guanine DNA sequence dna such as SEQ Shown in ID No.5.
- 5. a kind of preparation method of any fluorescence nano thermometers of claim 1-4, it is characterised in that including following step Suddenly：1) specific dna sequence is synthesized：The interlude of the specific dna sequence is Thermo-sensitive hairpin structure DNA sequence dna, and its both ends is divided Wei not be for the DNA profiling sequence for synthesizing silver nanoclusters and rich guanine DNA sequence dna；2) silver nanoclusters are synthesized in specific dna sequence：Specific dna sequence and silver nitrate are added in buffer solution, shaken, ice Bath, adds sodium borohydride afterwards, shakes, ice bath, produces.
- 6. preparation method according to claim 5, it is characterised in that the material of the specific dna sequence and silver nitrate Amount is than being 1:6-7.
- 7. preparation method according to claim 5, it is characterised in that the amount ratio of the material of the silver nitrate and sodium borohydride For 1:1-1.2.
- 8. preparation method according to claim 5, it is characterised in that the buffer solution is 10mM, and pH 7.0 phosphoric acid delays Fliud flushing.
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| CN112747833A (en) * | 2021-01-04 | 2021-05-04 | 中国海洋大学 | Temperature sensor based on noble metal nanoparticles and preparation method thereof |
| CN113465769A (en) * | 2021-07-06 | 2021-10-01 | 长春工业大学 | Preparation method of dual-emission nanometer fluorescence thermometer |
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| CN113465769B (en) * | 2021-07-06 | 2023-04-28 | 长春工业大学 | Preparation method of double-emission nano fluorescent thermometer |
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