CN107576418B

CN107576418B - Fluorescent nano thermometer based on DNA nano structure and preparation method thereof

Info

Publication number: CN107576418B
Application number: CN201710810329.8A
Authority: CN
Inventors: 穆丽璇; 卜聪聪; 师文生
Original assignee: Technical Institute of Physics and Chemistry of CAS
Current assignee: Technical Institute of Physics and Chemistry of CAS
Priority date: 2017-09-11
Filing date: 2017-09-11
Publication date: 2020-01-07
Anticipated expiration: 2037-09-11
Also published as: CN107576418A

Abstract

The invention discloses a fluorescent nano thermometer based on a DNA nano structure, which comprises a silver nano cluster, a temperature-sensitive hairpin structure DNA sequence and a guanine-rich DNA sequence; wherein, the silver nanocluster and the guanine-rich DNA sequence are respectively connected to two ends of the temperature-sensitive hairpin structure DNA sequence. The invention further discloses a preparation method of the fluorescent nano thermometer. The fluorescent nano thermometer overcomes the defects of the traditional thermometer that the hairpin structure thermometer modified by fluorescent molecules has cytotoxicity, is easy to bleach, is difficult to prepare, has sensitivity limited by energy transfer efficiency and the like, enhances the temperature sensitivity of silver nanoclusters, and has good development prospect.

Description

Fluorescent nano thermometer based on DNA nano structure and preparation method thereof

Technical Field

The invention relates to the field of nano thermometers. And more particularly, to a fluorescence nanothermometer based on DNA nanostructures and a method of making the same.

Background

Temperature is a very important thermodynamic parameter in the cellular environment, affecting the equilibrium of chemical reactions therein and the physical state of nucleic acids and proteins. Therefore, the method has important significance for recognizing the life process from the cellular level by accurately measuring the temperature of the micro system.

The DNA hairpin structure has temperature sensitivity. At lower temperatures the DNA hairpin structure folds, while at high temperatures the folded DNA hairpin structure opens. The fluorescence nanometer thermometer based on energy transfer is formed by utilizing the temperature-sensitive property of the DNA hairpin structure and further respectively connecting the fluorescent molecule and the quenching molecule at the two ends of the hairpin structure. The sensitive range is related to Tm (melting temperature). However, such hairpin structure thermometers have some unavoidable drawbacks, such as: cytotoxicity, easy bleaching, difficult preparation and the like. Furthermore, its sensitivity is limited by the efficiency of energy transfer.

Therefore, it is desirable to provide a fluorescence nanothermometer that overcomes the above disadvantages.

Disclosure of Invention

The invention aims to provide a fluorescent nano thermometer based on a DNA nano structure, which can be used for detecting the temperature of a micro system.

The invention also aims to provide a preparation method of the fluorescence nano thermometer.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention provides a fluorescent nano thermometer based on a DNA nano structure, which comprises a silver nano cluster, a temperature-sensitive hairpin structure DNA sequence and a guanine-rich DNA sequence; wherein, the silver nanocluster and the guanine-rich DNA sequence are respectively connected to two ends of the temperature-sensitive hairpin structure DNA sequence.

Preferably, the silver nanoclusters are synthesized by using CCCTTAATCCCC (shown as SEQ ID No. 1) as a DNA template.

Preferably, the temperature-sensitive hairpin DNA sequence is designed according to the desired response range, and may be, for example, ATCATAATTATTGTTTTTTTTTTTTTTTACTATTTTTTTGAT (shown as SEQ ID No. 2), ATCTAATCATTATTGTTTTTTTTTTTTTTTACTATTATGTTTAGAT (shown as SEQ ID No. 3), or ATATACATTTGTTTTTTTTTTTTACATATGTATAT (shown as SEQ ID No. 4).

Preferably, the guanine-rich DNA sequence is GGGTGGGGTGGGGTGGGG (shown as SEQ ID No. 5).

The invention also provides a preparation method of the fluorescent nano thermometer based on the DNA nano structure, which comprises the following steps:

1) designing and synthesizing a specific DNA sequence according to the requirement of a test temperature range:

the middle section of the specific DNA sequence is a temperature-sensitive hairpin structure DNA sequence, and the two ends of the specific DNA sequence are respectively a DNA template sequence for synthesizing the silver nanocluster and a guanine-rich DNA sequence;

2) synthesizing silver nanoclusters at a specific DNA sequence: and adding the specific DNA sequence and silver nitrate into a buffer solution, shaking, carrying out ice bath for 20min-30min, then adding sodium borohydride, shaking, and carrying out ice bath for 1-1.5h to obtain the DNA sequence.

Preferably, the mass ratio of the specific DNA sequence to the silver nitrate is 1: 6-7.

Preferably, the mass ratio of the silver nitrate to the sodium borohydride is 1: 1-1.2.

Preferably, the buffer is 10mM, pH 7.0 phosphate buffer.

Furthermore, the fluorescence nanometer thermometer based on the DNA nanometer structure is tested on the fluorescence spectrum, the excitation wavelength is 580nm, and the emission wavelength is 636 nm.

The fluorescence nano thermometer based on the DNA nano structure changes the distance between the silver nano cluster and the guanine-rich segment by utilizing the melting of the hairpin structure in the DNA sequence at a certain temperature, regulates the interaction between the silver nano cluster and the guanine-rich segment, and realizes the detection of the DNA nano structure on the temperature by utilizing the change of the fluorescence intensity.

The invention has the following beneficial effects:

the fluorescence nano thermometer based on the DNA nano structure is designed and synthesized by utilizing the principle that the red fluorescence is obviously enhanced when a silver nano cluster taking the DNA as a template is close to a guanine-rich sequence, and is suitable for measuring various temperature intervals. The fluorescent nano thermometer overcomes the defects of the traditional thermometer that the hairpin structure thermometer modified by fluorescent molecules has cytotoxicity, is easy to bleach, is difficult to prepare, has sensitivity limited by energy transfer efficiency and the like, enhances the temperature sensitivity of silver nanoclusters, and has good development prospect.

Drawings

The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.

FIG. 1 is a schematic diagram showing the structure and operation of the DNA nanostructure-based fluorescence nanothermometer according to examples 1-3.

Fig. 2 shows transmission electron micrographs of DNA nanostructures of examples 1 to 3, a being a transmission electron micrograph at low magnification, b being a high-resolution transmission electron micrograph, and d being a lattice spacing of a silver (111) plane at 0.23 nm.

FIG. 3 shows the fluorescence spectra of the DNA nanostructure-based fluorescence nanothermometer of example 1 as a function of temperature, ranging from 0 ℃ to 50 ℃ with 5 ℃ intervals.

FIG. 4 shows the fluorescence intensity of the DNA nanostructure-based fluorescence nanothermometer of example 1 as a function of temperature, ranging from 0 ℃ to 50 ℃ with 5 ℃ intervals.

FIG. 5 shows the fluorescence intensity of the DNA nanostructure-based fluorescence nanothermometers of examples 1-3 as a function of temperature, ranging from 0 ℃ to 50 ℃ with 5 ℃ intervals.

FIG. 6 shows the fluorescence intensity of the DNA nanostructure-based fluorescence nanothermometer of example 3 as a function of temperature, ranging from 30 ℃ to 50 ℃ with 5 ℃ intervals.

Detailed Description

In order to more clearly illustrate the invention, the invention is further described below with reference to preferred embodiments and the accompanying drawings. Similar parts in the figures are denoted by the same reference numerals. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and is not to be taken as limiting the scope of the invention.

EXAMPLE 1A fluorescent nanothermometer based on DNA nanostructures

A DNA nanostructure-based fluorescent nanothermometer comprising the steps of:

1) synthesis of a specific DNA sequence (T)_m22.4 ℃):

5‘-

ATCATAATTATTGTTTTTTTTTTTTTTTACTATTTTTTTGATGGGT GGGGTGGGGTGGGG-3' (as shown in SEQ ID No.6, the bold part is a DNA template sequence for synthesizing the silver nanoclusters, the middle section is a hairpin DNA sequence, and the underlined part is a guanine-rich DNA sequence).

2) Specific DNA sequence synthesis silver nanocluster construction nanometer thermometer:

adding a specific DNA sequence and silver nitrate into a 10mM phosphate buffer solution with the pH of 7.0 according to the mass ratio of 1:6, shaking for 20s, and then carrying out ice-bath reaction for 20min in the dark; and then adding newly-configured sodium borohydride with the mass ratio of silver nitrate to sodium borohydride being 1:1, oscillating for 20s, and keeping in an ice-water bath for 1h to obtain the fluorescent nano thermometer, wherein the structure is shown in figure 1, and the transmission electron microscope is shown in figure 2.

The fluorescence nano thermometer with the DNA nano structure prepared above is placed on a fluorescence spectrometer for testing, the excitation wavelength is 460nm and 580nm respectively, the emission wavelength is 543nm and 636nm respectively, the testing range is 0-50 ℃, and the temperature interval is 5 ℃. As can be seen from FIGS. 3 and 4, the fluorescence intensity decreased uniformly with temperature at 543nm, but the sensitivity was poor. The fluorescence intensity changes most obviously between 636nm and 15-30 ℃, which shows that the sensitivity is high in the temperature interval and is related to Tm of the hairpin structure, and the comparison shows that the designed fluorescence nano thermometer has enhanced temperature sensitivity to the silver nano cluster.

Example 2A fluorescent nanothermometer based on DNA nanostructures

A DNA nanostructure-based fluorescent nanothermometer comprising the steps of:

1) synthesis of a specific DNA sequence (Tm 38.7 ℃ C.)

5‘-ATCTAATCATTATTGTTTTTTTTTTTTTTTACTATTATGTTTAGATGGGTGGGGTGGGGTGGGG-3' (as shown in SEQ ID No.7, the bold part is the DNA template sequence for synthesizing the silver nanoclusters, the middle section is the hairpin DNA sequence, and the underlined part is the guanine-rich DNA sequence).

2) Nano thermometer constructed by silver nano cluster synthesized by specific DNA sequence

The mass ratio of the materials is 1: 7 and silver nitrate are added into a buffer solution, the mixture is shaken for 30s and then ice-bath reaction is carried out in the dark for 30 min; then adding newly configured sodium borohydride, wherein the mass ratio of silver nitrate to sodium borohydride is 1: and (3) oscillating for 30s, and keeping the temperature in the ice-water bath for 1.5h to obtain the fluorescence nano thermometer, wherein the structure is shown in figure 1, and the transmission electron microscope picture is shown in figure 2.

The fluorescence nano thermometer with the DNA nano structure prepared above is placed on a fluorescence spectrometer for testing, the excitation wavelength is 580nm, and the emission wavelength is 636 nm. The test range was 0-50 ℃ with a temperature interval of 5 ℃. The results are shown in FIG. 5, from which it can be seen that the fluorescence intensity changes most significantly between 25 ℃ and 40 ℃, indicating that the sensitivity is high in this temperature range, which is related to the Tm of the hairpin structure.

EXAMPLE 3A fluorescent nanothermometer based on DNA nanostructures

A DNA nanostructure-based fluorescent nanothermometer comprising the steps of:

1) synthesis of a specific DNA sequence (Tm of 48.7 ℃ C.)

5‘-

ATATACATTTGTTTTTTTTTTTTACATATGTATATGGGTGGGGTG GGGTGGGG-3' (shown in SEQ ID No.8, the bold part is a DNA template sequence for synthesizing the silver nanoclusters, the middle section is a hairpin DNA sequence, and the underlined part is a guanine-rich DNA sequence)

The mass ratio of the materials is 1:6 and silver nitrate are added into a buffer solution, the mixture is shaken for 25s and then ice-bath reaction is carried out in the dark for 25 min; then adding newly configured sodium borohydride, wherein the mass ratio of silver nitrate to sodium borohydride is 1: 1.1, shaking for 25s, and keeping in ice-water bath for 1.2h to obtain the fluorescence nanometer thermometer, wherein the structure is shown in figure 1, and the transmission electron microscope is shown in figure 2.

The fluorescence nano thermometer with the DNA nano structure prepared above is placed on a fluorescence spectrometer for testing, the excitation wavelength is 580nm, and the emission wavelength is 636 nm. The test range was 0-50 ℃ with a temperature interval of 5 ℃. The results are shown in FIG. 5, from which it can be seen that the fluorescence intensity changes most significantly between 30 ℃ and 50 ℃, indicating high sensitivity in this temperature interval. Further, only for the 30-50 ℃ test range, the test was performed on a fluorescence spectrometer, as shown in fig. 6, and it can be seen that the temperature has a good linear relationship with the fluorescence intensity.

Combining examples 1, 2 and 3 (fig. 5), it can be seen that the sensitivity ranges of the DNA nanostructure-based fluorescence nanotherms of different hairpin structures are different, indicating that the fluorescence nanotherms of the present invention can change the temperature sensitivity range by reasonable structural design to meet the temperature testing requirements of different systems.

It should be understood that the above-mentioned embodiments of the present invention are only examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention, and it will be obvious to those skilled in the art that other variations or modifications may be made on the basis of the above description, and all embodiments may not be exhaustive, and all obvious variations or modifications may be included within the scope of the present invention.

SEQUENCE LISTING

<110> research institute of physical and chemical technology of Chinese academy of sciences

<120> fluorescent nano thermometer based on DNA nano structure and preparation method thereof

<130> JLC17I0528E

<160> 8

<170> PatentIn version 3.3

<210> 1

<211> 12

<212> DNA

<213> Artificial Synthesis for synthesizing silver nanocluster sequence

<400> 1

cccttaatcc cc 12

<210> 2

<211> 42

<212> DNA

<213> artificially synthesized hairpin structure DNA sequence

<400> 2

atcataatta ttgttttttt ttttttttac tatttttttg at 42

<210> 3

<211> 46

<212> DNA

<213> artificially synthesized hairpin structure DNA sequence

<400> 3

atctaatcat tattgttttt tttttttttt actattatgt ttagat 46

<210> 4

<211> 35

<212> DNA

<213> artificially synthesized hairpin structure DNA sequence

<400> 4

atatacattt gttttttttt tttacatatg tatat 35

<210> 5

<211> 18

<212> DNA

<213> synthetic guanine-rich DNA sequence

<400> 5

gggtggggtg gggtgggg 18

<210> 6

<211> 72

<212> DNA

<213> Synthesis of the specific DNA sequence of example 1

<400> 6

cccttaatcc ccatcataat tattgttttt tttttttttt actatttttt tgatgggtgg 60

ggtggggtgg gg 72

<210> 7

<211> 76

<212> DNA

<213> Artificial Synthesis of the DNA sequence specified in example 2

<400> 7

cccttaatcc ccatctaatc attattgttt tttttttttt ttactattat gtttagatgg 60

gtggggtggg gtgggg 76

<210> 8

<211> 65

<212> DNA

<213> Artificial Synthesis of the specific DNA sequence of example 3

<400> 8

cccttaatcc ccatatacat ttgttttttt tttttacata tgtatatggg tggggtgggg 60

tgggg 65

Claims

1. A fluorescence nanometer thermometer based on DNA nanometer structure is characterized in that the fluorescence nanometer thermometer comprises silver nanometer clusters, temperature sensitive hairpin structure DNA sequences and guanine-rich DNA sequences; wherein, the silver nanocluster and the guanine-rich DNA sequence are respectively connected to two ends of the temperature-sensitive hairpin structure DNA sequence; the silver nanocluster is synthesized by a DNA template with a sequence shown as SEQ ID No. 1; the DNA sequence of the temperature-sensitive hairpin structure is shown as SEQ ID No.2, SEQ ID No.3 or SEQ ID No. 4; the guanine-rich DNA sequence is shown in SEQ ID No. 5.

2. A method for preparing the fluorescence nano thermometer according to claim 1, characterized by comprising the following steps:

1) synthesis of the DNA sequence: the middle section of the DNA sequence is a temperature-sensitive hairpin structure DNA sequence, and the two ends of the DNA sequence are respectively a DNA template sequence for synthesizing the silver nanocluster and a guanine-rich DNA sequence; wherein the DNA template sequence for synthesizing the silver nanocluster is shown as SEQ ID No. 1; the DNA sequence of the temperature-sensitive hairpin structure is shown as SEQ ID No.2, SEQ ID No.3 or SEQ ID No. 4; the guanine-rich DNA sequence is shown in SEQ ID No. 5;

2) synthesizing silver nanoclusters by using the DNA sequence obtained in the step 1): adding the DNA sequence obtained in the step 1) and silver nitrate into a buffer solution, oscillating, carrying out ice bath, then adding sodium borohydride, oscillating, and carrying out ice bath to obtain the DNA sequence.

3. The method according to claim 2, wherein the mass ratio of the DNA sequence obtained in step 1) to silver nitrate is 1: 6-7.

4. The method according to claim 2, wherein the mass ratio of silver nitrate to sodium borohydride is 1: 1-1.2.

5. The method according to claim 2, wherein the buffer is a 10mM phosphate buffer, pH 7.0.