DNA tweezers fluorescent probe as well as preparation method and application thereof

Abstract

The invention relates to the field of nano biosensing, in particular to a DNA (deoxyribonucleic acid) tweezers fluorescent probe as well as a preparation method and application thereof, and the DNA tweezers fluorescent probe is formed by hybridizing DNA single chains as shown in SEQ ID NO.1-SEQ ID NO.5. Five DNA single chains are designed, the DNA tweezers fluorescent probe with a tweezers type nanostructure is formed through complementary base pairing, and the probe has stimulation responsiveness to weak acid pH and high potassium ion concentration in a tumor microenvironment. Under the condition that two factors of weak acid pH and high potassium ion concentration exist at the same time, the fluorescent probe is completely folded, has an obvious fluorescence resonance energy transfer effect and emits obvious red fluorescence. Compared with a single-factor stimulation response imaging probe, the DNA tweezers fluorescent probe disclosed by the invention is simple and convenient in synthesis method, has higher specificity in tumor cell imaging, and has important significance in specific imaging of tumor cells.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of nanobiological sensing technology, and particularly relates to a DNA tweezers fluorescent probe and a preparation method and application thereof. BACKGROUND

[0002] Cancer is one of the diseases with the highest mortality rate worldwide, and accurate diagnosis and effective treatment of cancer is one of the biggest challenges in current clinical practice. Compared with normal tissues, tumor microenvironment has the following characteristics: acidic pH, hypoxia, up-regulated levels of oxidative and reductive substances such as hydrogen peroxide and glutathione, excessive metabolic biomolecules such as glucose, lactic acid and polyamine, and excessive enzymes. The energy required for the growth of normal cells in the human body mainly comes from mitochondrial oxidative phosphorylation, while tumor cells produce energy through the glycolytic pathway to meet the needs of rapid proliferation. However, the glycolytic pathway produces a large amount of lactic acid, which is discharged by tumor cells, thus resulting in a lower pH outside the tumor cells. Relative to the neutral pH of about 7.4 in normal tissues, tumor tissues exhibit micro-acidity with a pH of about 6.4~6.8. On the other hand, the rapid division of tumor cells in the competition for limited space resources will cause a lot of cell necrosis. The necrotic cells release potassium ions in the cells to the outside of the cells, resulting in an increase in the extracellular potassium ion concentration from 5mM in normal tissues to 40mM~50mM in tumor microenvironment. Therefore, the extracellular environment of tumor cells has the characteristics of weak acidity and high potassium ion concentration.

[0003] Based on the differences between tumor microenvironment and normal tissue microenvironment, many tumor microenvironment-responsive nanoprobes have been designed and developed in order to understand the progression of tumors by real-time monitoring of tumor microenvironment, and to provide favorable guidance and basis for efficient treatment of tumors. However, some tissues other than tumors, such as nodules, inflammatory tissues, etc., will also exhibit similar characteristics of tumor microenvironment, which will lead to false positive results of tumor imaging. The imaging effect of many current nanoprobes is limited by poor specificity for tumors, which seriously hinders their wide application. Therefore, it is urgent to develop an imaging probe with higher specificity for tumor tissues. SUMMARY

[0004] The present application aims to overcome the deficiencies in the prior art and provide a DNA tweezers fluorescent probe capable of simultaneously responding to weak acidic pH and high potassium ion concentration in tumor microenvironment, which can be applied to tumor cell imaging.

[0005] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows: In a first aspect, the present application provides a DNA tweezers fluorescent probe, which is formed by hybridization of DNA single strands represented by SEQ ID NO. 1~SEQ ID NO. 5; the DNA tweezers fluorescent probe is a tweezers-type nanostructure.

[0006] Preferably, the 3' end of SEQ ID NO. 1 contains a Cy5 modification; the 5' end of SEQ ID NO. 2 contains a Cy3 modification.

[0007] The present application utilizes the fluorescence resonance energy transfer effect mechanism to make the DNA tweezers fluorescent probe produce a signal. The fluorescence resonance energy transfer effect refers to that in two different fluorescent groups, if the emission spectrum of one fluorescent group has a certain overlap with the absorption spectrum of another group, when the distance between the two fluorescent groups is appropriate, generally less than 10 nm, the phenomenon of fluorescence energy transfer from the donor to the acceptor can be observed. Under the stimulation of high potassium ion concentration in the tumor microenvironment, the spatial distance between the two fluorescent groups Cy5 and Cy3 on the DNA tweezers fluorescent probe is reduced after the complete folding of the DNA tweezers, and a significant fluorescence resonance energy transfer effect occurs, emitting obvious red fluorescence, thereby realizing specific imaging of tumor cells.

[0008] Preferably, the 5' end and the 3' end of SEQ ID NO. 5 contain a cholesterol modification.

[0009] The present application designs to add cholesterol modification at the 5' end and the 3' end of SEQ ID NO. 5, and the cholesterol group can be inserted into the cell membrane to anchor the DNA tweezers fluorescent probe on the cell membrane surface.

[0010] In the second aspect, the present application provides a preparation method of the above-mentioned DNA tweezers fluorescent probe, which is prepared by mixing and hybridizing the single-stranded DNAs shown in SEQ ID NO. 1~SEQ ID NO. 5 and then annealing.

[0011] Preferably, the molar ratio of the mixed single-stranded DNAs is SEQ ID NO. 1: SEQ ID NO. 2: SEQ ID NO. 3: SEQ ID NO. 4: SEQ ID NO. 5 = 1: 1: 1: 1: 2.

[0012] The five single-stranded DNAs are mixed in the above-mentioned specific molar ratio, which can ensure the correct assembly of the DNA tweezers fluorescent probe.

[0013] Preferably, the hybridization annealing program is 95℃, 5min; 65℃, 30min; 50℃, 30min; 37℃, 30min; 22℃, 30min.

[0014] In the third aspect, the present application provides the use of the above-mentioned DNA tweezers fluorescent probe in the preparation of a product for imaging tumor cells.

[0015] Preferably, the tumor cell imaging is applied to an environment with pH 6.4~6.8 and potassium ion concentration of 40mM~50mM.

[0016] Preferably, the tumor cell comprises human esophageal cancer cell ECA109.

[0017] In a fourth aspect, the present application provides application of the above-mentioned DNA tweezers fluorescent probe in preparation of a product for cancer diagnosis and / or cancer prognosis.

[0018] The present application has the following advantages: In view of the poor specificity of the current fluorescent probes responding to single factor of tumor microenvironment, the present application provides a DNA tweezers fluorescent probe, which is formed by hybridization of DNA single strands shown in SEQ ID NO. 1~SEQ ID NO. 5. The DNA tweezers fluorescent probe designed in the present application has the responsiveness to pH and K + , and only when both the weak acidity and the high potassium ion concentration are met, the structure of the fluorescent probe can be completely folded to emit fluorescence, thereby ensuring the specificity of imaging. Compared with other fluorescent probes prepared through complex chemical synthesis or material preparation process, the DNA tweezers fluorescent probe of the present application can be prepared by mixing five DNA single strands in a certain proportion and then annealing, and the synthesis method is simple. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 FIG. 4 is a schematic diagram of structure transition of the DNA tweezers fluorescent probe.

[0020] Figure 2 FIG. 5 is a detailed structure schematic diagram of G-quadruplex and C-quadruplex, wherein a represents G-quadruplex, and b represents C-quadruplex.

[0021] Figure 3 FIG. 6 is an assembly diagram of the DNA tweezers fluorescent probe and identification thereof, wherein a diagram is a schematic diagram of assembly process of the DNA tweezers fluorescent probe, and b diagram is a result of polyacrylamide gel electrophoresis proving successful synthesis of the DNA tweezers fluorescent probe.

[0022] Figure 4 FIG. 6 is an assembly diagram of the DNA tweezers fluorescent probe and identification thereof, wherein a diagram is a schematic diagram of assembly process of the DNA tweezers fluorescent probe, and b diagram is a result of polyacrylamide gel electrophoresis proving successful synthesis of the DNA tweezers fluorescent probe.

[0023] Figure 5Figure 1 is a result graph for verifying the responsiveness of the DNA tweezer fluorescent probe to weak acidic pH; a is a schematic diagram of the structural transformation of the DNA tweezer fluorescent probe under pH stimulation; b is a fluorescence spectrum graph under different pH conditions; c is a relationship curve of A / D and pH, wherein A: Accept, indicating the emission intensity of rhodamine red at a wavelength of 590 nm; D: Donor, indicating the emission intensity of rhodamine green at a wavelength of 525 nm; d is a schematic diagram of the structural transformation of the DNA tweezer fluorescent probe anchored on the cell membrane under pH stimulation; e is a confocal image of ECA109 cells incubated under different pH conditions after the DNA tweezer fluorescent probe is anchored on the cell membrane surface; f is a relationship curve of the normalized A / D and pH calculated according to the results of e, wherein A indicates the average fluorescence intensity of the confocal image of the FRET channel, and D indicates the average fluorescence intensity of the confocal image of the rhodamine green channel.

[0024] Figure 6 Figure 2 is a result graph for verifying the responsiveness of the DNA tweezer fluorescent probe to weak acidic pH and potassium ions in solution; a is a schematic diagram of the complete folding of the C-quadruplex and G-quadruplex in the DNA tweezer under the conditions of pH 6.4-6.8 and potassium ion concentration of 40 mM-50 mM, and the obvious FRET effect is emitted, and the reaction equilibrium moves to the right side in the graph; b-d are fluorescence emission spectrum graphs of the DNA tweezer fluorescent probe measured under the conditions of pH 6.4, 6.6, 6.8 respectively and different potassium ion concentrations; e is a structural change diagram of the DNA tweezer fluorescent probe under the conditions of pH 7.4 and potassium ion concentration of 0 mM-50 mM, and the reaction equilibrium moves to the left side in the graph; f is a fluorescence emission spectrum graph of the DNA tweezer fluorescent probe measured under the conditions of pH 7.4 and different potassium ion concentrations; g is a relationship curve of A / D and potassium ion concentration obtained according to b, c, d and f, wherein A indicates the emission intensity of Cy5 at a wavelength of 670 nm, and D indicates the emission intensity of Cy3 at a wavelength of 565 nm.

[0025] Figure 7 Figure 3 is a result graph for investigating the responsiveness of the DNA tweezer fluorescent probe to weak acidic pH and potassium ions on the cell membrane surface; a is a schematic diagram of the folding process of the DNA tweezer fluorescent probe anchored on the cell membrane surface under the conditions of pH 6.4-6.8 and potassium ion concentration of 40 mM-50 mM, and the reaction equilibrium moves to the right side in the graph; b is a confocal fluorescence imaging graph of the ECA-109 cell membrane surface after the DNA tweezer fluorescent probe is anchored and incubated under different conditions. DETAILED DESCRIPTION

[0026] The application will be further described in the following with specific examples, but the scope of the application is not limited thereto. The details and forms of the technical solutions of the application can be modified or replaced without departing from the spirit and scope of the application, and these modifications or replacements all fall within the protection scope of the application.

[0027] The inventive concept of the application is as follows: The application designs five DNA single strands to form a DNA tweezers fluorescent probe with tweezers type nanostructure through base complementary pairing, wherein the SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.5 contain sequences forming G-quadruplex; the SEQ ID NO.3 contains sequences forming C-quadruplex; and the SEQ ID NO.4 plays a supporting role. The DNA tweezers fluorescent probe contains nucleic acid structures responsive to pH and potassium ions: C-quadruplex structure and G-quadruplex structure. The C-quadruplex structure is folded from cytosine-rich DNA. Under acidic conditions, the semi-protonated cytosine and another unprotonated cytosine form a C-C base pair through Hoogsteen hydrogen bonding, the C-C base pairs interpenetrate each other, and finally form a stable four-stranded nucleic acid structure. Since it needs to be protonated to form, the C-quadruplex thus has a response to hydrogen ions or pH. The G-quadruplex structure is folded from guanine-rich nucleic acid sequences. Four guanine bases form a complementary pairing structure, which is stacked layer by layer under the action of alkali metals such as K + , Na + , Li + , etc., to form a G-quadruplex structure; therefore, the G-quadruplex structure has a response to K + . Under the stimulation of the weakly acidic conditions of the tumor microenvironment, the C-rich single-stranded DNA sequence in the middle of the DNA tweezers fluorescent probe designed by the application folds to form a C-quadruplex, which shortens the two handles of the DNA tweezers, so that the G-rich sequences on the two ends of the DNA tweezers are close to each other. Under the stimulation of the high potassium ion concentration conditions of the tumor microenvironment, the G-quadruplex folds. The working principle diagram is shown in Figure 1 .

[0028] In order for those skilled in the art to better understand the technical solutions of the application and implement them, the application will be further described in the following with specific examples. In the description of the application, if not specifically stated, the reagents used are all commercially available, and the methods used are all conventional techniques in the art.

[0029] The English-Chinese translation table and abbreviation table in the application are shown in Table 1.

[0030] English-Chinese translation table and abbreviation table in Table 1 Example 1 A preparation method of a DNA tweezers fluorescent probe, specifically as follows: 1. Five DNA single strands, namely S1, S2, S3, S4 and S5, which constitute the DNA tweezers fluorescent probe, are prepared respectively, and the corresponding base sequences and the modifications contained therein are shown in Table 2, Figure 2 and Figure 3 The five DNA single strands are prepared into stock solutions with a concentration of 100 μM respectively.

[0031] Table 2 Sequence and explanation of the fluorescent probe 2. Synthesis of the fluorescent probe.

[0032] 2 μL of S1 stock solution, 2 μL of S2 stock solution, 2 μL of S3 stock solution, 2 μL of S4 stock solution and 4 μL of S5 stock solution are taken respectively and added to 88 μL of phosphate buffer solution to mix uniformly, so that the final concentration of S1:S2:S3:S4:S5 is 1:1:1:1:2 in molar ratio, to ensure the correct assembly of the DNA tweezers fluorescent probe. The formula of the phosphate buffer solution is: 10 mM Na2HPO4, 137 mM NaCl, pH 7.0.

[0033] The mixed system is placed in a 200 μL PCR tube, mixed uniformly with a vortex shaker, and then placed in a PCR instrument to perform programmed annealing and hybridization, so as to obtain 2 μM 100 μL of the DNA tweezers fluorescent probe. The annealing and hybridization program in the PCR instrument is set as: 95℃, 5 min; 65℃, 30 min; 50℃, 30 min; 37℃, 30 min; 22℃, 30 min.

[0034] Example 2 Application of a DNA tweezers fluorescent probe, specifically as follows: 1. Polyacrylamide gel electrophoresis is used to verify the successful synthesis of the DNA tweezers fluorescent probe.

[0035] (1) Preparation of experimental materials.

[0036] Group 1, denoted as DNA structure ①: DNA containing only S1.

[0037] Group 2, denoted as DNA structure ②: DNA structure obtained after hybridization of S1 and S4.

[0038] Group 3, denoted as DNA structure ③: DNA structure obtained after hybridization of S1, S2 and S4.

[0039] Group 4, denoted as DNA structure ④: DNA structure obtained after hybridization of S1, S2, S3 and S4.

[0040] Group 5, denoted as DNA structure ⑤: the DNA tweezers fluorescent probe prepared in Example 1.

[0041] Groups 1-4 are control groups, which are only different from the DNA tweezers fluorescent probe of Example 1 in that the single-stranded raw materials are different in composition, and are prepared according to the method of Example 1, with the hybridization annealing reaction system being prepared by lacking part and being supplemented with water, and using the hybridization annealing procedure of Example 1.

[0042] (2) The experimental materials prepared above are subjected to electrophoretic separation and analysis in a non-denaturing polyacrylamide gel.

[0043] The electrophoresis process is carried out at a constant voltage of 140 V in 1xTBE buffer for 1 h. After the electrophoresis is completed, the gel is placed in nucleic acid dye SybrGold, and is subjected to light-shielded and low-speed shaking for 30 min for dyeing, and finally is imaged by using a Bio-Rad Gel-Doc gel imager.

[0044] The results are shown in Figure 4 According to the principle of nucleic acid gel electrophoresis, it can be known that the smaller the molecular weight is, the faster the migration is, and therefore, the size relationship of the migration rate is: ①>②>③>④>⑤. By sequentially increasing the number of DNA single strands constituting the DNA tweezers fluorescent probe, the migration rate of the product is reduced, which indicates the successful assembly of the DNA tweezers fluorescent probe.

[0045] 2. Verification of the responsiveness of the DNA tweezers fluorescent probe to weak acidic pH in a solution.

[0046] In this example, first, Shanghai Shengong Biological Company is commissioned to modify the 5' end of the DNA tweezers fluorescent probe structure of SEQ ID NO. 4 prepared in Example 1 with a fluorescent group rhodamine red, and to modify the 3' end with rhodamine green. When the two fluorescent groups are close in space, a fluorescence resonance energy transfer effect will be produced, as shown in Figure 5 a.

[0047] The above DNA tweezers fluorescent probe is placed in phosphate buffer solutions with pH values of 6.4, 6.6, 6.8, 6.9, 7.0, 7.1, 7.2 and 7.4, respectively, and is incubated at 37℃ for 60 min, after which the emission spectrum is detected at room temperature: the excitation light wavelength is 480 nm, the emission spectrum of the interval of 510 nm-680 nm is collected, the excitation light bandwidth is 5 nm, and the emission light bandwidth is 5 nm. The results are shown in Figure 5 b. As the pH value gradually decreases from 7.4 to 6.4, the characteristic emission peak of rhodamine green continuously decreases, the characteristic emission peak of rhodamine red continuously increases, and an obvious FRET effect is formed. The relationship between A / D and pH is plotted as a curve, and the midpoint of the transition is at pH 6.86, as shown in Figure 5The value of 'c' indicates that the DNA tweezers fluorescent probe is in a folded state under weakly acidic pH conditions, i.e., within the pH range of 6.4 to 6.8.

[0048] 3. Verify the responsiveness of the DNA tweezers fluorescent probe to weakly acidic pH at the cellular level.

[0049] ECA109 cells were cultured in RPMI 1640 medium containing 10% v / v inactivated fetal bovine serum, 100 U / mL penicillin, and 100 ng / mL streptomycin at 37°C and 5% CO2. ECA109 cells were seeded at a density of 70,000 cells / mL in 35 mm confocal dishes and cultured for another 24 h. The medium was then aspirated, and the cells were washed three times with PBS.

[0050] A 0.5 μM DNA tweezers fluorescent probe solution was prepared by adding the probe to PBS solution and then adding it to a confocal dish containing cells. The solution was incubated at room temperature for 10 min. Excess solution was aspirated, and the cells were washed three times with PBS solution. Then, PBS solutions with pH values ​​of 7.4, 7.2, 7.0, 6.8, 6.6, and 6.4 were added to the confocal dishes, and the dishes were incubated at 37 °C for 20 min. The samples were imaged using a laser confocal scanning microscope. Rhodamine Green and FRET emission signals were continuously excited and collected using a 488 nm laser. The raw laser confocal data were processed using ImageJ software, and the average fluorescence intensity was analyzed. The final results of each experimental group were normalized.

[0051] like Figure 5 As shown in Figure e, FRET exhibits significant fluorescence at pH 6.4–6.8, while almost no red fluorescence is observed at neutral pH. Further quantitative analysis of the confocal plot yields the A / D versus pH curve, as shown below. Figure 5 As shown in f, the midpoint of the transition of the curve is at pH 6.90, and the curve shape is almost the same as... Figure 5 The c-overlap indicates that the DNA tweezer fluorescent probe prepared in this invention has the same pH responsiveness on the cell membrane surface as it does in solution, suggesting that under weakly acidic pH conditions, the DNA tweezer fluorescent probe anchored on the cell membrane surface is in a folded state, with the structure as shown in the figure. Figure 5 d.

[0052] 4. Investigate the response of the DNA tweezers fluorescent probe in solution to weak acidity pH and potassium ions.

[0053] Experimental materials: DNA tweezers fluorescent probes with a concentration of 0.1 μM prepared in Example 1.

[0054] Experiments were carried out with pH and potassium ions as two variables, and solutions with different potassium ion concentrations and pH of 6.4, 6.6, 6.8 and 7.4 were prepared, and the potassium ion concentrations were 0 mM, 5 mM, 10 mM, 20 mM, 30 mM, 40 mM and 50 mM, respectively.

[0055] The DNA tweezers fluorescent probe was incubated in the prepared solution at 37 DEG C for 1 h, and then the fluorescence spectrum was measured at room temperature, the excitation wavelength was 530 nm, and the emission wavelength was 550 nm-750 nm. The excitation light bandwidth was 5 nm, and the emission light bandwidth was 5 nm.

[0056] As shown in the results, Figure 6 with the increase of the potassium ion concentration from 0 mM to 50 mM, the emission peak of Cy5 in the range of 650 nm-700 nm of the DNA tweezers fluorescent probe under the condition of pH of 6.4-6.8 was obviously enhanced, and under the condition of pH of 7.4, the emission peak of Cy5 was almost unchanged. It is indicated that only under the weak acid condition, the C-quadruplex in the middle of the DNA tweezers is folded, and the G-quadruplex at the end of the DNA tweezers is easier to fold and induce the FRET effect, that is, to enhance the emission light of Cy5. Under the condition of pH of 7.4, the C-quadruplex in the DNA tweezers fluorescent probe cannot be folded, and the end of the DNA tweezers is difficult to approach, so that the G-quadruplex at the end is difficult to fold, and thus the FRET effect is not obvious.

[0057] The emission light intensity at 670 nm of each emission spectrum was taken as A value, the emission light intensity at 565 nm of each emission spectrum was taken as D value, the ratio of A and D, that is, A / D value was calculated, and the relationship between A / D value and potassium ion concentration was plotted, and the results as shown in Figure 6 The A / D value of the DNA tweezers fluorescent probe under the condition of pH of 6.4-6.8 and potassium ion concentration of 40 mM-50 mM in the dotted line box in the figure is higher than the A / D value under other conditions. The above results show that the DNA tweezers fluorescent probe prepared by the application has the responsiveness to weak acid pH and high potassium ion concentration, and can induce the FRET effect under the condition that the two factors meet the specific conditions, and emit strong Cy5 fluorescence.

[0058] 5、Investigate the responsiveness of the DNA tweezers fluorescent probe on the cell membrane surface to weak acid pH and potassium ions.

[0059] ECA109 cells were cultured in RPMI1640 medium containing v / v 10% inactivated fetal bovine serum, 100 U / mL penicillin and 100 ng / mL streptomycin at 37℃ in the presence of 5% CO2. ECA109 cells were seeded in a 35 mm confocal dish at a cell density of 70000 cells / mL and continued to be cultured in the incubator for 24 h; the culture medium was aspirated and the cells were washed with PBS for three times.

[0060] The DNA tweezers fluorescent probe was added to a PBS solution to prepare a DNA tweezers fluorescent probe solution with a concentration of 0.5 μM, and 0.5 mL of the DNA tweezers fluorescent probe solution was added to four confocal dishes containing cells, respectively, and incubated at room temperature for 10 min to anchor the DNA tweezers fluorescent probe on the cell membrane surface.

[0061] Meanwhile, four solutions were prepared, respectively: (1) a phosphate buffer solution with a pH of 7.4 and containing 5 mM KCl.

[0062] (2) a phosphate buffer solution with a pH of 6.8 and containing 5 mM KCl.

[0063] (3) a phosphate buffer solution with a pH of 7.4 and containing 40 mM KCl.

[0064] (4) a phosphate buffer solution with a pH of 6.8 and containing 40 mM KCl.

[0065] The excess solution in the four confocal dishes was aspirated, and the four prepared solutions were added to the four confocal dishes, respectively. Incubation was carried out at 37℃ for 20 min. The samples were imaged by laser confocal scanning microscopy. The Cy3 and FRET emission signals were collected using 488 nm laser continuous excitation, respectively.

[0066] According to the structure of the DNA tweezers fluorescent probe designed in the present application, only when the pH is weakly acidic, the C-quadruplex in the middle of the structure of the DNA tweezers fluorescent probe is folded and the two "handles" of the DNA tweezers fluorescent probe are close to each other; under the condition of the existence of both weakly acidic pH and potassium ions, the G-quadruplex at the end of the DNA tweezers fluorescent probe is folded, so that the two fluorescent molecules of Cy5 and Cy3 are close to each other, thereby triggering the FRET effect. As shown in FIG. 1a. Figure 7

[0067] The present embodiment was verified by experiments, and the results are shown in FIG. 2. Figure 7 ​As shown in FIG. 6B, only the solution with pH of 6.8 and potassium ion concentration of 40 mM can induce FRET effect to emit obvious fluorescence. The experimental results are consistent with the settings of the present application, indicating that the DNA tweezers fluorescent probe prepared in the present application has the responsiveness to weak acid pH and high potassium ion concentration, can induce FRET effect in the presence of the two factors, and emit strong Cy5 fluorescence, indicating that the DNA tweezers fluorescent probe prepared in the present application can specifically image cancer cells under the condition of weak acid pH and high potassium ion concentration.

[0068] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application and not to limit the protection scope of the present application. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present application.

Claims

1. A DNA tweezer fluorescent probe, characterized in that, The DNA tweezers fluorescent probe is formed by hybridization of the DNA single strands shown in SEQ ID NO. 1~SEQ ID NO.

5.

2. The DNA tweezer fluorescent probe of claim 1, wherein, The 3' end of SEQ ID NO. 1 contains Cy5 modification; The 5' end of SEQ ID NO. 2 contains Cy3 modification; The 5' end and 3' end of SEQ ID NO. 5 contain cholesterol modification.

3. The DNA tweezer fluorescent probe of claim 1, wherein, The response condition of the DNA tweezers fluorescent probe is the pH and potassium ion concentration of the tumor microenvironment.

4. The DNA tweezer fluorescent probe of claim 3, wherein, The pH ranges from 6.4 to 6.8; The potassium ion concentration is 40mM~50mM.

5. The method for preparing the DNA tweezers fluorescent probe as described in claim 1, characterized in that, The method comprises the following steps: Synthesizing and modifying the sequences shown in SEQ ID NO. 1~SEQ ID NO. 5; Mixing and hybridizing the sequences and then annealing to obtain the DNA tweezers fluorescent probe.

6. The production method according to claim 5, wherein The molar ratio of the DNA single strands shown in SEQ ID NO. 1~SEQ ID NO. 5 is: SEQ ID NO. 1: SEQ ID NO. 2: SEQ ID NO. 3: SEQ ID NO. 4: SEQ ID NO. 5 = 1: 1: 1: 1:

2.

7. The production method according to claim 5, wherein The hybridization and annealing program is: 95℃, 5min; 65℃, 30min; 50℃, 30min; 37℃, 30min; 22℃, 30min.

8. The use of the DNA tweezer fluorescent probe according to claim 1, wherein, The application refers to at least one of the following: 1) in the preparation of products for imaging tumor cells; 2) in the preparation of products for diagnosing cancer and / or cancer prognosis.

9. Use according to claim 8, wherein the compound is ###0002### The tumor cells are esophageal cancer cells; The cancer is esophageal cancer.

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