DNA tweezers fluorescent probe, and preparation method and application thereof

Abstract

The present application relates to the field of nanobiological sensing, and particularly relates to a DNA tweezers fluorescent probe and a preparation method and application thereof, wherein the DNA tweezers fluorescent probe is formed by hybridization of DNA single strands shown in SEQ ID NO. 1 to SEQ ID NO. 5. Five DNA single strands are designed to form a DNA tweezers fluorescent probe with tweezers type nanostructure through base complementary pairing, and the DNA tweezers fluorescent probe has a stimulation responsiveness to weak acidic pH and high potassium ion concentration in a tumor microenvironment. In the case that both weak acidic pH and high potassium ion concentration exist, the fluorescent probe is completely folded, has a clear fluorescence resonance energy transfer effect, and emits clear red fluorescence. Compared with imaging probes with single factor stimulation responsiveness, the DNA tweezers fluorescent probe has a simple synthesis method, and has higher specificity in tumor cell imaging, and has important significance for specific imaging of tumor cells.

Description

Technical Field

[0001] This invention relates to the field of nanobiosensing technology, and in particular to a DNA tweezers fluorescent probe, its preparation method, and its application. Background Technology

[0002] Cancer is one of the leading causes of death worldwide, and accurate diagnosis and effective treatment remain among the greatest challenges in clinical practice. Compared to normal tissues, the tumor microenvironment is characterized by: acidic pH, hypoxia, upregulated levels of redox substances such as hydrogen peroxide and glutathione, and excessive metabolic biomolecules and enzymes such as glucose, lactate, and polyamines. Normal human cells primarily derive their energy from mitochondrial oxidative phosphorylation, while tumor cells generate energy through glycolysis to meet their rapid proliferation needs. However, glycolysis produces large amounts of lactate, which is expelled from the tumor cells, resulting in a lower extracellular pH. Compared to the neutral pH of approximately 7.4 in normal tissues, tumor tissues are slightly acidic, with a pH of approximately 6.4–6.8. Furthermore, rapidly dividing tumor cells compete for limited space, leading to significant cell death. Necrotic cells release intracellular potassium ions, increasing the extracellular potassium concentration from 5 mM in normal tissues to 40–50 mM in the tumor microenvironment. Therefore, the extracellular environment of tumor cells is characterized by weak acidity and high potassium ion concentration.

[0003] Based on the differences between the tumor microenvironment and the normal tissue microenvironment, researchers have designed and developed many nanoprobes responsive to the tumor microenvironment, hoping to understand tumor progression through real-time monitoring and provide valuable guidance and evidence for efficient tumor treatment. However, some tissues other than tumors, such as nodules and inflammatory tissues, also exhibit similar characteristics to the tumor microenvironment, which can lead to false positive results in tumor imaging. Currently, the imaging performance of many nanoprobes is limited by their poor tumor specificity, severely hindering their widespread application. Therefore, there is an urgent need to develop an imaging probe with higher tumor tissue specificity. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the existing technology and provide a DNA tweezer fluorescent probe that can respond simultaneously to the weakly acidic pH and high potassium ion concentration in the tumor microenvironment, and can be applied to tumor cell imaging.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a DNA tweezer fluorescent probe, which is formed by hybridization of DNA single strands shown in SEQ ID NO.1 to SEQ ID NO.5; the DNA tweezer fluorescent probe is a tweezer-type nanostructure.

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

[0007] This invention utilizes the fluorescence resonance energy transfer (FRET) mechanism to generate signals using a fluorescent probe on DNA tweezers. FRET refers to the phenomenon where, between two different fluorophores, if the emission spectrum of one fluorophore overlaps with the absorption spectrum of the other, and the distance between the two fluorophores is appropriate (generally less than 10 nm), the transfer of fluorescence energy from the donor to the acceptor can be observed. Under the stimulation of high potassium ion concentration conditions in the tumor microenvironment, the spatial distance between the two fluorophores Cy5 and Cy3 on the fluorescent probe on the DNA tweezers decreases after the DNA tweezers are fully folded, resulting in a significant FRET effect and emitting a distinct red fluorescence, thereby achieving specific imaging of tumor cells.

[0008] Preferably, the 5' and 3' ends of SEQ ID NO.5 contain cholesterol modifications.

[0009] The present invention is designed to add cholesterol modification to the 5' and 3' ends of SEQ ID NO.5. The cholesterol group can be inserted into the cell membrane, and the DNA tweezers fluorescent probe is anchored to the cell membrane surface.

[0010] Secondly, the present invention provides a method for preparing the above-mentioned DNA tweezer fluorescent probe, which is formed by annealing the mixed hybridization of DNA single strands shown in SEQ ID NO.1 to SEQ ID NO.5.

[0011] Preferably, the molar ratio of the mixed DNA single strands 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] Mixing five DNA single strands in the specific molar ratio described above ensures the correct assembly of the DNA tweezers fluorescent probe.

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

[0014] Thirdly, the present invention provides the application of the above-mentioned DNA tweezers fluorescent probe in the preparation of products for tumor cell imaging.

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

[0016] Preferably, the tumor cells include human esophageal cancer cells ECA109.

[0017] Fourthly, the present invention provides the application of the above-described DNA tweezer fluorescent probe in the preparation of products for cancer diagnosis and / or cancer prognosis.

[0018] The beneficial effects of this invention are as follows: To address the poor specificity of current fluorescent probes that respond to single factors in the tumor microenvironment, this invention provides a DNA tweezer fluorescent probe, which is formed by hybridization of single-stranded DNA as shown in SEQ ID NO.1~SEQ ID NO.5. The DNA tweezer fluorescent probe designed in this invention exhibits specificity for pH and K... + The fluorescent probe exhibits high responsiveness; its structure can only fully fold and emit fluorescence when both weak acidity and high potassium ion concentration are simultaneously met, ensuring the specificity of imaging. Compared to other fluorescent probes that require complex chemical synthesis or material preparation processes, this invention provides a simple synthesis method: mixing five DNA single strands in a specific ratio followed by annealing to obtain the desired DNA tweezer fluorescent probe. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the structural transformation of a DNA tweezers fluorescent probe.

[0020] Figure 2 This is the composition of the fluorescent probe for DNA tweezers; the arrows for each DNA strand indicate the 5' to 3' direction of the DNA.

[0021] Figure 3 Detailed structural diagrams of the G-quadruplex and the C-quadruplex are shown. a: G-quadruplex; b: C-quadruplex.

[0022] Figure 4 Figure 1 shows the assembly diagram and identification of the DNA tweezers fluorescent probe; Figure 2a is a schematic diagram of the assembly process of the DNA tweezers fluorescent probe; Figure 3b shows the polyacrylamide gel electrophoresis results proving the successful synthesis of the DNA tweezers fluorescent probe.

[0023] Figure 5To verify the responsiveness of the DNA tweezers fluorescent probe to weakly acidic pH, the following figures are presented: Figure a shows a schematic diagram of the structural transformation of the DNA tweezers fluorescent probe under pH stimulation; Figure b shows the fluorescence spectra under different pH conditions; Figure c shows the A / D ratio versus pH curve, where A: Accept, representing the emission intensity of Rhodamine Red at 590 nm; D: Donor, representing the emission intensity of Rhodamine Green at 525 nm; Figure d shows a schematic diagram of the structural transformation of the DNA tweezers fluorescent probe after anchoring it to the cell membrane under pH stimulation; Figure e shows confocal images of ECA109 cells incubated under different pH conditions after anchoring the DNA tweezers fluorescent probe to the cell membrane surface; Figure f shows the normalized A / D ratio versus pH curve calculated based on the results in Figure e, where A represents the average fluorescence intensity of the confocal image of the FRET channel, and D represents the average fluorescence intensity of the confocal image of the Rhodamine Green channel.

[0024] Figure 6 To verify the responsiveness of the DNA tweezers fluorescent probe to weakly acidic pH and potassium ions in solution, the following graphs are presented: Figure a shows the complete folding of the C and G quadruple strands in the DNA tweezers under pH 6.4–6.8 and potassium ion concentrations of 40 mM–50 mM, exhibiting a significant FRET effect; the reaction equilibrium shifts to the right in the figure. Figures b–d show the fluorescence emission spectra of the DNA tweezers fluorescent probe measured at pH 6.4, 6.6, and 6.8, and different potassium ion concentrations, respectively. Figure e shows the structural changes of the DNA tweezers fluorescent probe under pH 7.4 and potassium ion concentrations of 0 mM–50 mM, with the reaction equilibrium shifting to the left in the figure. Figure f shows the fluorescence emission spectra of the DNA tweezers fluorescent probe measured at pH 7.4 and different potassium ion concentrations. Figure g shows the A / D ratio versus potassium ion concentration curve obtained from figures b, c, d, and f; where A represents the emission intensity of Cy5 at 670 nm, and D represents the emission intensity of Cy3 at 565 nm. The intensity of emitted light at a wavelength of nm.

[0025] Figure 7 To investigate the response of DNA tweezers fluorescent probes on the cell membrane surface to two factors: weakly acidic pH and potassium ions; Figure a shows a schematic diagram of the folding process of DNA tweezers fluorescent probes anchored on the cell membrane surface under conditions of pH 6.4–6.8 and potassium ion concentration of 40 mM–50 mM, where the reaction equilibrium shifts to the right; Figure b shows a confocal fluorescence image of DNA tweezers fluorescent probes anchored on the ECA-109 cell membrane surface and incubated under different conditions. Detailed Implementation

[0026] The present invention will be further illustrated below with specific embodiments, but these embodiments do not limit the scope of the invention. Modifications or substitutions to the details and form of the technical solutions of the present invention may be made without departing from the spirit and scope of the invention, but all such modifications or substitutions fall within the protection scope of the present invention.

[0027] The inventive concept of this invention is as follows: This invention designs five DNA single strands, forming a tweezer-like fluorescent probe with a tweezer-like nanostructure through base complementary pairing. SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.5 contain sequences forming a G quadruple strand; SEQ ID NO.3 contains a sequence forming a C quadruple strand; and SEQ ID NO.4 provides support. The DNA tweezer-like fluorescent probe of this invention contains nucleic acid structures responsive to pH and potassium ions: a C quadruple strand structure and a G quadruple strand structure. The C quadruple strand structure is formed by folding cytosine-rich DNA. Under acidic conditions, a half-protonated cytosine and another unprotonated cytosine form a C-C base pair through Hoogsteen hydrogen bonds. These C-C base pairs interpenetrate, ultimately forming a stable four-stranded nucleic acid structure. Because protonation is required for its formation, the C quadruple strand is responsive to hydrogen ions or pH. The G quadruple strand structure is formed by folding guanine-rich nucleic acid sequences. Four guanine bases form a complementary pairing structure, such as K... + Na + Li + Under the influence of alkali metals, layers stack to form a G quadruple chain structure; therefore, the G quadruple chain structure has the property of resisting K. + The responsiveness of the DNA tweezers designed in this invention is as follows: Under the stimulation of the weakly acidic conditions of the tumor microenvironment, the C-rich single-stranded DNA sequence in the middle of the fluorescent probe folds into a C-quadruplex, bringing the two "handles" of the DNA tweezers closer together, causing the G-rich sequences at the two ends of the DNA tweezers to move closer. Under the stimulation of the high potassium ion concentration conditions of the tumor microenvironment, the G-quadruplex folds. (See the working principle diagram below.) Figure 1 .

[0028] To enable those skilled in the art to better understand and implement the technical solutions of this invention, the invention will be further described below with reference to specific embodiments. Unless otherwise specified, all reagents used in this invention are commercially available, and all methods used are conventional techniques in the art.

[0029] The English-Chinese glossary and abbreviation list of this invention are shown in Table 1.

[0030] Table 1. Chinese-English Glossary and List of Abbreviations Example 1 A method for preparing a fluorescent probe for DNA tweezers is as follows: 1. Prepare five single-stranded DNA molecules, S1, S2, S3, S4, and S5, which constitute the fluorescent probe of the DNA tweezers. Their corresponding base sequences and modifications are shown in Table 2. Figure 2 and Figure 3 As shown, five DNA single strands were used to prepare stock solutions with a concentration of 100 μM.

[0031] Table 2. Sequences and descriptions of the fluorescent probes 2. Synthesis of fluorescent probes.

[0032] Take 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 respectively, and add them to 88 μL of phosphate buffer solution. Mix thoroughly to achieve a final molar ratio of S1:S2:S3:S4:S5 of 1:1:1:1:2 to ensure correct assembly of the DNA tweezers fluorescent probe. The phosphate buffer solution formula is: 10 mM Na2HPO4, 137 mM NaCl, pH 7.0.

[0033] Place the mixture in a 200 μL PCR tube, vortex to mix, and then place in a PCR instrument for programmed annealing hybridization to obtain 2 μM 100 μL DNA tweezer fluorescent probe. Set the annealing hybridization program in the PCR instrument as follows: 95℃, 5 min; 65℃, 30 min; 50℃, 30 min; 37℃, 30 min; 22℃, 30 min.

[0034] Example 2 The application of a DNA tweezers fluorescent probe is as follows: 1. Polyacrylamide gel electrophoresis was 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: DNA tweezers fluorescent probe prepared in Example 1.

[0041] Groups 1 through 4 served as control groups. The only difference between them and the DNA tweezers fluorescent probes in Example 1 was the composition of the raw material single strands. All groups were prepared according to the method in Example 1, with water used to make up any missing parts. They were prepared using the hybridization annealing procedure in Example 1.

[0042] (2) The experimental materials prepared above were separated and analyzed by electrophoresis in a non-denaturing polyacrylamide gel.

[0043] Electrophoresis was performed at a constant voltage of 140V for 1 hour in 1×TBE buffer. After electrophoresis, the gel was placed in SybrGold nucleic acid dye to shield it from light and swirled at low speed for 30 minutes. Finally, it was imaged using a Bio-Rad Gel-Doc gel imaging system.

[0044] The results are as follows Figure 4 As shown, according to the principle of nucleic acid gel electrophoresis, the smaller the molecular weight, the faster the migration. Therefore, the migration rate is in the following order: 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 decreases, indicating the successful assembly of the DNA tweezers fluorescent probe described in this invention.

[0045] 2. Verify the responsiveness of the DNA tweezers fluorescent probe to weakly acidic pH in solution.

[0046] In this embodiment, Shanghai Sangon Biotech Co., Ltd. was commissioned to modify the 5' end of the DNA tweezers fluorescent probe structure SEQ ID NO.4 prepared in Example 1 with the fluorescent group Rhodamine Red and the 3' end with Rhodamine Green. When the two fluorescent groups are spatially close, a fluorescence resonance energy transfer effect will occur, see... Figure 5 a.

[0047] The aforementioned DNA tweezers fluorescent probes were 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 incubated at 37°C for 60 min. The emission spectra were then detected at room temperature: the excitation wavelength was 480 nm, and the emission spectra in the 510 nm–680 nm range were collected, with an excitation bandwidth of 5 nm and an emission bandwidth of 5 nm. The results are as follows: Figure 5 As shown in Figure b, as the pH value gradually decreases from 7.4 to 6.4, the characteristic emission peak of Rhodamine Green continuously decreases, while the characteristic emission peak of Rhodamine Red continuously increases, forming a significant FRET effect. Plotting the A / D ratio against pH, the midpoint of the transition is observed at pH 6.86. 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 conducted using pH and potassium ions as two variables. Solutions with different potassium ion concentrations and pH values ​​of 6.4, 6.6, 6.8, and 7.4 were prepared, with potassium ion concentrations of 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°C for 1 hour, and then the fluorescence spectrum was measured at room temperature. The excitation wavelength was 530 nm, and the emission wavelength was 550 nm to 750 nm. The excitation bandwidth was 5 nm, and the emission bandwidth was 5 nm.

[0056] The results are as follows Figure 6 As shown, with the potassium ion concentration gradually increasing from 0 mM to 50 mM, the emission peak of Cy5 at 650 nm to 700 nm was significantly enhanced in the DNA tweezers fluorescent probe under pH conditions of 6.4–6.8; however, the emission peak at Cy5 remained almost unchanged under pH conditions of 7.4. This indicates that only under weakly acidic conditions, after the C quadruple strand in the middle of the DNA tweezers folds, can the G quadruple strand at the end of the DNA tweezers fold more easily and induce the FRET effect, i.e., enhance the emission of Cy5. Under pH conditions of 7.4, changing the potassium ion concentration does not prevent the C quadruple strand in the DNA tweezers fluorescent probe from folding, making it difficult for the DNA tweezers to approach, thus hindering the folding of the G quadruple strand at the end, and therefore the FRET effect is not obvious.

[0057] The emission intensity at 670 nm in each emission spectrum is taken as the A value, and the emission intensity at 565 nm in each emission spectrum is taken as the D value. The ratio of A to D, i.e., the A / D value, is calculated. A graph is plotted between the A / D value and the potassium ion concentration to obtain the following results: Figure 6 The results are shown in g. The dashed box in the figure represents the A / D value of the DNA tweezers fluorescent probe under conditions of pH 6.4–6.8 and potassium ion concentration of 40 mM–50 mM, which is higher than the A / D value under other conditions. These results demonstrate that the DNA tweezers fluorescent probe prepared in this invention exhibits responsiveness to both weakly acidic pH and high potassium ion concentration, and can induce the FRET effect and emit strong Cy5 fluorescence under specific conditions where both factors are simultaneously satisfied.

[0058] 5. Investigate the response of the DNA tweezers fluorescent probe on the cell membrane surface to weakly acidic pH and potassium ions.

[0059] 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.

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

[0061] Four solutions were prepared simultaneously: (1) A phosphate buffer solution with pH 7.4 containing 5 mM KCl.

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

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

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

[0065] Excess solution was aspirated from the four confocal dishes, and the four solutions prepared above were added to the four confocal dishes respectively. The samples were incubated at 37°C for 20 min. The samples were imaged using a laser confocal scanning microscope. Emission signals from Cy3 and FRET were continuously excited and collected using a 488 nm laser.

[0066] According to the structure of the DNA tweezer fluorescent probe designed according to the present invention, only when the pH is weakly acidic, the C quadruplex in the middle of the DNA tweezer fluorescent probe folds and brings the two "handles" of the DNA tweezer fluorescent probe closer together; under the conditions of weakly acidic pH and potassium ions, the G quadruplex at the end of the DNA tweezer fluorescent probe folds, causing the two fluorescent molecules Cy5 and Cy3 to come closer together, thereby triggering the FRET effect. Figure 7 As shown in a.

[0067] This embodiment has been verified through experiments, and the results are as follows: Figure 7As shown in b, among the four solutions, only at pH 6.8 and a potassium ion concentration of 40 mM could the FRET effect be triggered, emitting significant fluorescence. This experimental result is consistent with the specifications of this invention, indicating that the DNA tweezers fluorescent probe prepared in this invention simultaneously exhibits responsiveness to both weakly acidic pH and high potassium ion concentration, and can trigger the FRET effect and emit strong Cy5 fluorescence in the presence of both factors. This suggests that the DNA tweezers fluorescent probe prepared in this invention can specifically image cancer cells under conditions of weakly acidic pH and high potassium ion concentration.

[0068] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A DNA tweezers fluorescent probe, characterized in that, The DNA tweezers fluorescent probe is formed by hybridization of single-stranded DNA as shown in SEQ ID NO.1 to SEQ ID NO.5; The conditions for the response of the DNA tweezers fluorescent probe are the pH and potassium ion concentration of the tumor microenvironment; The pH range is 6.4 to 6.8; the potassium ion concentration is 40 mM to 50 mM.

2. The DNA tweezers fluorescent probe as described in claim 1, characterized in that, The 3' end of SEQ ID NO.1 contains a Cy5 modification; The 5' end of SEQ ID NO.2 contains a Cy3 modification; The 5' and 3' ends of SEQ ID NO.5 contain cholesterol modifications.

3. The method for preparing the DNA tweezers fluorescent probe as described in claim 1, characterized in that, Includes the following steps: Synthesize and modify the sequences shown in SEQ ID NO.1 to SEQ ID NO.5; The DNA tweezers fluorescent probe is obtained by mixing the sequences, hybridizing, and then annealing.

4. The preparation method according to claim 3, characterized in that, The molar ratio of the DNA single strands shown in SEQ ID NO.1 to SEQ ID NO.5 is as follows: 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.

5. The preparation method according to claim 3, characterized in that, The procedure for hybridization annealing is as follows: 95℃, 5min; 65℃, 30min; 50℃, 30min; 37℃, 30min; 22℃, 30min.

6. The application of the DNA tweezers fluorescent probe as described in claim 1, characterized in that, The application refers to at least one of the following: 1) In products used for preparing tumor cell imaging; 2) Prepare products for diagnosing cancer and / or predicting cancer prognosis.

7. The application as described in claim 6, characterized in that, The tumor cells were esophageal cancer cells; The cancer in question is esophageal cancer.

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