DNA nanoprobes MB and application thereof

Abstract

The present application relates to a kind of DNA nanoprobes MB and its application, belong to the field of biotechnology.The present application is recognized telomerase by telomerase primer on DNA hairpin probe, catalyzes DNA hairpin probe to open and self-assembles by the extension activity of telomerase itself, to realize signal output, greatly reduce the interference of background signal, improve signal-to-noise ratio, while guarantee the specificity in detection process, high fidelity amplification detection signal, and the probe has structural stability and recognition specificity, its dissociation is only related to telomerase activity, not affected by other DNA, protein and other biological background.Further, the present application combines DNA nano self-assembly detection platform and liposome nanoparticle targeted delivery platform, the method shows good biocompatibility, can be easily taken up by cell, realizes real-time in situ monitoring of telomerase activity in living cell.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to a DNA nanoprobe MB and its applications. Background Technology

[0002] Telomeres are short DNA-protein complexes located at the ends of linear chromosomes in eukaryotic cells, playing a crucial role in cell proliferation and differentiation. They gradually shorten with DNA replication, eventually causing cells to cease proliferation, leading to aging and death. Telomerase, a ribonucleoprotein reverse transcriptase, adds telomeric DNA to the ends of eukaryotic chromosomes, repairing and lengthening telomeres lost during DNA replication, thus indirectly regulating cell proliferation. In normal human cells, telomerase activity is tightly regulated. Disorders of telomerase activity often induce diseases. For example, malignant tumors are caused by excessive telomerase activity, leading to unlimited cell proliferation. Conversely, insufficient telomerase activity results in a slow rate of new cell generation, potentially causing a range of diseases such as cardiovascular disease, hereditary bone marrow failure syndrome, and chronic nephritis.

[0003] Current literature reports that telomerase activity is significantly elevated in approximately 85% of gastrointestinal malignancies, 80%–85% of hepatocellular carcinomas, 94% of glial cell carcinomas, 80% of lung cancers, 93.3% of head malignancies, and 87% of pancreatic cancers. The mechanism lies in the fact that when certain tumor suppressor genes, such as p53, mutate, cells can skip the M1 phase and continue dividing, causing telomeres to continue shortening until a critical threshold is reached. At this point, most cells undergo senescence and apoptosis, while some cells can reactivate telomerase at this stage, restoring telomere function and maintaining chromosome stability, thus enabling tumor cells to continuously proliferate and differentiate. Studies have found that telomerase activity is also closely related to disease stage, lymph node metastasis rate, tumor volume, and drug sensitivity. Therefore, the difference in telomerase activity between normal somatic cells and tumor cells makes telomerase a reliable biomarker for early tumor diagnosis.

[0004] Accurate quantitative analysis of telomerase activity plays a crucial role in clinical diagnosis and the development of anticancer drugs. Currently, the most commonly used method is telomerase activity determination using polymerase chain reaction (PCR)-based telomere repeat amplification (TRAP) assay, which offers high specificity but suffers from amplification instability, high error rates, and long processing times. Therefore, various biosensor methods have been developed, which can be categorized as follows: chemiluminescence immunoassay, electrochemical detection, fluorescence analysis, and visual detection. Despite significant progress in telomerase activity detection, certain limitations remain in practical applications. For example, the enzyme in chemiluminescence immunoassay is prone to degradation and environmental interference, resulting in high detection costs. Electrochemical methods for telomerase activity detection suffer from unstable signal probes and require the addition of additional redox media. Fluorescence analysis often requires complex synthesis steps for signal probes, has poor biocompatibility, and generates background noise that significantly reduces detection sensitivity. Colorimetric methods provide clear detection phenomena, but generally have low sensitivity and are difficult to implement in situ detection. Meanwhile, most telomerase activity assays are still performed in vitro, which makes it impossible to accurately understand the activity of telomerase in real living cell samples in actual use. Summary of the Invention

[0005] To further improve the sensitivity and reliability of telomerase activity detection and reduce detection costs, this invention provides a DNA nanoprobe MB, which has good selectivity for telomerase and can more sensitively monitor telomerase activity in living cells in real time, distinguishing the differences in telomerase activity among different types of cancer cells.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] This invention provides a DNA nanoprobe MB, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0008] Preferably, the DNA nanoprobe MB is labeled with CY5.

[0009] The present invention also provides a kit for detecting telomerase activity, the kit containing the aforementioned DNA nanoprobe MB.

[0010] This invention also provides the application of the above-mentioned DNA nanoprobe MB in the preparation of telomerase detection products.

[0011] The present invention also provides a probe liposome composition for in situ detection of telomerase activity, the composition comprising a DNA nanoprobe (MB), a lipid membrane, and a tumor-targeting aptamer.

[0012] Preferably, the tumor-targeting aptamer is AS1411.

[0013] More preferably, the DNA nanoprobe MB is labeled with CY5 and AS1411 is labeled with a fluorescent group.

[0014] The present invention also provides an application of the above composition in the preparation of products for in situ detection of telomerase activity.

[0015] The present invention also provides a method for preparing the above composition, the method comprising the following steps:

[0016] (1) Dissolve DMPC and cholesterol in anhydrous ethanol at a mass ratio of 2:1 to 5:1, remove the solvent, and dry to obtain a dry lipid membrane;

[0017] (2) Add the lipid membrane to the buffer solution and then hydrate it;

[0018] (3) Dissolve MB in 1 ml of the above buffer solution to a final concentration of 200 nM. Before repeated extrusion through 800, 400, 200 and 100 nm polycarbonate filters using a small extruder, perform ultrasonic treatment. After co-extruding the above MB with hydrated lipids, remove unencapsulated MB by ultrafiltration to obtain Lipo@MB.

[0019] (4) Finally, the tumor-targeting aptamer AS1411 and Lipo@MB were incubated at room temperature to obtain AS1411-Lipo@MB.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] (1) Compared with the telomerase repeat amplification method based on polymerase chain reaction (PCR), the present invention uses a DNA hairpin probe labeling and hybridization substitution that is lower in cost, more stable and more specific, which reduces molecular mismatch problems, greatly simplifies the telomerase activity detection steps and improves the reliability of the detection results.

[0022] (2) This invention identifies telomerase by using telomerase primers on DNA hairpin probes. The extension activity of telomerase itself catalyzes the opening of DNA hairpin probes and self-assembly, thereby achieving signal output. This greatly reduces the interference of background signals, improves the signal-to-noise ratio, and ensures the specificity of the detection process, while amplifying the detection signal with high fidelity.

[0023] (3) The detection probe of the present invention utilizes the complementary relationship of bases to form a stable dimer structure, which has structural stability and recognition specificity. The dissociation of the probe is only related to telomerase activity and is not affected by other biological backgrounds such as DNA and proteins.

[0024] (4) The direct detection method for telomerase activity described in this invention combines a DNA nano-self-assembly detection platform and a liposome nanoparticle targeted delivery platform. This method exhibits good biocompatibility and can be easily taken up by cells, realizing real-time in-situ monitoring of telomerase activity in living cells. It overcomes the cumbersome procedures of traditional detection methods and has the advantage of simple operation, which is conducive to the application of nanosensors in the detection of telomerase activity in cells. Attached Figure Description

[0025] Figure 1 This is a schematic diagram of the experiment of the present invention.

[0026] Figure 2 This is the verification result of the reaction between the probe and telomerase in Example 1.

[0027] Figure 3 The results of liposome characterization and liposome functionalization analysis in Example 1 are shown.

[0028] Figure 4 The results show the sensitivity and specificity of telomerase activity detection in Example 1.

[0029] Figure 5 The results show the influence of the telomerase activity detection reaction system in Example 1 on cell viability.

[0030] Figure 6 This is an in situ imaging characterization diagram of telomerase activity in Example 1. Detailed Implementation

[0031] Example 1

[0032] 1. Probe sequence design

[0033] The main probe sequence structures involved in this study are shown in Table 1. The probe sequences of the reaction systems in all experiments were designed in-house. All nucleic acid probe sequences used in this study were purchased from Shanghai Sangon Biotech Co., Ltd. (China).

[0034] Table 1. Oligonucleotide probe sequences involved in this invention.

[0035]

[0036] 2. Feasibility verification of the probe system

[0037] The in vitro feasibility verification experiment of the telomerase detection system in this study yielded the following results: First, single-stranded oligonucleotides (MB) and telomerase-mimicking extended probes (E-MB) were added to TM buffer (10 mM Tris-HCl, 50 mM NaCl, 10 mM MgCl2, pH 7.4) to prepare MB and E-MB solutions with a final concentration of 100 nM each. The reaction solution was heated and maintained at 95°C for 5 minutes, then slowly cooled to room temperature. To ensure the self-assembly of the telomerase-mimicking extended probe, the annealed MB and E-MB were mixed and incubated for 1 hour. The experimental results are as follows: Figure 2 As shown in Figure a, a band indicating hybridization of MB and E-MB appeared above lane 3. Subsequently, 5 μL of telomerase Te (500 cells / μL) extracted from the cells was incubated at 37°C for 1.5 hours in TRAP reaction buffer (20 mM Tris-HCl, 1.5 mM MgCl2, 63 mM KCl, 0.05% Tween 20, 1 mM MEGTA, pH 8.3) containing 100 nMMB and 0.1 mM dNTPs. The experimental results are as follows: Figure 2 As shown in b, lane 3 exhibits a ladder-like product, and the MB band below disappears. To verify whether MB is extended by telomerase, 5 μL of telomerase extracted from cells was heat-inactivated to form H-Te, which was then incubated in TRAP reaction buffer containing 100 nMMB and 0.1 mM dNTPs at 37°C for 1.5 hours. The experimental results are as follows. Figure 2 The ladder-like product disappeared in lane 4 (shown in b), and the probe MB band reappeared, verifying that MB can be extended by telomerase and self-assemble into dendritic products by opening hairpins. This result validated the feasibility of the probe-telomerase reaction using polypropylene gel electrophoresis (PAGE).

[0038] 3. Polyacrylamide gel electrophoresis

[0039] The feasibility of the telomerase detection system needs to be verified using PAGE gel electrophoresis. Prepare a 12% polyacrylamide gel in a clean beaker, with the following composition: 4 mL acrylamide, 2 mL 5×TBE buffer, 4 mL ultrapure water, 20 μL LTEMED, and 150 μL 10% APS solution (prepared before the experiment; this solution should be used immediately). Use a 1 mL pipette to thoroughly mix the above mixture and slowly add it to the fixed gel plate mold. After adding the mixed sample, immediately insert a clean and dried comb mold as needed. Incubate the entire gel plate at 37°C for 30 minutes to allow the gel to fully solidify. Finally, disassemble the solidified gel plate sample and assemble the experimental electrophoresis tank. Slowly add clean 1×TBE buffer to the electrophoresis tank as the electrophoresis buffer. Add the indicator band DNA ladder (20-500 bp in size) to the first well of the gel plate. Mix all experimental samples and control samples thoroughly with DNA loading buffer at a ratio of 1:5. After thoroughly mixing the sample with the DNA Loading Buffer using a pipette, slowly add it to the wells. Assemble the electrophoresis apparatus. Perform electrophoresis at 80V for 5 minutes, followed by 40 minutes at 100V. When the DNA Ladder indicator band reaches two-thirds of the way across the gel, turn off the program and power to stop the electrophoresis. Disassemble the apparatus and remove the gel. Stain the gel with nucleic acid dye at room temperature for approximately 20 minutes, gently shaking it on a shaker to ensure even staining. This process requires protection from light. Finally, observe the results using a gel imaging system.

[0040] Figure 2 In Figure a, nucleic acid probe after telomerase extension and probe reaction characterization are PAGE electrophoresis images; in Figure b, nucleic acid probe and cell-extracted telomerase reaction characterization are PAGE electrophoresis images.

[0041] 4. Liposome preparation and functionalization

[0042] DMPC and cholesterol (3:1 by mass) were dissolved in anhydrous ethanol to a final lipid concentration of 4 mg / mL. The solvent was removed by rotary evaporation, and the membrane was further dried overnight under high vacuum to obtain a dried lipid membrane. The membrane was rehydrated in buffer (10 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, pH 8.0) to a final concentration of 4 mg lipid / mL. To prepare Lipo@MB, MB was dissolved in 1 mL of buffer to a final concentration of 200 nM. Before repeated extrusion using a small extruder through 800, 400, 200, and 100 nm polycarbonate filters, the membrane was sonicated for 10 minutes. After co-extruding the MB with the hydrated lipids, unencapsulated MB was removed by ultrafiltration. Finally, the tumor-targeting aptamer AS1411 (10 μM, 0.5 μL) and Lipo@MB (1 mM vesicle particle, 100 μL) (both the aptamer and Lipo@MB were dissolved in a buffer solution of 10 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, pH 8.0) were incubated at room temperature for 1 hour to obtain AS1411-Lipo@MB. Laser confocal microscopy was used to observe whether AS1411 and MB were successfully loaded onto the liposomes. Individual liposomes were stained with the fluorescent dye Dil for 10 minutes at room temperature. MB was linked to the CY5 fluorescent group, and AS1411 was linked to the FAM fluorescent group. The samples were mixed and collected in a confocal dish. This part of the experiment required protection from light. Finally, the results were observed and recorded using a laser confocal scanning microscope.

[0043] Figure 3 For liposome characterization and liposome functionalization analysis, (a) transmission electron microscopy (TEM) image of AS1411-Lipo@MB prepared by this method, scale bar 100 nm; (b) nanoparticle tracking analysis (NTA) results of AS1411-Lipo@MB; (c) laser confocal image of dye-labeled AS1411-Lipo@MB, scale bar (10 μm).

[0044] 5. Cell Culture

[0045] DMEM and 1640 medium containing 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin were prepared under sterile conditions. Cervical cancer cells (HeLa), normal breast epithelial cells (MCF-10A), normal liver cells (HL-7702), breast cancer cells (MCF-7), liver cancer cells (HepG2), and non-small cell lung cancer cells (A549) were cultured in the prepared medium at a humid environment of 5% CO2 and at a constant temperature of 37°C.

[0046] 6. Detection of telomerase activity in cell extracts

[0047] Telomerase was extracted using the CHAPS method. Specifically, 4 mL of culture medium was added to a cell culture dish, and approximately 300 μL of cell solution from a centrifuge tube was added to the dish. The dish was gently agitated to ensure even cell distribution, and the cells were then placed in an incubator for growth to maintain HeLa, MCF-7, HL-7702, HepG2, and A549 cells. Cells were collected during the exponential growth phase and washed twice with ice-cold PBS buffer (0.01 M, pH 7.4). The cells were then resuspended in 200 μL of ice-cold CHAPS lysis buffer (10 mM pH 7.5 Tris-HCl, 1 mM MgCl2, 1 mM EGTA, 0.5% CHAPS, 10% glycerol, 0.1 mM PMSF). The lysate was incubated in an ice-water bath for 30 minutes, followed by centrifugation at 15,000 rpm for 20 minutes at 4°C. Finally, the supernatant (cell extract) was collected and carefully transferred to fresh RNase-free test tubes and stored at -80°C for later use.

[0048] To detect telomerase activity, 20 μL of MB (final concentration 100 nM, dissolved in TM buffer), 10 μL of cell extract, 10 μL of dNTP (final concentration 0.1 μM, dissolved in TM buffer), and 160 μL of TRAP buffer were mixed and incubated at 37°C for 2 h, followed by fluorescence measurement. For the control experiment without telomerase, 10 μL of CHAPS lysis buffer was used instead of the cell extract. The experimental results are shown below. Figure 2 As shown in Figure c, MB exhibits a strong fluorescent signal under the action of telomerase. However, when heat-inactivated telomerase H-Te is co-incubated with MB, the fluorescence signal is almost indistinguishable from the background signal. To investigate the probe sensitivity, telomerase extracts from different amounts of HeLa cells were incubated with MB at 37°C for 2 hours, with HeLa cell concentrations ranging from 0 to 1.2 x 10⁻⁶. 5 At that time, the elongation of the telomerase primer sequence triggered by the presence of telomerase could open more MBs, recover the fluorescence signal, and record the fluorescence spectrum. The results showed that the fluorescence intensity at 670 nm increased with increasing cell concentration. Figure 4 a). At a cell concentration of 2x10 4 Up to 1.2x10 5 Good linearity can be obtained within the range. Figure 4 b) The linear equation is Y = 0.023*X + 839(R) 2=0.9939), where Y is the fluorescence intensity recorded at 670 nm, and X is the concentration of HeLa cell extract. To examine the selectivity of this detection system for telomerase activity, different types of cell extracts at a concentration of 500 cells / μL, including HeLa, MCF-10A, MCF-7, HL-7702, HepG2, and A549 cells, were compared and analyzed, with heat-inactivated HeLa cell extract (Heated-HeLa) and HeLa cell extract treated with the telomerase activity inhibitor (AZT) (AZT-HeLa). 10 μL of the cell extracts treated with different methods were co-incubated with 20 μL of LMB (final concentration 100 nM, dissolved in TM buffer) and 10 μL of dNTPs (final concentration 0.1 nM, dissolved in TM buffer) for 2 h. The results showed that when the nanoprobe reacted with cancer cell extracts (including MCF-7, HeLa, A549, and HepG2), the fluorescence intensity of CY5 was enhanced. However, when the probe reacted with extracts from normal somatic cells (including MCF-10A and HL-7702 cells), the fluorescence intensity of CY5 was almost no different from that of heat-inactivated and AZT-treated HeLa cell extracts. Figure 4 d) This is because telomerase exhibits high activity in cancer cells, while its activity is inhibited in normal somatic cells. Heating and telomerase inhibitors both denature reverse transcriptase proteins, leading to telomerase inactivation. The above results indicate that this detection system can effectively distinguish differences in telomerase activity among different types of cancer cells, demonstrating good selectivity. Next, to verify the stability of the proposed method for detecting telomerase activity in different media, we co-incubated MB (final concentration 100 nM) with different media, including DEPC water, cell lysis buffer (CHAPS), different cell culture media (1640 and DMEM), PBS, and human serum (serum) for 2 h. The results showed that the fluorescent probe MB exhibited good stability in all these media. Figure 4 c). Finally, to verify the specificity of this system for the detection of telomerase and other enzymes, we co-incubated other proteases such as bovine serum albumin (BSA), glutathione (GSH), lysozyme, and trypsin. MB (final concentration 100 nM) was incubated with 10 μL of each protease (final concentration 1 mM) for 2 h. The results showed that, compared with telomerase (TE), these proteases did not produce obvious fluorescent signals in the detection system, demonstrating that this method has good detection specificity. Figure 4 e). To measure Cy5 fluorescence, the excitation wavelength was set to 600 nm, the emission wavelength to 650–800 nm, and the voltage to 700 V. All in vitro fluorescence values ​​in this experiment were measured and statistically analyzed using a Hitachi F-4700 fluorescence spectrophotometer.

[0049] Figure 2 c represents the fluorescence characterization of the telomerase activity detection reaction. Figure 4 The sensitivity and specificity of the telomerase activity assay are shown below. (a) Fluorescence curves of the probe reaction with extracts of different cell numbers; (b) Linearity analysis; (c) Stability characterization of the probe in different media; (d) Specificity characterization of the probe reaction with different cell extracts; (e) Fluorescence intensity of the probe reaction with different interfering agents and telomerase.

[0050] 7. Cell viability assay

[0051] The cytotoxicity of the assay system was examined before cell imaging. HeLa and HL-7702 cells were seeded at a density of 4000 cells per well in 96-well cell culture plates and incubated for 24 hours. Figure 5 a) 100 μL of cell culture medium containing MB (final concentration 200 nM), Liposome (final concentration 0.4 mg / mL) and AS1411-Lipo@MB (final concentration of MB 200 nM, final concentration of AS1411 100 nM, final concentration of Liposome 0.4 mg / mL) was added to each well of a 96-well plate and incubated with the cells for 6 hours. Figure 5 b is the cell culture medium containing different concentrations (final concentrations of 10 nM, 50 nM, 100 nM, 200 nM, and 500 nM) of MB, which is co-incubated with the cells for 6 hours. Figure 5 c) 100 μL of cell culture medium containing AS1411-Lipo@MB (final MB concentration 200 nM, final AS1411 concentration 100 nM, final Liposome concentration 0.4 mg / mL) was added to 96-well plates and incubated with cells for 3, 6, 12, and 24 hours. After discarding the culture medium, all cells in the above three experiments were washed with PBS buffer, and then 100 μL of 5 mg / mL LMT was added. After incubation for 4 hours, 100 μL of Formazan dissolving solution was added to each well, mixed appropriately, and incubated further in a cell culture incubator until the formazan was completely dissolved under a regular optical microscope. The absorbance at 570 nm was measured using a microplate reader, and cell viability was calculated. The experimental results showed that the cell viability was higher than 90%, indicating that the detection system had low cytotoxicity.

[0052] Figure 5 The results show the influence of the telomerase activity assay system on cell viability. (a) shows the cell viability of HL-7702 cells and HeLa cells treated with different concentrations of MB; (b) shows the cell viability of HL-7702 cells and HeLa cells treated with different concentrations of MB; and (c) shows the cell viability of HL-7702 cells and HeLa cells at different time points as determined by the assay system.

[0053] 8. In situ imaging and flow cytometry analysis of telomerase activity

[0054] HeLa cells (or MCF-7, HepG2, A549, HL-7702 cells) were seeded in confocal culture dishes and incubated for 24 hours. After removing the culture medium, 100 μL of fresh culture medium containing AS1411-Lipo@MB (MB final concentration 200 nM, AS1411 final concentration 100 nM, Liposome final concentration 0.4 mg / mL) was added to the confocal culture dish. After incubation at 37°C for a period of time, the cells were washed with PBS buffer and incubated with 100 μL of fresh culture medium for confocal imaging. The confocal images showed almost no fluorescence in normal somatic cells (HL-7702), while the CY5 fluorescence signal was strong in cancer cells. The fluorescence signal was stronger in MCF-7, HeLa, and A549 cells, while the fluorescence was weaker in HepG2 cells, indicating relatively low telomerase activity. Figure 6 a). Figure 6 b is a cubic graph of fluorescence intensity measured along a straight line in the zoomed area of ​​the confocal image. Figure 6 c represents the statistical analysis results of fluorescence intensity in the confocal image. Next, we performed fluorescence analysis of telomerase activity detection in this assay system using flow cytometry. HeLa cells (or MCF-7, HepG2, A549, HL-7702 cells) were seeded in 12-well plates (1×10⁻⁶ cells / well). 5 After incubating in cells / well for 24 hours, 100 μL of fresh culture medium containing AS1411-Lipo@MB (MB final concentration 200 nM, AS1411 final concentration 100 nM, Liposome final concentration 0.4 mg / mL) was added to a 12-well plate and incubated at 37°C for 4 hours. Cells were then washed three times with PBS to remove uninternalized probes. Cells were then separated from the culture plate using trypsin, washed twice with PBS, and resuspended in PBS for flow cytometry analysis. Results showed that cancer cells had a stronger fluorescence signal than normal somatic cells. Figure 6 d). These results all indicate that the detection system can detect telomerase activity in cancer cells, image telomerase activity in living cells, and distinguish between normal somatic cells and cancer cells.

[0055] Figure 6The images show in situ imaging characterization of telomerase activity, where (a) is a confocal fluorescence image of telomerase activity in different live cells obtained by AS1411-Lipid@MB, with a scale bar of 50 μm; (b) is the fluorescence intensity along the straight line in the corresponding measurement zoom area; (c) is the confocal fluorescence intensity analysis of telomerase activity in different live cells obtained by AS1411-Lipid@MB; and (d) is the flow cytometry analysis of telomerase in different live cells obtained by AS1411-Lipid@MB.

[0056] All experimental results in this study are expressed as independent experimental mean ± standard error. All experimental data were analyzed using paired t-tests (two groups) or ANOVA (three or more groups), with P < 0.05 used as the significance threshold. All experimental data were corrected using Bonferroni. All statistical tests were performed using GraphPadPrism 8.02 software.

[0057] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A DNA nanoprobe MB, characterized in that, The nucleotide sequence of the DNA nanoprobe MB is shown in SEQ ID NO.

1.

2. The DNA nanoprobe MB according to claim 1, characterized in that, The DNA nanoprobe MB was labeled with CY5.

3. A kit for detecting telomerase activity, characterized in that, The kit contains the DNA nanoprobe MB as described in claim 1 or claim 2.

4. The application of the DNA nanoprobe MB as described in claim 1 or 2 in the preparation of telomerase detection products.

5. The use of the kit described in claim 3 in the preparation of telomerase detection products.

6. A probe liposome composition for in situ detection of telomerase activity, characterized in that, The composition comprises the DNA nanoprobe MB, lipid membrane, and tumor-targeting aptamer as described in claim 1.

7. The composition according to claim 6, characterized in that, The tumor-targeting aptamer is AS1411.

8. The composition according to claim 7, characterized in that, The DNA nanoprobe MB was labeled with CY5 and AS1411 with a fluorescent group.

9. The use of the composition according to any one of claims 6-8 in the preparation of products for in situ detection of telomerase activity.

10. A method for preparing the composition according to claim 8, characterized in that, The preparation method includes the following steps: (1) Dissolve DMPC and cholesterol in anhydrous ethanol at a mass ratio of 2:1 to 5:1, remove the solvent, and dry to obtain a dry lipid membrane; (2) Add the lipid membrane to the buffer solution and then hydrate it; (3) Dissolve MB in 1 ml of the above buffer solution to a final concentration of 200 nM. Before repeated extrusion through 800, 400, 200 and 100 nm polycarbonate filters using a small extruder, perform ultrasonic treatment. After co-extruding the above MB with hydrated lipids, remove unencapsulated MB by ultrafiltration to obtain Lipo@MB. (4) Finally, the tumor-targeting aptamer AS1411 and Lipo@MB were incubated at room temperature for 1 hour to obtain AS1411-Lipo@MB.

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