Kidney cancer marker detection method based on nucleic acid aptamer-three-dimensional DNA nanostructure

Abstract

The invention provides a kidney cancer marker detection method based on a nucleic acid aptamer-three-dimensional DNA nano-structure, and relates to the field of marker detection.A three-dimensional tetrahedral DNA nano-structure is synthesized through a hybridization chain reaction (HCR), a nucleic acid aptamer and the three-dimensional tetrahedral DNA nano-structure are combined to form the nucleic acid aptamer-three-dimensional DNA nano-structure, and the nucleic acid aptamer-three-dimensional DNA nano-structure is used for detecting kidney cancer markers based on the nucleic acid aptamer-three-dimensional DNA nano-structure. The kit is used for targeted detection of renal clear cell carcinoma markers; amplifying a fluorescence signal by using a hybridization chain reaction (HCR) technology; the nucleic acid aptamer-three-dimensional DNA nanostructure is applied to detection of a pathological section, and the targeted binding effect of the nucleic acid aptamer-three-dimensional DNA nanostructure in the pathological section is verified; and detecting a fluorescence signal generated by the amplification chain H2 by using a fluorescence signal detection technology.

Description

Technical Field

[0001] The present invention relates to the technical field of biomarker detection, and in particular to a method for detecting renal cancer biomarkers based on aptamer-three-dimensional DNA nanostructures, which is applicable to the pathological detection of clear cell renal cell carcinoma (ccRCC). Background Art

[0002] Clear cell renal cell carcinoma (ccRCC) is the most common type of renal cell carcinoma, accounting for 70-80% of all renal cancer cases. Surgical resection is one of the main treatment methods, and partial nephrectomy is widely used in surgeries for preserving renal function. During and after the surgery, how to quickly and accurately evaluate the nature of the tumor, the margin status, and the postoperative pathological features is of great significance for ensuring the complete resection of the tumor, reducing the recurrence risk, and guiding subsequent treatment. A rapid and accurate pathological diagnosis can not only help surgeons make timely decisions during the surgery, shorten the warm ischemia time, and protect renal function, but also provide an important basis for postoperative pathological analysis.

[0003] Frozen pathological section diagnosis is an important means for making rapid decisions during the surgery. Pathologists perform a preliminary analysis of the frozen sections through a microscope to evaluate the nature of the tumor and the margin conditions. However, due to the low quality of the frozen sections and the urgent surgery time, the accuracy of the diagnosis is limited to a certain extent. After the surgery, it is usually necessary to further confirm the nature of the tumor through conventional immunohistochemistry (IHC) technology.

[0004] Immunohistochemistry (IHC) technology is the standard method for detecting tumor biomarkers and is commonly used to detect the renal clear cell carcinoma biomarker carbonic anhydrase IX (CAIX). However, the process of immunohistochemistry technology is complex and time-consuming. The complete process of immunohistochemistry of renal cancer specimens usually includes: formalin fixation for 12-24 hours (depending on the size and thickness of the sample), dehydration, paraffin embedding and sectioning for 3 hours, antigen retrieval and blocking for 1-2 hours, primary antibody incubation for 12-18 hours (usually overnight), secondary antibody incubation and color development for 2-4 hours, counterstaining, coverslipping and drying for 1-2 hours, plus 1 hour of microscopic observation, with a total time consumption of up to 30-54 hours. Such a long detection time limits the application of immunohistochemistry technology in intraoperative real-time detection, and its dependence on antibody quality and batch consistency further affects the reliability of the detection.

[0005] Current tumor marker detection methods, especially traditional immunohistochemistry (IHC) techniques, although widely used in postoperative pathological analysis, have significant limitations in rapid diagnosis. The immunohistochemistry technique not only takes a long time (usually more than 24 hours), but also has a high detection cost, unstable signal intensity, and batch differences. In addition, during intraoperative rapid detection, pathologists need to rely on frozen sections for preliminary diagnosis. However, due to the low quality of frozen sections, tissue deformation is common, the staining effect may not be ideal, which easily leads to unclear pathological results and affects the accuracy of diagnosis. Therefore, there is an urgent need for a rapid, sensitive, stable and low-cost tumor marker detection method, especially for intraoperative rapid detection and postoperative accurate diagnosis. Summary of the Invention

[0006] The present invention provides a method for detecting renal cancer markers based on aptamer-three-dimensional DNA nanostructures, and the method includes the following steps:

[0007] (1) Synthesize three-dimensional tetrahedral DNA nanostructures through hybridization chain reaction (HCR), and bind aptamers to the three-dimensional tetrahedral DNA nanostructures to form aptamer-three-dimensional DNA nanostructures for targeted detection of renal clear cell carcinoma markers;

[0008] (2) Amplify the fluorescence signal using the hybridization chain reaction (HCR) technique;

[0009] (3) Apply the aptamer-three-dimensional DNA nanostructures to the detection of pathological sections to verify the targeted binding effect of the aptamer-three-dimensional DNA nanostructures in pathological sections;

[0010] (4) Detect the fluorescence signal generated by the amplified strand H2 using fluorescence signal detection techniques.

[0011] In a preferred embodiment, in the step (2), the fluorescence signal is amplified by providing multiple binding sites and using the hybridization chain reaction (HCR) technique.

[0012] In a preferred embodiment, the pathological section is a frozen section or a paraffin section.

[0013] In a preferred embodiment, verifying the targeted binding effect of the aptamer-three-dimensional DNA nanostructures in frozen sections includes:

[0014] Put the sample tissue into a cryoembedding agent, store it at -80 °C, then section it in a cryostat, incubate the frozen section with the aptamer-three-dimensional DNA nanostructures, and use a fluorescence microscope to detect the Cy3-labeled red fluorescence signal for the detection and identification of the target.

[0015] In a preferred embodiment, verifying the targeting binding effect of the aptamer-three-dimensional DNA nanostructure in paraffin sections includes:

[0016] The fixed tissue is dehydrated and cleared, and then embedded in paraffin to form paraffin sections;

[0017] The paraffin sections are subjected to antigen retrieval in a buffer solution;

[0018] After antigen retrieval, the sections are incubated with the aptamer-three-dimensional DNA nanostructure, and the red fluorescence signal labeled with Cy3 is observed using a fluorescence microscope for target detection.

[0019] Technical effects:

[0020] The present invention significantly improves the detection speed and sensitivity, and is particularly suitable for rapid intraoperative detection and postoperative accurate diagnosis of clear cell renal cell carcinoma (ccRCC). Especially in the identification of tumor margins, the present invention has high clinical application value. By introducing the aptamer-three-dimensional DNA nanostructure and hybridization chain reaction (HCR) technology, the present invention shortens the detection time while enhancing the fluorescence signal intensity, reducing the detection cost, and greatly improving the detection efficiency.

[0021] The detection efficiency of the present invention is improved by about 33.8% compared with immunohistochemistry (IHC), and the detection cost is reduced by about 65.6%. The fluorescence signal intensity is enhanced by 3.276 times compared with using only aptamers for imaging, and the signal duration is extended by about 140%. This method is particularly suitable for rapid intraoperative detection of tumor margins and postoperative accurate diagnosis, significantly improving the detection efficiency, reducing the detection cost, and having broad clinical application potential.

[0022] The present invention combines aptamers, three-dimensional tetrahedral DNA nanostructures with hybridization chain reaction (HCR), and integrates the HCR initiator strand into the CAIX aptamer. When the aptamer binds to the CAIX marker, the aptamer triggers the HCR reaction, inducing continuous amplification of the amplification strands H1 and H2 to generate a long-chain double helix structure, realizing enzyme-free sequence-specific signal amplification.

[0023] The aptamer not only realizes target recognition but also activates the HCR reaction as an initiator strand carrier. The present invention combines the target recognition of the aptamer with the multiple signal amplification ability of the three-dimensional DNA nanostructure, significantly enhancing the fluorescence signal and improving the detection sensitivity and specificity.

[0024] The present invention solves the problems of long detection time, high cost, and low sensitivity in the prior art. Compared with the traditional immunohistochemistry (IHC) method, it has the advantages of rapid detection and lower cost, and can quickly identify tumor margins during surgery, providing immediate assistance for surgical decisions. Description of the Drawings

[0025] To more clearly illustrate the technical solutions in the embodiments of the present application, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the drawings in the following description are only some embodiments of the present application. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0026] Figure 1 It is a complete flowchart of the detection method of aptamer - three - dimensional DNA nanostructure in the detection of clear cell renal cell carcinoma;

[0027] Figure 2 It is the distribution of CAIX in different parts of clear cell renal cell carcinoma;

[0028] Figure 3 It is the effect comparison of aptamer - three - dimensional DNA nanostructure with immunohistochemistry, immunofluorescence and HE staining on paraffin sections;

[0029] Figure 4 It is the comparison of fluorescence duration and fluorescence intensity of aptamer - three - dimensional DNA nanostructure and aptamer applied to frozen sections of clear cell renal cell carcinoma;

[0030] Figure 5 It is the application of aptamer - three - dimensional DNA nanostructure in the recognition of lymphoma cells. Specific Embodiments

[0031] To make the objectives, technical solutions and advantages of the embodiments of the present application clearer, the following will clearly and completely describe the technical solutions in the embodiments of the present application with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all of them. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present application.

[0032] Embodiment 1

[0033] A rapid detection method for renal cancer markers based on aptamer - three - dimensional DNA nanostructure, comprising:

[0034] (1) Synthesize three - dimensional tetrahedral DNA nanostructure through hybridization chain reaction (HCR), and combine the aptamer with the three - dimensional tetrahedral DNA nanostructure to form aptamer - three - dimensional DNA nanostructure for targeted detection of clear cell renal carcinoma markers;

[0035] (2) Amplify the fluorescence signal using the hybridization chain reaction (HCR) technology;

[0036] (3) Apply the nucleic acid aptamer - three - dimensional DNA nanostructure to the detection of pathological sections, verify the targeted binding effect of the nucleic acid aptamer - three - dimensional DNA nanostructure in pathological sections, and use it for targeted recognition and detection of renal clear cell carcinoma markers;

[0037] In a preferred embodiment, the pathological section is a frozen section. Apply the nucleic acid aptamer - three - dimensional DNA nanostructure to the detection of frozen sections, verify the targeted binding effect of the nucleic acid aptamer - three - dimensional DNA nanostructure in frozen sections. The verification process specifically includes:

[0038] Preparation of frozen sections: Use the tissue after renal clear cell carcinoma surgery, and cut tumor tissue, border tissue, and normal tissue.

[0039] Sample treatment: Put the tissue into a cryo - embedding agent (OCT), store it at - 80 °C, and then section it (thickness 5 - 10 μm) in a cryostat.

[0040] Incubation conditions:

[0041] Blocking: Block the sections with a 1 μM random nucleic acid sequence solution at room temperature for 5 minutes to avoid non - specific binding.

[0042] Incubation: Incubate the Cy3 - labeled CAIX nucleic acid aptamer - three - dimensional DNA nanostructure complex (250 nM) with the frozen sections at room temperature for 30 minutes.

[0043] Washing: Wash the sections three times with PBS buffer, 10 minutes each time, to remove the unbound complex.

[0044] Fluorescence signal detection: Use a fluorescence microscope to detect the Cy3 - labeled red fluorescence signal. The distribution of the fluorescence signal in the tumor tissue is uniform, and the strong red signal shows the successful binding of the CAIX marker. No obvious fluorescence signal is observed in the normal tissue, indicating the high specificity of the aptamer.

[0045] In a preferred embodiment, the pathological section is a paraffin section. Apply the nucleic acid aptamer - three - dimensional DNA nanostructure to the detection of paraffin sections, verify the targeted binding effect of the nucleic acid aptamer - three - dimensional DNA nanostructure in paraffin sections. This embodiment aims to verify the targeted binding ability of the nucleic acid aptamer - three - dimensional DNA nanostructure in paraffin sections, and evaluate the effect of the complex in detecting the tumor marker CAIX by comparison with traditional immunohistochemistry (IHC) / and HE staining methods.

[0046] The verification process specifically includes:

[0047] a. Preparation and treatment of paraffin sections:

[0048] Tissue sampling and fixation

[0049] Tumor tissues were obtained from patients with clear cell renal cell carcinoma during surgery and immediately placed in 10% neutral formalin solution for fixation for 24 hours.

[0050] Paraffin embedding and sectioning

[0051] The fixed tissues were dehydrated and cleared, and then embedded in paraffin. The embedded tissues were cut into sections with a thickness of 3 - 5 μm using a microtome. The sections were placed on glass slides and dried in an oven at 60 °C for 30 minutes to ensure firm adhesion of the tissues to the slides.

[0052] Deparaffinization and hydration

[0053] The paraffin sections were successively immersed in xylene for 3 minutes each time, and repeated twice to remove paraffin.

[0054] Subsequently, the sections were successively immersed in 100%, 95%, and 75% ethanol solutions for 1 minute each step, and then washed with distilled water 3 times, 1 minute each time, to complete the hydration process.

[0055] b. Incubation of aptamer - three - dimensional DNA nanostructure

[0056] Antigen retrieval

[0057] The deparaffinized paraffin sections were placed in Tris - EDTA buffer (pH 8.0) at 95 °C for antigen retrieval for 15 minutes. After antigen retrieval, the sections were cooled to room temperature and washed with distilled water 3 times, 2 minutes each time.

[0058] Blocking

[0059] To reduce non - specific binding, the sections were blocked with a 1 μM random nucleic acid sequence solution and incubated at room temperature for 5 minutes.

[0060] Complex incubation

[0061] After blocking, Cy3 - labeled aptamer - three - dimensional DNA nanostructure (concentration 250 nM) was added to the sections and incubated at 37 °C for 30 minutes.

[0062] Washing

[0063] After incubation, the sections were washed 3 times with PBS buffer, 5 minutes each time, to remove unbound complexes and ensure the specificity of the detection signal.

[0064] c. Fluorescence signal detection and imaging

[0065] After the incubation and washing steps were completed, a fluorescence microscope was used to observe the Cy3-labeled red fluorescence signal, with a focus on observing the distribution of the CAIX marker in tumor tissues, and the results were compared with those of traditional IHC, IF, and HE staining.

[0066] d. Experimental results

[0067] The experimental results showed that the aptamer - three-dimensional DNA nanostructure could successfully bind to the CAIX marker in paraffin sections of tumor tissues, generating a strong and uniform red fluorescence signal, especially in the tumor cell membrane region. By comparing with traditional IHC and HE staining, the fluorescence signal was highly consistent with the expression location of the CAIX marker, verifying the accuracy and specificity of the method of the present invention.

[0068] (4) The fluorescence signal generated by the amplified strand H2 was detected using fluorescence signal detection technology. Figure 1 This is the detection flow chart of the present invention, which shows the application of the aptamer - three-dimensional DNA nanostructure in the detection of clear cell renal cell carcinoma (ccRCC), demonstrating its potential in clinical applications, including the identification of renal cancer tissues and the determination of resection margins.

[0069] The initiator strand of HCR activates the H1 and H2 strands, enabling them to generate long-chain double-helical structures through sequence-specific enzyme-free hybridization reactions, thereby continuously amplifying the fluorescence signal.

[0070] The presence of the CAIX aptamer not only achieved target recognition but also served as a carrier for the initiator strand, activating the HCR reaction. This design ingeniously combined the target recognition ability of the aptamer with the stability of the three-dimensional DNA nanostructure to achieve the effect of multiple signal amplification. The multivalent binding sites on the tetrahedral structure and the HCR amplification mechanism significantly enhanced and maintained the stability of the fluorescence signal, thereby significantly improving the sensitivity and specificity of the detection. Figure 1 This shows the complete process of the aptamer - three-dimensional DNA nanostructure detection method in the detection of clear cell renal cell carcinoma, including its application from preoperative diagnosis, intraoperative resection margin assessment to postoperative review. Compared with traditional immunohistochemistry, this method has higher sensitivity and specificity, can quickly identify tumor tissues and accurately label the boundary between tumor and normal tissues, providing strong support for the determination of surgical resection margins. The application of this detection method in clear cell renal cell carcinoma surgery can not only improve the accuracy and thoroughness of the surgery but also reduce the recurrence risk of patients, providing more reliable data support for personalized treatment.

[0071] Example 2

[0072] The fluorescence intensity and duration of the nucleic acid aptamer - three - dimensional DNA nanostructure and the simple aptamer were compared and analyzed on frozen sections.

[0073] This example aims to evaluate the differences in the fluorescence signal intensity and duration of the nucleic acid aptamer - three - dimensional DNA nanostructure and the aptamer used alone in frozen sections. By comparing with the traditional method of using aptamers alone, the signal amplification effect and specificity of the method of the present invention were verified.

[0074] Experimental design

[0075] The preparation and treatment process of the frozen sections were the same as those described in Example 1 and will not be elaborated here.

[0076] a. Incubation of aptamer and nucleic acid aptamer - three - dimensional DNA nanostructure

[0077] Incubation conditions

[0078] Experimental group: Incubate the frozen sections with Cy3 - labeled nucleic acid aptamer - three - dimensional DNA nanostructure (concentration: 250 nM).

[0079] Control group: Incubate the frozen sections with Cy3 - labeled single CAIX aptamer (concentration: 250 nM).

[0080] The incubation conditions were incubation at room temperature for 30 minutes, followed by washing the sections 3 times with PBS buffer for 5 minutes each time to remove unbound complexes or aptamers.

[0081] b. Fluorescence signal detection, imaging, and analysis of fluorescence intensity and duration

[0082] After the incubation and washing steps were completed, the Cy3 - labeled red fluorescence signals of the two groups of samples were observed using a fluorescence microscope. To further evaluate the signal intensity and duration, the fluorescence intensity of the two groups of samples was quantitatively analyzed using ImageJ software, and the attenuation of the fluorescence signal was recorded.

[0083] c. Experimental results

[0084] Comparison of fluorescence intensity

[0085] The experimental results showed that the initial fluorescence intensity of the nucleic acid aptamer - three - dimensional DNA nanostructure group was significantly stronger than that of the control group (aptamer used alone). The fluorescence intensity data showed that the fluorescence signal intensity of the complex was increased by about 3.276 times compared with that of the single aptamer, indicating that the nucleic acid aptamer - three - dimensional DNA nanostructure has a significant signal amplification effect.

[0086] Duration of fluorescence signal

[0087] The fluorescence signal duration of the complex was significantly prolonged, maintaining the fluorescence signal stability for a long time. In contrast, the fluorescence signal of the single aptamer group began to decay significantly after 30 minutes and there was almost no detectable signal at 60 minutes, while the fluorescence signal duration of the complex was extended by about 140%.

[0088] Example 3

[0089] Application of nucleic acid aptamer - three - dimensional DNA nanostructure in lymphoma cell recognition.

[0090] This example verified the targeting binding ability of the nucleic acid aptamer - three - dimensional DNA nanostructure in hematological malignancies (lymphoma) cells. Through confocal microscopy observation, it was further demonstrated the expandability and targeting recognition ability of the complex in different types of tumor cells.

[0091] Experimental design

[0092] The human acute lymphoblastic leukemia T - lymphocyte line (CCRF - CEM) used in the experiment was used as the experimental subject to evaluate the performance of the complex in the detection of hematological malignancy markers.

[0093] a. Cell culture and treatment

[0094] Cell culture

[0095] The CCRF - CEM cell line was cultured in a medium containing RPMI - 1640 medium, 10% fetal bovine serum and 1% penicillin - streptomycin solution, and incubated in an incubator at 37°C and 5% CO2.

[0096] Cell treatment

[0097] After the cultured cells reached the logarithmic growth phase, they were washed 3 times with PBS buffer to remove the residual medium and prepared for complex incubation.

[0098] b. Incubation of nucleic acid aptamer - three - dimensional DNA nanostructure

[0099] Incubation conditions

[0100] The Cy3 - labeled nucleic acid aptamer - three - dimensional DNA nanostructure (concentration 250 nM) was incubated with CCRF - CEM cells under the incubation conditions of 37°C for 30 minutes.

[0101] Washing

[0102] After the incubation, the cells were washed 3 times with PBS buffer for 5 minutes each time to remove the unbound complex and ensure the specificity of the detection signal.

[0103] c. Confocal microscopy detection and imaging

[0104] After the incubation and washing steps were completed, a confocal microscope was used to observe the red fluorescence signal on the cell membrane, with a focus on detecting the binding of the aptamer - three - dimensional DNA nanostructure to lymphoma cells. The distribution of the red fluorescence signal in the cell membrane region was recorded through microscope imaging.

[0105] Experimental results

[0106] The confocal microscope results showed that the aptamer - three - dimensional DNA nanostructure produced a strong red fluorescence signal on the CCRF - CEM cell membrane, and the signal was concentrated in the cell membrane region, indicating that the complex could accurately recognize the CAIX marker on the surface of lymphoma cells. The experimental results verified the targeting binding ability of this material in hematological malignancies.

[0107] Figure 2 For the distribution of CAIX in different parts of clear cell renal cell carcinoma of the kidney, Figure 2 A shows the staining of clear cell renal cell carcinoma tissue; Figure 2 B shows the staining at the junction of clear cell renal cell carcinoma tissue and normal renal tissue; Figure 2 C shows the staining of normal renal tissue, demonstrating the targeting recognition ability of this nanostructure on frozen sections. Especially, the fluorescence signal was significantly enhanced at the junction of tumor and normal tissues, showing its advantage in tumor margin recognition.

[0108] Figure 3 The figure shows the staining results of renal carcinoma tissues from three renal cancer patients, comparing the distribution of CAIX markers in clear cell renal cell carcinoma by different methods. Figure 3 The first three columns respectively correspond to the results of HE staining, immunohistochemical (IHC) staining, and the labeling of aptamer - three - dimensional DNA nanostructure + DAPI. The results showed that the aptamer - three - dimensional DNA nanostructure had good targeting recognition ability on paraffin sections, presenting fluorescence signals consistent with the immunohistochemical staining position in the tumor cell region, with clear signals and low background noise, further demonstrating the application potential of this nanostructure in tumor marker detection.

[0109] Figure 4 This is the comparison of the fluorescence intensities of the aptamer - three - dimensional DNA nanostructure and the conventional aptamer on frozen sections from the same tissue source at different times. Figure 4 In A, the fluorescence signal of the aptamer - three - dimensional DNA nanostructure decayed slowly, and there was still a detectable fluorescence signal even after 12 hours; Figure 4In B, it is the fluorescence duration of the aptamer under high HV conditions. Although the aptamer can generate a strong fluorescence signal at a higher voltage, its persistence is poor, and the fluorescence signal decays rapidly over time, especially after 6 hours when the signal weakens significantly. Figure 4 C shows the fluorescence duration of the aptamer - three - dimensional DNA nanostructure material under low HV conditions. Compared with the high HV conditions, the material still shows good fluorescence persistence under low HV conditions, indicating that the material can maintain a long - time fluorescence output under different voltage conditions and has a wider adaptability. Figure 4 D shows the decay of the fluorescence signal of the aptamer under low HV conditions. Similar to the high HV conditions, the fluorescence signal of the aptamer starts to weaken significantly after 1 hour, decreases substantially at 3 hours, and there is almost no remaining fluorescence signal after 4 hours.

[0110] Figure 5 It is the experimental result of the binding of the aptamer - three - dimensional DNA nanostructure to CEM cells. Under the fluorescence field of view, the strong red fluorescence signal indicates that the complex has successfully targeted and bound to the cell membrane region. The dark field provides the cell morphology background information, and the merged image further confirms the overlap of the fluorescence signal and the cell structure. This result proves that the complex has good targeting recognition ability and can be used for the detection and imaging analysis of tumor markers.

[0111] The conventional techniques in the above - mentioned embodiments are the existing techniques well - known to those skilled in the art, so they will not be elaborated in detail here.

[0112] As mentioned above, the above are only the specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention, and all should be covered by the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope of the claims.

[0113] The above - mentioned embodiments and / or implementation manners are only used to illustrate the preferred embodiments and / or implementation manners for realizing the technology of the present invention, and do not impose any form of limitation on the implementation manners of the technology of the present invention. Any person skilled in the art, without departing from the scope of the technical means disclosed by the content of the present invention, can make some changes or modifications to other equivalent embodiments, but should still be regarded as the same technology or embodiment as the present invention in essence.

[0114] In this article, specific examples are used to illustrate the principle and implementation manner of the present application. The description of the above embodiments is only used to help understand the method and its core idea of the present application. The above is only the preferred implementation manner of the present application. It should be noted that due to the limitation of literal expression, and objectively there are infinite specific structures. For those of ordinary skill in the art in this technical field, without departing from the principle of the present application, several improvements, retouches or changes can also be made, or the above technical features can be combined in an appropriate manner; these improvements, retouches, changes or combinations, or directly applying the concept and technical solution of the invention to other occasions without improvement, shall all be regarded as the protection scope of the present application.

Claims

1. A method for detecting renal cancer markers based on aptamer - three - dimensional DNA nanostructures, characterized in that, The method includes the following steps: (1) Synthesize a three-dimensional tetrahedral DNA nanostructure through hybridization chain reaction (HCR), bind the nucleic acid aptamer to the three-dimensional tetrahedral DNA nanostructure to form a nucleic acid aptamer-three-dimensional DNA nanostructure for targeted detection of renal clear cell carcinoma markers; (2) Amplify the fluorescence signal using the hybridization chain reaction (HCR) technique; (3) Apply the nucleic acid aptamer-three-dimensional DNA nanostructure to the detection of pathological sections to verify the targeted binding effect of the nucleic acid aptamer-three-dimensional DNA nanostructure in pathological sections; (4) Detect the fluorescence signal generated by the amplified strand H2 using a fluorescence signal detection technique.

2. The method for detecting a renal cancer marker according to claim 1, wherein In step (2), the fluorescence signal is amplified by providing multiple binding sites and using the hybridization chain reaction (HCR) technique.

3. The method for detecting a renal cancer biomarker according to claim 1, wherein The pathological section is a frozen section or a paraffin section.

4. The method for detecting a renal cancer marker according to claim 3, wherein Verifying the targeted binding effect of the nucleic acid aptamer-three-dimensional DNA nanostructure in frozen sections includes: Put the sample tissue into a cryoembedding agent, store it at -80 °C, section it in a cryostat, incubate the frozen section with the nucleic acid aptamer-three-dimensional DNA nanostructure, and use a fluorescence microscope to detect the Cy3-labeled red fluorescence signal for target detection and identification.

5. The method for detecting a renal cancer marker according to claim 3, wherein Verifying the targeted binding effect of the nucleic acid aptamer-three-dimensional DNA nanostructure in paraffin sections includes: Dehydrate and clear the fixed tissue, and then embed it in paraffin to form a paraffin section; Perform antigen retrieval on the paraffin section in a buffer solution; After antigen retrieval, incubate the section with the nucleic acid aptamer-three-dimensional DNA nanostructure, and use a fluorescence microscope to observe the Cy3-labeled red fluorescence signal for target detection.

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