Colorectal cancer Septin9 gene methylation detection kit based on graded anchoring probe

Abstract

The invention discloses a colorectal cancer Septin9 gene methylation detection kit based on a graded anchoring probe, and belongs to the technical field of molecular diagnosis. The graded anchoring probe is composed of a specific capture probe (F1), a precise anchoring probe (R1), a synergistic blocking probe (F2 / R2) and a short chain complementary sequence (S1) which are Septin9 methylation specific probes and can be fixed in a conical pore channel of a nuclear track membrane in a graded manner to form a capture-anchoring-blocking functional chain; during application, a general accumulation body is formed by combining with a Septin9 methylation target, a nuclear track membrane pore channel is blocked, current drop is caused, and detection is realized. Through cooperation of the probe, the nuclear track membrane and the electrochemistry, the problems of insufficient sensitivity, poor stability and poor specificity of traditional detection are solved, and the kit is suitable for ultra-early diagnosis of colorectal cancer.

Description

Technical Field

[0001] This invention belongs to the field of molecular diagnostic technology, specifically relating to a colorectal cancer Septin9 gene methylation detection kit based on graded anchoring probes, which is suitable for early screening of colorectal cancer. Background Technology

[0002] Colorectal cancer is one of the leading causes of cancer-related deaths worldwide. According to data from the International Agency for Research on Cancer (IARC) of the World Health Organization in 2023, there were over 2 million new cases of colorectal cancer and over 1 million deaths globally. Early diagnosis and intervention are crucial for improving patient prognosis and reducing mortality. In the field of molecular biology, gene methylation, as an important mechanism of epigenetic regulation, is catalyzed by DNA methyltransferases and can regulate gene expression without altering the DNA nucleotide sequence. Abnormal modifications are closely related to the occurrence and development of tumors. Septin9 is an important tumor suppressor gene associated with colorectal cancer. Under normal physiological conditions, its promoter region is hypomethylated and can be normally expressed to exert its tumor-suppressive function. However, during the development and progression of colorectal cancer, the promoter region of this gene becomes abnormally hypermethylated, leading to gene silencing and promoting the proliferation, invasion, and metastasis of tumor cells. Therefore, Septin9 gene methylation detection has become a highly promising biomarker detection direction for the early diagnosis of colorectal cancer. Currently, the main detection technologies based on Septin9 gene methylation are methylation-specific PCR and traditional electrochemical detection. Both have significant technical defects in practical applications and are difficult to meet the detection needs of early diagnosis of colorectal cancer. The specific problems are as follows: (1) Insufficient specific recognition ability: In electrochemical detection, the thermodynamic stability difference between methylated and unmethylated cytosine base pairs is slight. Traditional probes cannot accurately distinguish between methylated and unmethylated sequences of Septin9 gene and it is difficult to achieve specific capture of Septin9 methylation sites. The cross-reaction rate is high and false positive results are easy to occur. Although PCR technology can identify methylation sites through methylation-specific primers, it is easy to encounter problems such as primer dimers and non-specific amplification when facing complex detection systems, which will also lead to an increase in the false positive rate. (2) Poor detection performance stability and accuracy: The electron transfer efficiency of the probe and electrode interface of traditional electrochemical detection is low and the charge transfer resistance is high. The electrical signal generated by the trace target is easily drowned out by background noise. Moreover, the probe fixation method is easy to cause molecular conformation disorder and insufficient exposure of active sites, which further reduces the detection accuracy. The technology is easily affected by the inhibitors in the detection system, the amplification efficiency is unstable, and the reliability of the detection results is limited; (3) Insufficient detection efficiency and operability: Traditional electrochemical detection has no standardized probe pre-fixation design, the operation process is cumbersome, it is easy to introduce human error, and the detection repeatability is poor; PCR technology detection process is complicated, requiring multiple steps such as sample processing, thermal cycling amplification, and product analysis, which takes a long time and has strict technical requirements for equipment and operators, making it difficult to meet the actual needs of rapid detection.

[0003] In summary, existing Septin9 gene methylation detection technologies face insurmountable technical bottlenecks in terms of specificity, stability, and detection efficiency. Developing novel Septin9 methylation-specific detection probes and optimizing detection systems to achieve accurate and efficient detection of Septin9 gene methylation in colorectal cancer has become an urgent technical problem to be solved in this field. Summary of the Invention

[0004] The core of this invention is to design the probe as a four-level component according to its "functional division of labor". By pre-fixing it to the nuclear pore membrane to form a sensing core, it is then integrated with standardized hardware and reagents to form a detection kit, achieving "ready to use". This solves the problems of insufficient sensitivity, poor stability and poor specificity of traditional detection methods, and is suitable for the ultra-early diagnosis of colorectal cancer.

[0005] To achieve the above objectives, the technical solution of this invention is specifically designed as follows: Using a nuclear pore membrane as the substrate, the membrane is immersed in a mixed solution of chloroauric acid and sodium citrate, and incubated in a constant-temperature water bath to grow gold nanoparticles on the pore surface; then, the membrane is activated with an activator; then, the outlet end of the membrane is sealed with waterproof tape, exposing only the inlet end; the membrane is immersed in a probe solution containing only F1, and after incubation in a constant-temperature water bath, it is rinsed with PBS buffer to remove unfixed free F1; subsequently, the membrane is immersed in a blocking agent... The membrane was sealed at room temperature to block non-specific binding sites outside the capture region. The waterproof tape at the outlet end of the nuclear pore membrane was removed, the membrane was inverted, and the inlet end was sealed with waterproof tape. The nuclear pore membrane was immersed in a mixed solution containing R1 and S1, incubated in a constant temperature water bath, rinsed with PBS buffer, and then sealed with a blocking agent. The membrane was kept inverted, and the waterproof tape at the inlet end was removed. The membrane was immersed in a mixed solution containing F2 and R2, incubated in a constant temperature water bath, rinsed with PBS buffer, and finally sealed with a blocking agent to obtain a functionalized nuclear pore membrane sensor.

[0006] The nuclear pore membrane described in the technical solution is a tapered channel nuclear pore membrane with an inlet of 50-60 nm and an outlet of 100-150 nm.

[0007] The technical solution involves the synthesis of F1, R1, F2, R2, and S1, wherein F1, R1, F2, and R2 are modified with thiohexaalkyl groups at their 5' ends.

[0008] The connection between the probe and the channel in the described technical solution is a covalent connection of Au-S bonds;

[0009] The probe structure and function of the described technical solution are as follows: The four-level components of the hierarchical anchoring probe are a specific capture probe, a precise anchoring probe, a co-blocking probe, and a short complementary sequence. Detailed information for each component is as follows: Specific capture probe F1: Its 5' end is modified with a thiohexaalkyl-modified -S-(CH2)6, which can form an Au-S covalent bond with gold nanoparticles within the nuclear pore membrane channel, ensuring stable probe fixation. The core nucleotide sequence is shown in SEQ ID NO.1, with a sequence length of 18 bp. F1 is designed for the methylated CpG islands in the chr17:77373630-77373647 region of the Septin9 gene, binding only to the 5' end of the Septin9 methylation target to achieve initial target anchoring. Precise anchoring probe R1: Its 5' end is modified with a thiohexaalkyl-modified -S-(CH2)6, used to form an Au-S covalent bond with gold nanoparticles within the nuclear pore membrane channel. The core nucleotide sequence is shown in SEQ ID NO.2, with a sequence length of 19 bp. bp; R1 is designed for the methylated CpG islands in the chr17:77373710-77373728 region of the Septin9 gene, binding only to the 3' end of the Septin9 methylation target, forming a "pincer" binding mode with F1 to prevent target dissociation during detection; the co-blocking probe F2 has a thiohexaalkyl-modified -S-(CH2) at its 5' end. 6，The core nucleotide sequence of the probe, F2, is 22 bp in length and is designed to form Au-S covalent bonds with gold nanoparticles within the nuclear pore membrane channels. It is shown in SEQ ID NO.3. F2 targets the methylated CpG islands in the chr17:77373522-77373543 region of the Septin9 gene, binding to the middle segment of the Septin9 methylation target. The longer sequence enhances the binding strength, laying the foundation for the subsequent formation of large-volume polymers. The co-blocking probe R2, with a thiohexaalkyl-modified -S-(CH2)6 at its 5' end, is used to form Au-S covalent bonds with gold nanoparticles within the nuclear pore membrane channels. Its core nucleotide sequence is shown in SEQ ID NO.4. R2 is designed to target the methylated CpG island in the chr17:77373647-77373663 region of the Septin9 gene. It binds to another intermediate segment of the Septin9 methylation target and forms an "intermediate wrapping" binding mode with F2, ensuring that the final complex can completely block the nuclear pore membrane pores. The short complementary sequence S1 has the following nucleotide sequence as shown in SEQ ID NO.5, with a sequence length of 13 bp. S1 is divided into two functional regions: the 5' end 6 bp is complementary to the 5' end non-target binding region of F1, and the 3' end 6 bp is complementary to the 3' end non-target binding region of R1. The two functional regions are connected by a single T base. This design can flatten the binary complex formed by "F1-target-R1", expose the F2 and R2 binding sites in the intermediate segment of the target, reduce costs due to the unmodified design, and does not interfere with the specific binding of the target and probe.

[0010] The specificity guarantee mechanism of the probe in the above technical solution is as follows: After the Septin9 gene is transformed by sodium bisulfite, the unmethylated cytosine (C) will be transformed into uracil (U), while the methylated cytosine (mC) will remain unchanged. The probe base sequence of the present invention is designed for methylated CpG islands. Its guanine (G) can only form a stable GC base pair with the unchanged mC in the target. Moreover, since the complementary base of U is adenine (A), the G on the probe cannot form an effective pair with the U of the unmethylated target after transformation, thereby achieving specific differentiation between methylated and unmethylated targets.

[0011] The hierarchical synergistic mechanism of the technical solution is as follows: the binding process of the hierarchical anchoring probe and the Septin9 methylation target is divided into three steps, forming a complete "capture-anchoring-blocking" functional chain, and the functional region completely covers the pore: (1) Specific capture: F1 is pre-fixed in the functional region at the entrance of the nuclear pore membrane (pore size 50-60 nm). When the Septin9 methylation target diffuses to the entrance of the nuclear pore membrane with the solution, F1 preferentially binds to the 5' end of the target; the non-methylated target cannot bind due to the mismatch of the base sequence and is directly discharged from the nuclear pore membrane outlet, realizing the initial screening of the target; (2) Precise anchoring: the target bound to F1 continues to diffuse to the functional region in the middle section of the nuclear pore membrane (pore size 60-100 nm). (3) Cooperative blocking: The rigid complex diffuses to the functional region of the nuclear pore membrane (pore size 100-120 nm). The pre-fixed R1 in this region binds to the 3' end of the target to form a binary complex of “F1-target-R1”. At the same time, the short chain complementary sequence S1 binds to the non-target binding regions of F1 and R1 through base complementarity, pulling the originally possibly bent binary complex into a rigid straight structure, exposing the two methylation sites (F2 and R2 binding regions) in the middle section of the target; (4) Cooperative blocking: The rigid complex diffuses to the functional region of the back section of the nuclear pore membrane (pore size 100-120 nm). The pre-fixed F2 and R2 in this region bind to the two methylation sites in the middle section of the target to form a six-component polymer of “F1-target-R1-S1-F2-R2”. The diameter of the polymer is about 100-150 nm, which is precisely matched with the diameter of the pores in the back section of the nuclear pore membrane (120-150 nm), which can achieve complete blocking of the pores, thereby causing a significant decrease in current during the electrochemical detection process; (5) Exit sealing: The pore size of this region is 120-150 nm. nm, a sealing agent, blocks the back diffusion of non-specific impurities, ensuring that unbound impurities are completely removed and avoiding interference with detection results.

[0012] A colorectal cancer Septin9 gene methylation detection kit based on hierarchical anchoring probes, the core component of which is a functionalized nuclear pore membrane sensor, is prepared as follows: (1) The conical pore nuclear pore membrane with an inlet of 50-60 nm and an outlet of 100-150 nm is immersed in a mixed solution of 0.4-0.6 mM chloroauric acid + 0.10-0.12 M sodium citrate and incubated in a constant temperature water bath at 36-38℃ for 30-35 min to grow gold nanoparticles on the pore surface and form a conductive network; (2) The outlet end of the nuclear pore membrane is sealed with waterproof tape, exposing only the inlet end, and then the nuclear pore membrane is activated with an activation solution. After activation, the modification device and the nuclear pore membrane are cleaned with 1×PBS solution 2-3 times, each time for 3-4 min; (3) The nuclear pore membrane is immersed in a probe solution containing only F1 (concentration 0.5-0.6 μM) and incubated in a constant temperature water bath at 37-40℃ for 4-6 min. h; After incubation, rinse the nuclear pore membrane 2-3 times with 1×TE buffer to remove unfixed free F1; then immerse the nuclear pore membrane in blocking solution and block at 2℃ for 18-20 h to block non-specific binding sites outside the capture area. After blocking, wash with washing solution 3-4 times; (4) Remove the waterproof tape from the outlet end of the nuclear pore membrane, invert the nuclear pore membrane, and seal the inlet end with waterproof tape; immerse the nuclear pore membrane in a mixed solution containing R1 (0.4-0.5 μM) and S1 (0.4-0.5 μM) and incubate in a constant temperature water bath at 37-40℃ for 4-6 h; after incubation, rinse the nuclear pore membrane 2-3 times with 1×TE buffer to remove unfixed free R1 and S1; then immerse the nuclear pore membrane in blocking solution and block at 2℃ for 18-20 h. h, block non-specific binding sites outside the capture area, and wash with cleaning solution 3-4 times after sealing; (5) keep the nuclear pore membrane inverted and remove the waterproof tape at the inlet end; immerse the nuclear pore membrane in a mixed solution containing F2 (0.3-0.4 μM) and R2 (0.3-0.4 μM) and incubate in a constant temperature water bath at 37-40℃ for 4-6 h; after incubation, rinse the nuclear pore membrane with 1×TE buffer 2-3 times to remove unfixed free F2 and R2; then immerse the nuclear pore membrane in sealing solution and block at 2℃ for 18-20 h to block non-specific binding sites outside the capture area, and wash with cleaning solution 3-4 times after sealing; (6) take the modified nuclear pore membrane out of the device, spread it flat on a sterile culture dish, and put it in a forced-air drying oven at 40-45℃ for 1-2 h.

[0013] A colorectal cancer Septin9 gene methylation detection kit based on hierarchical anchored probes is designed with the core concept of "hardware standardization + probe pre-fixation + reagent matching" to ensure high compatibility between components. The specific compatibility design of each module is as follows: Sensing core module: The core component is a functionalized nuclear pore membrane sensor with an area of ​​1-2 cm². 2The sensor has a thickness of 10-15 μm and a conical pore structure (inlet 50-60 nm / outlet 100-150 nm). The sensor dimensions precisely match the sealing groove in the middle of the H-type electrolytic cell, ensuring no leakage after installation. Pre-fixed hierarchical anchoring probes are functionally distributed in the corresponding pore areas, allowing direct hierarchical binding with the target without manual probe fixing, simplifying the operation process and ensuring uniform probe distribution. The electrode module consists of two double-silver-silver chloride reference electrodes, both saturated KCl type, providing a stable potential reference. The electrodes are compatible with the electrode mounting holes of the H-type electrolytic cell, ensuring smooth insertion and sealing within the chamber. The electrode leads are compatible with standard interfaces on electrochemical workstations, enabling signal transmission without additional adapters. The reaction vessel module is an H-type electrolytic cell with a total volume of 6-10 mL, divided into two independent chambers (3-5 mL each), compatible with 2.5-4.5 mL reactors. The electrochemical detection buffer solution volume is specified in mL / chamber to avoid over- or under-concentration affecting detection. A sealing slot is located in the center of the electrolytic cell to secure the nuclear pore membrane sensor. The chamber has electrode mounting holes and a drain valve at the bottom for easy cleaning and waste removal after detection. The reagent module includes electrochemical detection buffer, quality control kit, standards, and auxiliary reagents. The electrochemical detection buffer has a pH of 7.0-7.4 and contains 0.05-0.06 M KCl; this formulation maintains probe activity while ensuring suitable conductivity of the electrolyte solution. The quality control kit has a concentration of 10... 5 -10 6 The dosage is per μL, matching the volume of the electrolytic cell chamber to avoid reagent waste and effectively verify the validity of the test kit; the standards are in gradient concentrations (10 μL / μL). 2 -10 6 (copy / μL), used to plot quantitative standard curves; auxiliary reagents are activation solution, blocking solution, and washing solution.

[0014] A colorectal cancer Septin9 gene methylation detection kit based on graded anchoring probes, the application steps of which are as follows when detecting colorectal cancer Septin9 gene methylation: (1) I0 detection: ① Take the graded anchoring probe functionalized nuclear pore membrane sensor from the detection kit, remove the sealed packaging, fix it in the middle sealed slot of the H-type electrolytic cell with tweezers, install the silicone sealing ring, tighten the slot cover to ensure no leakage in the chamber, rinse the two Ag / AgCl electrodes with deionized water, soak them in the electrode cleaning solution, rinse them with deionized water again, and use them as the anode and cathode of the electrolytic cell. The anode is connected to the working electrode clamp of the electrochemical workstation, and the cathode is connected to the counter electrode and the reference electrode clamp to assemble the electrolytic cell. Add the electrochemical detection buffer and prepare for testing; ② Use the cyclic voltammetry method, set the upper limit voltage to 1 V, the lower limit voltage to -1 V, the starting voltage to 0 V, the scan rate to 100-150 mV / s, and the number of cycles to 5-6 times, and start the test; ③ Record 1 V and -1 V. The current value under V is taken as I0; (2) I1 detection: ① Take 50-100 μL of DNA standard, add 50-100 μL of 10×Bst buffer and 800-900 μL of 1×PBS solution, and shake to mix evenly in the sample tube; place the sample tube in a 90-95℃ water bath and heat for 10-15 min to promote DNA double strand unwinding; after heating, sonicate the solution for 25-30 s, and then place it in a 50-55℃ water bath and keep warm for 25-30 min; ② Add 4.0-4.5 mL of electrochemical detection buffer to the left chamber of the H-type electrolytic cell, add 4.0-4.475 mL of electrochemical detection buffer to the right chamber, and then add 25-50 μL of the standard solution treated above; apply a pre-polarization voltage of 0.1-0.2 V to activate for 3-5 min, and incubate at 37-40℃ for 30-40 min; ③ Use cyclic voltammetry, set the upper limit voltage to 1 V and the lower limit voltage to -1 V. V, the initial voltage is 0 V, the scan rate is 100-150 mV / s, the number of cycles is 5-6, and the test begins; ④ Record the current values ​​at 1 V and -1 V as I1, and calculate ΔI; ⑤ Measure ΔI at different concentrations of standard products, and plot the concentration-ΔI quantization curve based on the concentration.

[0015] This invention addresses the core pain points of existing Septin9 methylation detection technologies, including high false positive rates, insufficient sensitivity, cumbersome operation, and poor stability. Through innovative hierarchical anchoring probe systems, functionalized nuclear pore membrane sensing design, and standardized adaptation across the entire system, it achieves accurate, efficient, and convenient detection of Septin9 gene methylation in colorectal cancer. The beneficial technical effects are as follows: 1. Ultra-high detection specificity: The probes are specifically designed for the CpG islands of Septin9 gene methylation, accurately distinguishing methylated and unmethylated sequences at the base level. Through a hierarchical "capture-anchor-blocking" synergistic system, only fully matched methylated targets can specifically bind, effectively eliminating interfering sequences. The kit cross-reactivity rate is <5%, completely solving the problem of high false positive rates caused by non-specific binding and amplification in traditional technologies. 2. Ultra-high detection sensitivity: Relying on the signal cascade amplification effect of complete blockage of nuclear pore membrane channels, single-molecule target binding events are converted into accurately detectable current changes, solving the industry problem of trace target electrical signals being easily drowned out by background noise. The kit's detection limit is as low as 10. 2 With a detection capacity of 1 copy / μL, it can accurately capture ultra-low abundance methylation targets in peripheral blood; the linear detection range covers 10 2 -10 6 copy / μL, linear fitting correlation coefficient R 2 ≥0.9992, perfectly suited for ultra-early diagnosis of colorectal cancer; 3. Excellent stability and repeatability: All functional probes are irreversibly immobilized through Au-S covalent bonds, avoiding the defects of traditional probes such as easy detachment and disordered molecular conformation; combined with the full-channel partitioned sealing design, the background noise is greatly reduced, the anti-interference ability is strong, the relative standard deviation (RSD) of 5 parallel detections of the sensor is <5%, and the performance is highly stable between batches; 4. Simple and efficient operation: The core sensing component is a pre-functionalized nuclear pore membrane sensor, which can be used immediately after installation, without the need for users to manually perform tedious operations such as probe immobilization and electrode modification; at the same time, it eliminates the complex steps of thermal cycling amplification and product analysis in traditional PCR technology, and the entire detection process can be completed within 2 hours; 5. Standardized adaptation of the whole system: Adopting an integrated design of "hardware standardization + probe pre-immobilization + reagent matching", the core sensing component, electrodes, reaction container and matching reagents are precisely adapted in all dimensions. Attached Figure Description

[0016] Figure 1 This is a schematic diagram of the grouped structure of the probe system;

[0017] Figure 2 A schematic diagram of the fabrication process for a functionalized nucleopore membrane sensor;

[0018] Figure 3 This is a schematic diagram illustrating the mechanism of the "capture-anchor-block" functional chain.

[0019] Figure 4 The current response curve during the preparation process is shown.

[0020] Figure 5 This is a sensitivity quantization curve;

[0021] Figure 6 This is a graph showing the results of specific detection.

[0022] Figure 7 The graph shows the results of stability and repeatability tests. Detailed Implementation

[0023] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, the scope of protection of the present invention is not limited thereto. Any changes made to the technical solutions of the present invention by those skilled in the art should fall within the scope of protection of the present invention.

[0024] The nucleotide sequence listing involved in the following embodiments is shown in Table 1.

[0025] Table 1

[0026] Example 1: Synthesis of hierarchical anchoring probes and fabrication of functionalized nuclear pore membrane sensors

[0027] 1. Reagent preparation

[0028] (1) Activation solution (solution A): Weigh 0.39 g MES (2-morpholinoethanesulfonic acid), add 100 mL deionized water, and stir until completely dissolved; add 5 mL of 10 mg / mL EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) solution, adjust the pH to 5.5 with 0.1 M HCl and NaOH, mix thoroughly and pour into the modification device, and store at room temperature; (2) Probe binding solution (solution B series, divided into 3 groups according to the fixed region): Capture region probe solution (B1): Take 100 mL of 1×PBS solution, add 100 μL of 5 μM F1 probe solution (final concentration 0.5 μM), adjust the pH to 7.4, mix thoroughly and store at 2℃; Anchoring region probe solution (B2): Take 100 mL of 1×PBS solution, add 80 μL of 5 μM R1 probe solution + 80 μL of 5 μM S1 sequence solution (final concentration 0.4 μM for both). (3) Blocking solution (solution C): Weigh 3.75 g glycine, add 100 mL 1×TE solution (10 mM Tris-HCl + 1 mM EDTA), adjust pH to 7.4, mix well, and store at 2℃ for later use; (4) Cleaning solution (solution D): Weigh 1.74 g NaCl, add 100 μL Tween-20, and make up to 200 mL with 1×TE solution, stir until completely dissolved, adjust pH to 7.4, mix well, and store at 2℃ for later use.

[0029] 2. Probe Synthesis

[0030] (1) Probe synthesis: F1, R1, F2, R2 and S1 were synthesized by Sangon Biotech (Shanghai) Co., Ltd., of which F1, R1, F2 and R2 were modified with thiohexaalkyl (-S-(CH2)6) at the 5' end. The synthesis process adopted solid-phase phosphoramide method to ensure the accuracy of probe sequence and the stability of modified group; (2) Probe solution preparation: The probe was dissolved in deionized water and the concentration was calculated according to the molar ratio of F1:R1:F2:R2:S1=1.2:1:1:1:1. The solutions B1, B2 and B3 were prepared respectively to ensure that each probe remains active in the binding solution.

[0031] 3. Fabrication of Functionalized Nuclear Pore Membrane Sensors

[0032] (1) Nuclear pore membrane substrate treatment: Select a nuclear pore membrane (diameter 13 mm, thickness 10 μm) with a conical structure of pores with an inlet of 50 nm and an outlet of 150 nm; immerse the nuclear pore membrane in a mixed solution of 0.6 mM chloroauric acid + 0.12 M sodium citrate, incubate in a constant temperature water bath at 38℃ for 35 min, and grow 3 nm gold nanoparticles on the inner wall of the pores to form a conductive network; after removal, rinse with deionized water 3 times and dry with nitrogen for later use; (2) Pre-fixation of hierarchical probes: ① Fixation of the capture area (F1): Seal the outlet end of the nuclear pore membrane with waterproof tape, exposing only the inlet end, and fix it in the modification device to ensure that the nuclear pore membrane is completely immersed in solution A (activation solution) and there are no bubbles on the surface; place the modification device on a transfer decolorization shaker and activate it at a oscillation frequency of 2 S / cycle for 1 h at room temperature; after activation, pour out solution A and wash the modification device and nuclear pore membrane twice with 1×PBS solution, 3 times each time. min; Pour in solution B1 (capture zone probe solution), ensuring the nuclear pore membrane is completely submerged and free of bubbles; Return the modification device to the shaker and continue modification at 37℃ for 4 h with an oscillation frequency of 2 S / cycle; After modification, pour out solution B1 and wash twice with 1×TE solution; Pour in solution C (blocking solution), shake on the shaker at 2 S / cycle for 30 min, then transfer the modification device to a 2℃ refrigerator and seal overnight for 18 h; After sealing, pour out solution C, take 100 mL of solution D (washing solution) and pour it into the modification device, shake on the shaker at 2 S / cycle for 1 min, repeat the washing 3 times, and check that the pH of the solution after washing is 7.4, which is considered as the washing is complete; ② Anchoring zone fixation (R1+S1): Remove the waterproof tape at the outlet end of the nuclear pore membrane, invert the nuclear pore membrane, seal the inlet end with waterproof tape, and re-fix it in the modification device; Pour in solution A (activation solution), ensuring the nuclear pore membrane is completely submerged and free of bubbles, shake on the shaker at a oscillation frequency of 2 S / cycle, and activate at room temperature for 1 1 h; Pour out solution A, wash twice with 1×PBS solution; Pour in solution B2 (anchoring probe solution), and modify with shaking at 37℃ for 2 S / cycle for 4 h; After modification, pour out solution B2, wash twice with 1×TE solution; Pour in solution C (blocking solution), shake for 30 min, and then block overnight at 2℃ for 18 h; After blocking, wash three times with solution D, and confirm pH=7.4 before use; ③ Blocking region fixation (F2+R2): Keep the nuclear pore membrane inverted, remove the waterproof tape at the inlet end, and fix it in the modification device; Pour in solution A (activation solution), and activate with shaking at room temperature for 1 h; Pour out solution A, wash twice with 1×PBS solution; Pour in solution B3 (blocking probe solution), and modify with shaking at 37℃ for 2 S / cycle for 4 h; After modification, pour out solution B3, wash twice with 1×TE solution; Pour in solution C (blocking solution), shake for 30 min, and then block overnight at 2℃ for 18 h. h; After sealing, wash three times with solution D to confirm pH=7.4; (3) Drying: Remove the modified nuclear pore membrane from the device, spread it evenly on a sterile petri dish, and place it in a forced-air drying oven at 40℃ for 1 h.

[0033] Example 2: Assembly and Quantitative Performance Verification of the Detection System

[0034] 1. Preparation of reagents and equipment

[0035] Detection system components: H-type electrolytic cell, double silver-silver chloride reference electrode, functionalized nuclear pore membrane sensor (prepared in Example 1), electrochemical detection buffer (1×PBS, pH 7.4, containing 0.06 M KCl); Standard series: Septin9 methylation positive standard (gradient concentration: 10... 2 copy / μL, 10 3 copy / μL, 10 4 copy / μL, 10 5 copy / μL, 10 6 copy / μL); Septin9 methylation positive control (10 5 copy / μL), Septin9 unmethylated anion control (10 5 (copy / μL); Auxiliary equipment: electrochemical workstation, 37℃ incubator, pipette, lint-free cloth, anhydrous ethanol, deionized water, 0.1 M nitric acid solution; Electrolyte: 1×PBS solution.

[0036] 2. System Assembly

[0037] (1) Preparation of electrolytic cell and nuclear pore membrane: Take an H-type electrolytic cell, wipe the inner wall of the chamber with a lint-free cloth dipped in anhydrous ethanol to remove impurities; cut the functionalized nuclear pore membrane sensor into a circle with a diameter of 13 mm, take it out of the sealed package, and carefully fix it in the middle sealing slot of the electrolytic cell with tweezers, install a silicone sealing ring, tighten the slot cover plate, and verify by adding a small amount of deionized water to ensure that there is no leakage in the left and right chambers; (2) Electrode treatment and installation: Take a double silver-silver chloride reference electrode, rinse the electrode tip with deionized water for 1 min; immerse it in 0.1 M nitric acid solution for 1 min to remove the surface oxide layer; rinse it with deionized water 3 times, 1 min each time, and blow it dry with nitrogen for later use; insert the treated electrode into the electrode mounting holes of the left and right chambers of the electrolytic cell respectively, adjust the electrode height so that the tip can be immersed in the buffer solution, and the electrode is parallel to the nuclear pore membrane sensor; connect the electrode wires to the working electrode clamp, counter electrode and reference electrode clamp of the electrochemical workstation respectively; (3) Baseline test and buffer addition: add 4.5 to each of the left and right chambers. Add 1 mL of 1×PBS electrolyte, start the electrochemical workstation, and perform baseline testing using cyclic voltammetry (CV). Set the parameters as follows: upper limit voltage 1 V, lower limit voltage -1 V, starting voltage 0 V, scan rate 100 mV / s, and 5 cycles. Record the current values ​​at 1 V and -1 V as baseline currents to ensure the stability of the conductivity of the electrode and nuclear pore membrane. Drain the electrolyte and add 4.5 mL of electrochemical detection buffer to the left chamber. Add 4.5 mL of the same detection buffer to the right chamber.

[0038] 3. Quantitative performance testing

[0039] (1) Sensor activation: Start the electrochemical workstation, select the "potential constant" mode, apply a pre-polarization voltage of 0.2V based on the left chamber reference electrode, and activate at room temperature for 3 min; after activation, switch to cyclic voltammetry (CV) mode, set the potential range to -1~1 V, the scan rate to 100 mV / s, the number of cycles to 5, start the test, and record the initial current value (I0); (2) Target binding and current detection: Take 100 μL of DNA standard, add 100 μL of 10×Bst buffer and 800 μL of 1×PBS solution, and shake to mix evenly in the sample tube; place the sample tube in a 90℃ water bath and heat for 10 min to promote DNA double-strand unwinding; after heating, sonicate the solution for 30 s, and then place it in a 55℃ water bath and keep warm for 30 min; add 4.5 mL of electrochemical detection buffer to the left chamber of the H-type electrolytic cell, add 4.475 mL of electrochemical detection buffer to the right chamber, and then add 25 μL of the standard solution treated above; apply 0.2V. V pre-polarization voltage activation for 3 min, incubation at 37℃ for 30 min, then detection by cyclic voltammetry (CV), record the current value (I1) after incubation; calculate the current change ΔI=I0-I1; (3) Quantization curve plotting: according to the above steps, test 10 in sequence. 2 copy / μL, 10 3 copy / μL, 10 4 copy / μL, 10 5 copy / μL, 10 6 Five positive standards at concentrations of 5 μL were tested, with each concentration tested three times, and the average ΔI was taken. A "concentration-ΔI quantification curve" was plotted with the standard concentration as the x-axis and the corresponding average ΔI as the y-axis. Linear fitting was performed to obtain the regression equation: y=a×lgx+b (where y is ΔI, x is the standard concentration, and a and b are fitting coefficients), with a correlation coefficient R²≥0.9992.

[0040] Example 3: Current response results during the preparation process are as follows Figure 4As shown, a current response of 415 μA was observed in the blank membrane in the electrochemical detection buffer; a current response of 585 µA was observed after modification with gold nanoparticles; a current response of 570 µA was observed after activation with EDC; a current response of 560 µA was observed after modification with F1; a current response of 545 µA was observed after modification with R1 and S1; a current response of 525 µA was observed after modification with blocking agent; a current response of 506 µA was observed after incubation with the target agent; and a current response of 350 µA was observed after incubation with the target agent, which was significantly lower than the current before incubation. This indicates that the target agent specifically recognizes the probe, causing a change in current.

[0041] Example 4: To evaluate the sensitivity of this technical solution in detecting the target analyte, the current of the target analyte at different concentrations was measured under optimal experimental conditions. The results are as follows: Figure 5 The current decreases as the concentration of the target increases, and there is a significant functional relationship between the change in current and the concentration of the target. The detection limit can reach 10² copy / μL, which meets the requirements for low abundance target detection.

[0042] Example 5: To investigate the specificity of this electrochemical biosensor, a specificity experiment was conducted using four mismatched sequences (interferants 1-4) and the specific target compound as controls; the results are as follows. Figure 6 The results showed that, under the same reaction conditions, the current difference of the target analyte was much greater than that of the non-target analyte, while the negative control showed no significant current change, demonstrating that the kit has excellent specificity and a cross-reactivity rate of <5%.

[0043] Example 6: To investigate the stability of this electrochemical biosensor, five parallel current responses were performed in the electrochemical detection buffer. The results are as follows: Figure 7 The current response of this electrochemical biosensor exhibits small fluctuations, with a relative standard deviation (RSD) of <5%, demonstrating excellent stability.

[0044] It should be noted that the above examples are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the given examples, those skilled in the art can make modifications or equivalent substitutions to the technical solutions of the present invention as needed, without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. A kit for detecting Septin9 gene methylation in colorectal cancer based on hierarchical anchoring probes, characterized in that, The hierarchical anchoring probes include a specific capture probe F1, a precise anchoring probe R1, a co-blocking probe F2, a co-blocking probe R2, and a short complementary sequence S1. The nucleotide sequences of each probe / sequence are as follows: (1) Specific capture probe F1: Its 5' end is modified with thiohexaalkyl-S-(CH2)6, and the core nucleotide sequence is shown in SEQ ID NO.1, with a sequence length of 18 bp; F1 is designed for the methylated CpG islands in the chr17:77373630-77373647 region of the Septin9 gene and binds only to the 5' end of the Septin9 methylation target; (2) Precise anchoring probe R1: Its 5' end is modified with thiohexaalkyl-S-(CH2)6, and the core nucleotide sequence is shown in SEQ ID NO.2, with a sequence length of 19 bp; R1 is designed for the methylated CpG islands in the chr17:77373710-77373728 region of the Septin9 gene and binds only to the 5' end of the Septin9 gene methylation target. (3) Co-blocking probe F2: Its 5' end is modified with thiohexaalkyl-S-(CH2)6, and the core nucleotide sequence is shown in SEQ ID NO.3, with a sequence length of 22 bp; F2 is designed for the methylation CpG island in the chr17:77373522-77373543 region of Septin9 gene, and binds to the middle segment of Septin9 methylation target; (4) Co-blocking probe R2: Its 5' end is modified with thiohexaalkyl-S-(CH2)6, and the core nucleotide sequence is shown in SEQ ID NO.4, with a sequence length of 17 bp; R2 is designed for the methylation CpG island in the chr17:77373647-77373663 region of Septin9 gene, and binds to another middle segment of Septin9 methylation target; (5) Short complementary sequence S1: Its nucleotide sequence is shown in SEQ ID NO.5, with a sequence length of 13 S1 is divided into two functional regions: the 5' end 6bp is complementary to the 5' end non-target binding region of F1, and the 3' end 6bp is complementary to the 3' end non-target binding region of R1. The two functional regions are connected by one T base.

2. The colorectal cancer Septin9 gene methylation detection kit based on hierarchical anchoring probes according to claim 1, characterized in that, The 5'-terminal thiohexaalkyl modification of F1, R1, F2, and R2 is used to form Au-S covalent bonds with gold nanoparticles on the inner wall of the nuclear pore membrane channel, thereby achieving hierarchical fixation of the probe within the channel.

3. The colorectal cancer Septin9 gene methylation detection kit based on hierarchical anchoring probes according to claim 1, characterized in that, The kit is an integrated system of hardware components and reagent components, including: (1) core sensing hardware: ① functionalized nuclear pore membrane sensor: nuclear pore membrane, conical channel, gold nanoparticle conductive network on the inner wall of the channel, hierarchical anchoring probes are pre-fixed to the corresponding functional area of ​​the channel and sealed for preservation; ② double silver-silver chloride reference electrode: saturated KCl type, adapted to electrochemical workstation interface; ③ H-type electrolytic cell: divided into left and right independent chambers, with a sealing slot in the middle, and electrode mounting holes in the chambers; (2) matching reagent components: ① electrochemical detection buffer: PBS, KCl, refrigerated; ② quality control kit: Septin9 methylated positive quality control and Septin9 non-methylated positive quality control, both frozen for preservation; ③ standard: Septin9 methylated positive standard; ④ auxiliary reagents: activation solution, blocking solution, cleaning solution.

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