Methylated DNA Sequence Detection System Based on Covalent Organic Framework Materials, Detection Method Thereof and Application

Abstract

The present invention discloses a methylation DNA sequence detection system based on covalent organic framework materials, its detection method and application. The detection system includes a covalent organic framework nanoprobe, a DNA sample processing unit and a detection unit. The detection process does not rely on composite materials and the preparation process is simple. The present invention uses an organic small molecule containing a triphenylamine structure as a monomer raw material, and prepares a covalent organic framework nanoprobe through a Schiff base reaction. The DNA is subjected to bisulfite treatment, asymmetric PCR and fluorescence labeling by the DNA sample processing unit to form a DNA to be detected. Then, the detection unit measures the fluorescence of the combination of the DNA sequence to be detected and the covalent organic framework nanoprobe, so as to realize the methylation detection of the DNA sequence. This system can efficiently and conveniently detect the methylation DNA sequence, and the operation process is simple and easy to implement.

Description

Technical Field

[0001] The present invention relates to the technical field of nanomaterials and nucleic acid detection, and particularly relates to a methylation DNA sequence detection system based on covalent organic framework materials, a detection method thereof, and an application thereof. Background Art

[0002] Covalent Organic Frameworks (COFs for short) are a class of porous crystalline polymers formed by covalently connecting organic molecules. Due to their highly ordered pore structures, adjustable pore sizes, and surface chemical properties, they have been widely studied in the field of biosensing. Especially in nucleic acid detection, the large specific surface area and abundant active sites of COFs can interact with nucleic acids through non-covalent or covalent forces, showing great application potential in the field of DNA sequence recognition and detection.

[0003] DNA methylation detection is a technique for evaluating the DNA expression status and abnormalities by analyzing the methylation patterns of DNA, which can discover the methylation abnormal sites and expression profiles of tumor-related DNA, thereby enabling early diagnosis, prevention, and treatment of diseases, and is of great significance in the fields of biology, medicine, and life sciences. However, there have been no relevant reports on directly detecting methylation DNA sequences using covalent organic framework materials in the existing technologies.

[0004] Therefore, developing a DNA methylation detection scheme based on covalent organic framework materials has important research and application significance. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a methylation DNA sequence detection system based on covalent organic framework materials, a detection method thereof, and an application thereof in view of the deficiencies in the above-mentioned existing technologies.

[0006] To solve the above technical problems, the technical solution adopted by the present invention is: In the first aspect of the present invention, a methylation DNA sequence detection system based on covalent organic framework materials is provided, including:

[0007] A covalent organic framework nanoprobe, which is prepared by a Schiff base reaction using an organic small molecule containing a triphenylamine structure as a monomer raw material, and the covalent organic framework nanoprobe has different adsorption abilities for different bases;

[0008] A DNA sample processing unit, which obtains a DNA sequence to be detected by performing bisulfite treatment, asymmetric PCR amplification, and fluorescence labeling of PCR products on the DNA sample; after the DNA sequence to be detected is adsorbed by the covalent organic framework nanoprobe, the fluorescence of the fluorescent substance labeled on the DNA sequence to be detected can be quenched by the covalent organic framework nanoprobe;

[0009] and a detection unit that measures the fluorescence intensity of the product after the DNA sequence to be detected binds to the covalent organic framework nanoprobe; the fluorescence intensity of the product is positively correlated with the proportion of methylated DNA in the sample to be detected.

[0010] Preferably, the covalent organic framework nanoprobe is a single-layer or multi-layer nanosheet.

[0011] Preferably, the covalent organic framework nanoprobe is a single crystal or polycrystal.

[0012] Preferably, the monomer raw materials include tris(4-aminophenyl)amine and tris(4-benzoyl)amine.

[0013] Preferably, the covalent organic framework nanoprobe is prepared by the following method:

[0014] 1) Using tris(4-aminophenyl)amine and tris(4-benzoyl)amine as monomer raw materials, adding them to a solvent, and subjecting to ultrasonic treatment until clear;

[0015] 2) Adding an acetic acid solution to the solution in step 1), mixing evenly and then degassing;

[0016] 3) Sealing after degassing, heating for reaction, after the reaction ends, collecting the product, washing, and drying;

[0017] 4) Taking the powder product obtained in step 3) and adding it to absolute ethanol for ultrasonic exfoliation to obtain an ethanol dispersion of the covalent organic framework nanoprobe;

[0018] 5) Allowing the ethanol dispersion of the covalent organic framework nanoprobe in step 4) to stand and precipitate at room temperature, centrifuging the upper suspension, and redispersing the nanosheets in deionized water to obtain a dispersion of the covalent organic framework nanoprobe.

[0019] Preferably, in step 3), the temperature of the heating reaction is 80 - 120 °C, and the reaction time is 24 - 96 hours. More preferably, the temperature of the heating reaction is 120 °C, and the reaction time is 72 hours.

[0020] Preferably, in the dispersion of the covalent organic framework nanoprobe, the concentration of the covalent organic framework nanoprobe is 30 - 40 mg / mL. More preferably, the concentration is 33 mg / mL.

[0021] Preferably, the covalent organic framework nanoprobe is prepared by the following method:

[0022] 1) Take a mixture of 10 - 40 mg of tris(4-aminophenyl)amine and 10 - 40 mg of tris(4-benzoyl)amine as raw material monomers, add it to 3 - 12 mL of a solvent composed of o-dichlorobenzene and ethanol in a volume ratio of 4:1, and ultrasonically treat for 2 - 10 minutes to obtain a clear solution;

[0023] 2) Add 120 - 480 mL of acetic acid solution with a concentration of 1.5 - 6 mol / L to the product of step 1), mix evenly, freeze in liquid nitrogen at 77 K, and degas through 2 - 6 freeze-thaw cycles;

[0024] 3) After degassing, seal and react at 80 - 120 °C for 24 - 96 hours. After the reaction is completed, collect the product, wash it with tetrahydrofuran, and dry it;

[0025] 4) Take 0.5 - 2 mg of the powder product obtained in step 3) and add it to 15 - 60 mL of absolute ethanol, and ultrasonically exfoliate it at 50 - 220 W for 3 - 12 h to obtain an ethanol dispersion of the covalent organic framework nanoprobe;

[0026] 5) Let the ethanol dispersion of the covalent organic framework nanoprobe in step 4) stand and precipitate at room temperature for 12 h, discard the bottom precipitate, centrifuge the upper suspension at 7000 - 15000 rpm for 3 - 15 min, discard the upper ethanol, and redisperse the obtained covalent organic framework nanoprobe in the same volume of deionized water to obtain the dispersion of the covalent organic framework nanoprobe.

[0027] Preferably, in the DNA sample processing unit, the method for fluorescently labeling the PCR product is: hybridizing with a complementary sequence labeled with a fluorescent substance or using terminal deoxynucleotidyl transferase for terminal labeling.

[0028] Preferably, in the DNA sample processing unit, the fluorescent substance used in the fluorescent labeling process is a quantum dot or at least one of the following fluorescent molecules or their derivatives: Texas Red fluorescent dye, cyanine series fluorescent dyes, rhodamine fluorescent dyes, Alexa series fluorescent dyes, Atto series fluorescent dyes.

[0029] In the second aspect of the present invention, there is provided an application of the methylation DNA sequence detection system based on covalent organic framework materials as described above in detecting methylation DNA sequences, and the application is for non-disease diagnosis and treatment purposes.

[0030] In the third aspect of the present invention, there is provided a method for detecting methylation DNA sequences using the methylation DNA sequence detection system based on covalent organic framework materials as described above, and the method includes the following steps:

[0031] S1. First, use bisulfite to transform the sample DNA by the DNA sample processing unit, then perform asymmetric PCR amplification, and then use a fluorescent substance to fluorescently label the PCR product to obtain the DNA sequence to be detected;

[0032] S2. Mix the dispersion of the covalent organic framework nanoprobe with the DNA sequence to be detected and incubate it. Use a fluorescence spectrometer to detect the fluorescence emission spectrum of the incubated product, obtain the fluorescence intensity at the emission peak position of the fluorescent substance labeled in the DNA sequence to be detected, and judge the proportion of methylated DNA in the sample to be detected according to the value of this fluorescence intensity; wherein, the value of this fluorescence intensity is positively correlated with the proportion of methylated DNA in the sample to be detected.

[0033] The beneficial effects of the present invention are as follows:

[0034] 1. The present invention provides a detection system for methylated DNA sequences based on covalent organic framework materials, its detection method and application. In the detection system of the present invention, there is no need to rely on composite materials. Only an organic small molecule containing a triphenylamine structure is used as a monomer raw material, and a covalent organic framework nanoprobe can be prepared as the main detection material through a Schiff base reaction, simplifying the preparation process;

[0035] 2. By combining the covalent organic framework nanoprobe, the DNA sample processing unit and the detection unit, the present invention can realize the detection of methylated DNA sequences, and the process is simple and easy to implement;

[0036] 3. The detection system of the present invention can perform qualitative and quantitative analysis on methylated DNA sequences by measuring fluorescence, and has the advantages of high efficiency and convenience. Description of the Drawings

[0037] Figure 1 is the chemical structural formula of TBA-COF prepared in Example 1;

[0038] Figure 2 is the SEM image of the TBA-COF nanosheets prepared in Example 1;

[0039] Figure 3 is the TEM image of the TBA-COF nanosheets prepared in Example 1;

[0040] Figure 4 is the capillary electrophoresis map of the experimental group DNA and the control group DNA in Example 1 after bisulfite treatment and asymmetric PCR;

[0041] Figure 5 is the fluorescence emission spectrum of the DNA of the experimental group to be detected and the DNA of the control group to be detected in Example 1 after incubation with TBA-COF nanosheets for 20 min, and the excitation wavelength is 590 nm;

[0042] Figure 6 It is a linear relationship diagram of the fluorescence intensity at 612 nm and the proportion of methylated DNA in Example 2;

[0043] Figure 7 It is the fluorescence emission spectra of the DNA of the experimental group to be detected and the DNA of the control group to be detected in Example 3 after incubation with TBA-COF nanosheets for 20 min, and the excitation wavelength is 590 nm;

[0044] Figure 8 It is the fluorescence emission spectra of the Cy3-labeled DNA of the experimental group to be detected and the DNA of the control group to be detected in Example 6 after incubation with TBA-COF nanosheets for 20 min, and the excitation wavelength is 550 nm. Detailed implementation manners

[0045] The present invention will be further described in detail below in conjunction with examples, so that those skilled in the art can implement it according to the description in the specification.

[0046] It should be understood that the terms such as "having", "comprising" and "including" used herein do not exclude the presence or addition of one or more other elements or their combinations.

[0047] Unless otherwise specified, the test methods used in the following examples are all conventional methods. The materials and reagents used in the following examples, unless otherwise specified, can all be obtained from commercial channels. For those conditions not specified in the following examples, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For those reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase. Example 1

[0048] A methylation DNA sequence detection system based on covalent organic framework materials, comprising:

[0049] A covalent organic framework nanoprobe, which is prepared by a Schiff base reaction using an organic small molecule containing a triphenylamine structure as a monomer raw material, and the covalent organic framework nanoprobe has different adsorption abilities for different bases;

[0050] A DNA sample processing unit, which obtains the DNA sequence to be detected by performing bisulfite treatment, asymmetric PCR amplification and fluorescence labeling of PCR products on the sample DNA; after the DNA sequence to be detected is adsorbed by the covalent organic framework nanoprobe, the fluorescence of the fluorescent substance labeled on the DNA sequence to be detected can be quenched by the covalent organic framework nanoprobe;

[0051] And a detection unit that measures the fluorescence intensity of the product after the DNA sequence to be detected binds to the covalent organic framework nanoprobe; the fluorescence intensity of the product is positively correlated with the proportion of methylated DNA in the sample to be detected.

[0052] Among them, the preparation method of the covalent organic framework nanoprobe is as follows:

[0053] Add 20 mg of tris(4-aminophenyl)amine, 20 mg of tris(4-benzoyl)amine, and 6 mL of a mixed solvent of o-dichlorobenzene and ethanol (volume ratio 4:1) into a reaction tube in sequence; after ultrasonic treatment for 5 minutes, a clear solution is obtained, and then 240 mL of acetic acid solution with a concentration of 3 mol / L is added; after mixing evenly, the reaction tube is frozen in liquid nitrogen at 77 K and degassed through three freeze-thaw cycles; after degassing, it is sealed and heated at 120°C for 72 hours. After the reaction is completed, the product is collected, washed with tetrahydrofuran, and vacuum dried to constant weight to obtain a powder product (denoted as TBA-COF powder);

[0054] Weigh 1 mg of TBA-COF powder, add 30 mL of absolute ethanol, and exfoliate it by ultrasonic treatment (power 110 w) for 6 h to form TBA-COF nanosheets, obtaining an ethanol dispersion of the covalent organic framework nanoprobe (i.e., TBA-COF nanosheets). After standing at room temperature for 12 h, the upper nanosheet suspension is centrifuged at 14000 rpm for 10 min, the ethanol is discarded, and the obtained nanosheets (i.e., the covalent organic framework nanoprobe) are redispersed in the same volume (30 mL) of deionized water to obtain a dispersion of the covalent organic framework nanoprobe (i.e., TBA-COF nanosheets), denoted as the dispersion of TBA-COF nanosheets, with a concentration of 33 mg / mL.

[0055] The method for detecting the methylated DNA sequence by the system of this embodiment includes the following steps:

[0056] S1. Prepare the DNA sequence to be detected through the DNA sample processing unit:

[0057] S1-1. First, use a DNA methylation conversion kit (Thermo Scientific TMThe bisulfite in ( ) is used to transform the DNA sample as follows: Add 20 μL of the test sample containing methylated DNA (10 ng / mL, experimental group, the sequence is shown in Table 1) and the test sample containing unmethylated DNA (10 ng / mL, control group) into two PCR tubes respectively, then add 120 μL of Modification Reagent Solution. After heat denaturation at 98 °C for 10 min, incubate at 60 °C for 150 min to complete the transformation of the two kinds of DNA. After that, use column chromatography to purify the transformed DNA. After the above treatment, the unmethylated cytosine in the DNA sequence can be transformed into uracil, while the methylated cytosine remains unchanged.

[0058] S1-2. Use the transformed and purified DNA as a template for the asymmetric PCR process to obtain single-stranded DNA. The specific method is as follows:

[0059] The total volume of the asymmetric PCR reaction solution is 50 μL, including 2 μL of template DNA, 3 μL of forward primer (100 mM, the sequence is shown in Table 1), 1.5 μL of reverse primer (10 mM), 25 μL of Phusion U PCR premix (Thermo Scientific TM ), and 18.5 μL of sterile and enzyme-free water. The program settings for asymmetric PCR are as follows: Denature at 98 °C for 30 s, denature at 98 °C for 10 s, anneal at 57 °C for 30 s, extend at 72 °C for 15 s, cycle 35 times, and finally extend at 72 °C for 5 min. Use column chromatography to purify the PCR product, and use the Qubit quantification kit (Invitroge TM ) to detect the concentration and perform capillary electrophoresis tests.

[0060] S1-3. Subsequently, the PCR product is fluorescently labeled to obtain the DNA sequence to be detected. The specific method is as follows: The PCR product is mixed with a fluorescent probe probe (the sequence is partially complementary to the PCR product, and the 5' end of the probe is labeled with Texas Red fluorescent dye, as shown in Table 1) at an equal concentration, and after mixing at a volume ratio of 1:1, it is heated at 95 °C for 5 min for denaturation, and then cooled to 25 °C at a rate of 0.1 °C / s and maintained for 10 min to hybridize the two, finally obtaining the DNA sequence to be detected. If the original sample used in step S1-1 is a test sample containing methylated DNA (i.e., the experimental group), then after bisulfite treatment, asymmetric PCR process, and fluorescent labeling, the obtained DNA sample is denoted as the DNA of the experimental group to be detected; if the original sample used in step S1-1 is a test sample containing non-methylated DNA (i.e., the control group), then after bisulfite treatment, asymmetric PCR process, and fluorescent labeling, the obtained DNA sample is denoted as the DNA of the control group to be detected.

[0061] S2. Detect the fluorescence emission spectrum of the DNA sequence to be detected through the detection unit:

[0062] The dispersion of TBA-COF nanosheets (50 mL, 33 mg / mL) is mixed with the DNA of the experimental group to be detected (2.5 mL, 10 mM) and the DNA of the control group to be detected (2.5 mL, 10 mM) respectively, and diluted to 500 mL with TE buffer solution. The final concentration of TBA-COF nanosheets is 3.3 mg / mL, and the final concentration of the DNA sequence to be detected is 50 nM. After incubation for 20 min, a fluorescence spectrometer is used to set 590 nm as the excitation wavelength to detect the fluorescence emission spectrum of the solution. The fluorescence intensity at 612 nm is obtained, and this fluorescence intensity value is correlated with the DNA methylation status in the original sample used in step S1-1.

[0063] Table 1 DNA sequences involved in Example 1

[0064]

[0065] Experimental results:

[0066] Refer to Figure 1 , which is the chemical structure of TBA-COF; after ultrasonic exfoliation of TBA-COF powder in absolute ethanol, the SEM image of the obtained TBA-COF nanosheets is as shown in Figure 2 . It can be seen from the SEM image that the nanosheets have a layered structure, and from the TEM image, it can be seen that the nanosheets have an ultrathin thickness and ultra-small pores ( Figure 3 ).

[0067] Bisulfite can convert unmethylated cytosine (C) in the DNA sequence into uracil (U), while methylated cytosine (C) remains unchanged. Therefore, after asymmetric PCR (the capillary electrophoresis results are as Figure 4 shown), the original methylated DNA (experimental group) is converted into a cytosine (C)-rich sequence, while the unmethylated DNA (control group) is converted into a thymine (T)-rich sequence. The covalent organic framework nanoprobe of the present invention has a strong adsorption force for the thymine (T)-rich DNA sequence. After adsorption and binding, it can effectively quench the fluorescence linked to the DNA sequence, while it has a weak adsorption force for the cytosine-rich DNA sequence. Therefore, compared with the fluorescent molecule linked to the DNA sequence of the test experimental group, the fluorescent molecule linked to the DNA sequence of the test control group will be quenched to a greater extent by the COF. As Figure 5 shown, the fluorescence intensity of the experimental group is significantly higher than that of the control group. Example 2

[0068] Effect of the ratio of methylated DNA to unmethylated DNA on the fluorescence intensity:

[0069] The DNA of the test experimental group and the DNA of the test control group processed by the DNA sample processing unit in Example 1 were mixed at a certain ratio, namely, test experimental group DNA / test control group DNA = 100% / 0%, 75% / 25%, 50% / 50%, 25% / 75% and 0% / 100%, to obtain 5 samples. The above 5 samples were incubated with the dispersion of TBA-COF nanosheets respectively according to the method of Example 1, and the fluorescence emission spectrum was detected using a fluorescence spectrometer. The results are as Figure 6 shown. As the proportion of the DNA of the test experimental group, that is, the methylated DNA, increases, the content of thymine (T) gradually decreases, the adsorption ability of the TBA-COF nanosheets decreases, and the fluorescence intensity at 612 nm (the maximum emission wavelength) increases linearly. Its linear equation is: y = 171.01x + 169.62, R 2 = 0.9842. Using this linear equation, the proportion of methylated DNA can be calculated according to the fluorescence intensity, so as to realize the qualitative and quantitative detection of methylated DNA. Example 3

[0070] The fluorescence labeling method for the PCR product in the DNA sample processing unit in Example 1 was replaced with: using terminal deoxynucleotidyl transferase to label the end of the PCR product with Texas red-dCTP, and purifying the DNA by column chromatography to remove the unreacted Texas red-dCTP. The remaining steps are the same as those in Example 1.

[0071] The experimental results are as Figure 7As shown, the COF of this embodiment can still achieve the detection of methylated DNA. Example 4

[0072] In the preparation process of the covalent organic framework nanoprobe in Example 1, change the reaction temperature to 80 °C, the reaction time to 96 hours, and the concentration of TBA-COF nanosheets dispersed in water to 40 mg / mL. The remaining procedures are the same as those in Example 1.

[0073] After testing, the COF of this embodiment can still achieve the detection of methylated DNA. Example 5

[0074] In the preparation process of the covalent organic framework nanoprobe in Example 1, change the reaction temperature to 120 °C, the reaction time to 24 hours, and the concentration of TBA-COF nanosheets dispersed in water to 30 mg / mL. The remaining procedures are the same as those in Example 1.

[0075] After testing, the COF of this embodiment can still achieve the detection of methylated DNA. Example 6

[0076] Change the fluorescent molecule used in the fluorescent labeling process of the DNA sample processing unit to Cy3. The remaining procedures are the same as those in Example 1.

[0077] The experimental results are as Figure 8 As shown, the COF of this embodiment can still achieve the detection of methylated DNA.

[0078] Although the embodiments of the present invention have been disclosed as above, they are not limited to only the applications listed in the specification and embodiments. It can be fully applied to various fields suitable for the present invention. For those familiar with the field, additional modifications can be easily made. Therefore, without departing from the general concept defined by the claims and the equivalent scope, the present invention is not limited to specific details.

Claims

1. A methylation DNA sequence detection system based on covalent organic framework materials, characterized in that, Comprising: A covalent organic framework nanoprobe, which is prepared by a Schiff base reaction using an organic small molecule containing a triphenylamine structure as a monomer raw material. This covalent organic framework nanoprobe has different adsorption capacities for different bases: its adsorption force for thymine is stronger than that for cytosine; A DNA sample processing unit, which obtains a DNA sequence to be detected by performing bisulfite treatment, asymmetric PCR amplification, and fluorescence labeling of the PCR product on the DNA sample; After the DNA sequence to be detected is adsorbed by the covalent organic framework nanoprobe, the fluorescence of the fluorescent substance labeled on the DNA sequence to be detected can be quenched by the covalent organic framework nanoprobe; And a detection unit, which measures the fluorescence intensity of the product after the DNA sequence to be detected binds to the covalent organic framework nanoprobe; the fluorescence intensity of this product is positively correlated with the proportion of methylated DNA in the sample to be tested; When this system detects the methylated DNA sequence, the dispersion of the covalent organic framework nanoprobe is mixed and incubated with the DNA sequence to be detected, and a fluorescence spectrometer is used to detect the fluorescence emission spectrum of the incubated product, obtain the fluorescence intensity at the emission peak position of the fluorescent substance labeled on the DNA sequence to be detected, and judge the proportion of methylated DNA in the sample to be tested according to the value of this fluorescence intensity; The monomer raw materials are tris(4-aminophenyl)amine and tris(4-benzoyl)amine.

2. The methylation DNA sequence detection system based on covalent organic framework materials according to claim 1, wherein The covalent organic framework nanoprobe is prepared by the following method: 1) Taking tris(4-aminophenyl)amine and tris(4-benzoyl)amine as monomer raw materials, adding them to a solvent, and performing ultrasonic treatment until it becomes clear; 2) Adding an acetic acid solution to the solution in step 1), mixing evenly, and then degassing; 3) Sealing after degassing, heating for reaction, after the reaction ends, collecting the product, washing, and drying; 4) Taking the powder product obtained in step 3) and adding it to absolute ethanol for ultrasonic exfoliation to obtain an ethanol dispersion of the covalent organic framework nanoprobe; 5) Allowing the product in step 4) to stand for precipitation, and redispersing the upper suspension obtained after centrifugation in water to obtain the dispersion of the covalent organic framework nanoprobe.

3. The methylation DNA sequence detection system based on covalent organic framework materials according to claim 2, wherein, In step 3), the temperature of the heating reaction is 80 - 120 °C, and the reaction time is 24 - 96 hours.

4. The methylation DNA sequence detection system based on covalent organic framework materials according to claim 3, wherein In the dispersion of the covalent organic framework nanoprobe, the concentration of the covalent organic framework nanoprobe is 30 - 40 mg / mL.

5. The methylation DNA sequence detection system based on covalent organic framework materials according to claim 4, wherein The covalent organic framework nanoprobe is prepared by the following method: 1) Taking a mixture of 10 - 40 mg of tris(4-aminophenyl)amine and 10 - 40 mg of tris(4-benzoyl)amine as the raw material monomer, adding it to 3 - 12 mL of a solvent composed of ortho-dichlorobenzene and ethanol in a volume ratio of 4:1, and performing ultrasonic treatment for 2 - 10 minutes to obtain a clear solution; 2) Adding 120 - 480 mL of an acetic acid solution with a concentration of 1.5 - 6 mol / L to the product in step 1), mixing evenly, freezing in liquid nitrogen at 77 K, and performing degassing through 2 - 6 freeze-thaw cycles; 3) Sealing after degassing, reacting at 80 - 120 °C for 24 - 96 hours, after the reaction ends, collecting the product, washing with tetrahydrofuran, and drying; 4) Take 0.5 - 2 mg of the powder product obtained in step 3) and add it to 15 - 60 mL of absolute ethanol. Ultrasonically exfoliate it at 50 - 220 W for 3 - 12 h to obtain the ethanol dispersion of the covalent organic framework nanoprobe; 5) After allowing the ethanol dispersion of the covalent organic framework nanoprobe in step 4) to stand and precipitate at room temperature for 12 h, discard the bottom precipitate. Centrifuge the upper suspension at 7000 - 15000 rpm for 3 - 15 min, discard the upper ethanol, and redisperse the obtained covalent organic framework nanoprobe in the same volume of deionized water to obtain the dispersion of the covalent organic framework nanoprobe.

6. The methylation DNA sequence detection system based on covalent organic framework materials according to claim 1, wherein In the DNA sample processing unit, the method for fluorescently labeling the PCR product is: using a complementary sequence labeled with a fluorescent substance or performing end - labeling using terminal deoxynucleotidyl transferase.

7. The methylation DNA sequence detection system based on covalent organic framework materials according to claim 1, wherein In the DNA sample processing unit, the fluorescent substance used in the fluorescent labeling process is a quantum dot or at least one of the following fluorescent molecules or their derivatives: Texas Red fluorescent dye, cyanine series fluorescent dyes, rhodamine - type fluorescent dyes, Alexa series fluorescent dyes, Atto series fluorescent dyes.

8. Use of a methylation DNA sequence detection system based on a covalent organic framework material as described in any one of claims 1 - 7 in detecting a methylation DNA sequence, wherein the use is for non - disease diagnosis and treatment purposes.

9. A method for detecting methylated DNA sequences using the methylated DNA sequence detection system based on covalent organic framework materials according to any one of claims 1-7, which is for non-diagnostic and non-therapeutic purposes, and is characterized in that, The method comprises the following steps: S1. First, use bisulfite to transform the sample DNA through the DNA sample processing unit, then perform asymmetric PCR amplification, and then fluorescently label the PCR product with a fluorescent substance to obtain the DNA sequence to be detected; S2. Mix and incubate the dispersion of the covalent organic framework nanoprobe with the DNA sequence to be detected, use a fluorescence spectrometer to detect the fluorescence emission spectrum of the incubated product, obtain the fluorescence intensity at the emission peak position of the fluorescent substance labeled in the DNA sequence to be detected, and judge the proportion of methylated DNA in the sample to be detected according to the value of this fluorescence intensity; wherein, the value of this fluorescence intensity is positively correlated with the proportion of methylated DNA in the sample to be detected.

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