Covalent organic framework material-based methylated DNA sequence detection system, detection method and application thereof

Abstract

The invention discloses a methylated DNA sequence detection system based on a covalent organic framework material and a detection method and application thereof, the detection system comprises a covalent organic framework nanoprobe, a DNA sample processing unit and a detection unit, the detection process does not need to depend on a composite material, and the preparation flow is simple and convenient. Small organic molecules containing a triphenylamine structure are adopted as monomer raw materials, a covalent organic framework nanoprobe is prepared through a Schiff base reaction, DNA is subjected to hydrosulfite treatment, asymmetric PCR and fluorescence labeling through a DNA sample treatment unit, and DNA to be detected is formed; and measuring fluorescence of a combination of the DNA sequence to be detected and the covalent organic framework nanoprobe by using a detection unit, thereby realizing methylation detection of the DNA sequence. The system can efficiently and conveniently detect the methylated DNA sequence, and is simple in operation process and easy to implement.

Description

Technical Field

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

[0002] Covalent Organic Frameworks (COFs) are a type of porous crystalline polymer formed by organic molecules connected by covalent bonds. They have been widely studied in the field of biosensing due to their highly ordered pore structure, adjustable pore size and surface chemical properties. In particular, in the field of nucleic acid detection, COF's huge specific surface area and abundant active sites 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 technology that evaluates DNA expression status and abnormalities by analyzing DNA methylation patterns. It can detect abnormal methylation sites and expression profiles of tumor-related DNA, thereby enabling early diagnosis, prevention and treatment of diseases. It is of great significance in the fields of biology, medicine and life sciences. However, there are no reports in the prior art on the direct detection of methylated DNA sequences using covalent organic framework materials.

[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 methylated DNA sequence detection system based on a covalent organic framework material, a detection method and an application thereof in view of the deficiencies in the above-mentioned prior art.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is: In a first aspect of the present invention, a methylated DNA sequence detection system based on a covalent organic framework material is provided, comprising: Covalent organic framework nanoprobes, which are prepared by Schiff base reaction using organic small molecules containing triphenylamine structure as monomer raw materials, and have different adsorption capacities for different bases; A DNA sample processing unit, which obtains a DNA sequence to be tested by performing bisulfite treatment, asymmetric PCR amplification and fluorescent labeling of PCR products on the DNA sample; after the DNA sequence to be tested is adsorbed by the covalent organic framework nanoprobe, the fluorescence of the fluorescent substance labeled on the DNA sequence to be tested 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 tested is combined with 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 tested.

[0007] Preferably, the covalent organic framework nanoprobe is a single-layer or multi-layer nanosheet.

[0008] Preferably, the covalent organic framework nanoprobe is single crystal or polycrystalline.

[0009] Preferably, the monomer raw material includes tris(4-aminophenyl)amine and tris(4-benzoyl)amine.

[0010] Preferably, the covalent organic framework nanoprobe is prepared by the following method: 1) Tris(4-aminophenyl)amine and tris(4-benzoyl)amine as monomer raw materials are added into a solvent and ultrasonically treated until clear; 2) adding acetic acid solution to the solution in step 1), mixing well and then degassing; 3) After degassing, seal and heat to react. After the reaction is completed, collect the product, wash and dry; 4) adding the powder product obtained in step 3) into anhydrous ethanol for ultrasonic exfoliation to obtain an ethanol dispersion of the covalent organic framework nanoprobe; 5) After the ethanol dispersion of the covalent organic framework nanoprobe in step 4) is allowed to settle at room temperature, the upper suspension is centrifuged and the nanosheets are redispersed in deionized water to obtain the dispersion of the covalent organic framework nanoprobe.

[0011] 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.

[0012] Preferably, in the dispersion of the covalent organic framework nanoprobe, the concentration of the covalent organic framework nanoprobe is 30-40 mg / mL, and more preferably, the concentration is 33 mg / mL.

[0013] Preferably, the covalent organic framework nanoprobe is prepared by the following method: 1) A mixture of 10-40 mg of tris(4-aminophenyl)amine and 10-40 mg of tris(4-benzoyl)amine as raw monomers is added to 3-12 mL of a solvent consisting of o-dichlorobenzene and ethanol in a volume ratio of 4:1, and ultrasonically treated for 2-10 minutes to obtain a clear solution; 2) adding 120-480 mL of 1.5-6 mol / L acetic acid solution to the product of step 1), mixing well, freezing in liquid nitrogen at 77 K, and degassing by 2-6 freeze-thaw cycles; 3) After degassing, seal the mixture 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; 4) taking 0.5-2 mg of the powder product obtained in step 3) and adding it to 15-60 mL of anhydrous ethanol, and ultrasonically exfoliating at 50-220 W for 3-12 h to obtain an ethanol dispersion of the covalent organic framework nanoprobe; 5) After the ethanol dispersion of the covalent organic framework nanoprobe in step 4) is allowed to settle at room temperature for 12 h, the bottom precipitate is discarded, the upper suspension is centrifuged at 7000-15000 rpm for 3-15 min, the upper ethanol is discarded, and the obtained covalent organic framework nanoprobe is re-dispersed in the same volume of deionized water to obtain the dispersion of the covalent organic framework nanoprobe.

[0014] Preferably, in the DNA sample processing unit, the method for fluorescently labeling the PCR product is: hybridization with a complementary sequence labeled with a fluorescent substance or terminal labeling using terminal deoxynucleotidyl transferase.

[0015] Preferably, in the DNA sample processing unit, the fluorescent substance used in the fluorescent labeling process is quantum dots or at least one of the following fluorescent molecules or their derivatives: Texas Red fluorescent dye, cyanine series fluorescent dye, rhodamine fluorescent dye, Alexa series fluorescent dye, Atto series fluorescent dye.

[0016] The second aspect of the present invention provides an application of the above-mentioned covalent organic framework material-based methylated DNA sequence detection system in detecting methylated DNA sequences, wherein the application is for the purpose of non-disease diagnosis and treatment.

[0017] In a third aspect of the present invention, there is provided a method for detecting methylated DNA sequences using the methylated DNA sequence detection system based on the covalent organic framework material as described above, the method comprising the following steps: S1. The sample DNA is first converted using bisulfite by a DNA sample processing unit, and then asymmetric PCR amplification is performed, and then the PCR product is fluorescently labeled using a fluorescent substance to obtain a DNA sequence to be tested; S2. Mix the dispersion of the covalent organic framework nanoprobe with the DNA sequence to be tested and incubate them, use a fluorescence spectrometer to detect the fluorescence emission spectrum of the incubated product, obtain the fluorescence intensity of the emission peak position of the fluorescent substance labeled in the DNA sequence to be tested, and judge the proportion of methylated DNA in the sample to be tested according to the value of the fluorescence intensity; wherein the value of the fluorescence intensity is positively correlated with the proportion of methylated DNA in the sample to be tested.

[0018] The beneficial effects of the present invention are: 1. The present invention provides a methylated DNA sequence detection system based on a covalent organic framework material, a detection method and an application thereof. The detection system of the present invention does not need to rely on composite materials, but only uses organic small molecules containing triphenylamine structure as monomer raw materials, and can prepare covalent organic framework nanoprobes as main detection materials through Schiff base reaction, thereby simplifying the preparation process; 2. The present invention can detect methylated DNA sequences by combining a covalent organic framework nanoprobe, a DNA sample processing unit, and a detection unit, and the process is simple and easy to implement; 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 being efficient and convenient. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is the chemical formula of TBA-COF prepared in Example 1; Figure 2 is a SEM image of the TBA-COF nanosheets prepared in Example 1; Figure 3 is a TEM image of the TBA-COF nanosheets prepared in Example 1; Figure 4 is the capillary electrophoresis pattern of the experimental group DNA and the control group DNA in Example 1 after bisulfite treatment and asymmetric PCR respectively; Figure 5 is the fluorescence emission spectrum of the experimental group DNA to be tested and the control group DNA to be tested in Example 1 after incubation with TBA-COF nanosheets for 20 min, with an excitation wavelength of 590 nm; Figure 6 is a linear relationship diagram of the fluorescence intensity at 612 nm and the proportion of methylated DNA in Example 2; Figure 7 is the fluorescence emission spectrum of the experimental group DNA and the control group DNA in Example 3 after incubation with TBA-COF nanosheets for 20 min, with an excitation wavelength of 590 nm; Figure 8It is the fluorescence emission spectrum of the Cy3-labeled experimental group DNA and the control group DNA in Example 6 after incubation with TBA-COF nanosheets for 20 min, with an excitation wavelength of 550 nm. DETAILED DESCRIPTION

[0020] The present invention is further described in detail below in conjunction with embodiments so that those skilled in the art can implement the invention with reference to the description.

[0021] It should be understood that the terms such as “having”, “including” and “comprising” used herein do not exclude the existence or addition of one or more other elements or combinations thereof.

[0022] The test methods used in the following examples are conventional methods unless otherwise specified. The materials and reagents used in the following examples are all commercially available unless otherwise specified. In the following examples, if no specific conditions are specified, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used, if the manufacturer is not specified, are all conventional products that can be purchased commercially. Example 1

[0023] A methylated DNA sequence detection system based on a covalent organic framework material, comprising: Covalent organic framework nanoprobes, which are prepared by Schiff base reaction using organic small molecules containing triphenylamine structure as monomer raw materials, and have different adsorption capacities for different bases; A DNA sample processing unit, which obtains a DNA sequence to be tested by performing bisulfite treatment, asymmetric PCR amplification and fluorescent labeling of PCR products on the sample DNA; after the DNA sequence to be tested is adsorbed by the covalent organic framework nanoprobe, the fluorescence of the fluorescent substance labeled on the DNA sequence to be tested 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 tested is combined with 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 tested.

[0024] Wherein, the preparation method of the covalent organic framework nanoprobe is: 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 of 4:1) were added to the reaction tube in sequence; a clear solution was obtained after ultrasonic treatment for 5 minutes, and then 240 mL of 3 mol / L acetic acid solution was added; after uniform mixing, the reaction tube was frozen in liquid nitrogen at 77 K and degassed through three freeze-thaw cycles; after degassed, the tube was sealed and heated at 120°C for 72 hours. After the reaction was completed, the product was collected, washed with tetrahydrofuran, and vacuum dried to constant weight to obtain a powder product (referred to as TBA-COF powder); 1 mg of TBA-COF powder was weighed, 30 mL of anhydrous ethanol was added, and ultrasonic (power 110 w) exfoliation was performed for 6 h to form TBA-COF nanosheets to obtain an ethanol dispersion of covalent organic framework nanoprobes (i.e., TBA-COF nanosheets). After standing at room temperature for 12 h, the upper nanosheet suspension was centrifuged at 14000 rpm for 10 min, the ethanol was discarded, and the obtained nanosheets (i.e., covalent organic framework nanoprobes) were redispersed in the same volume (30 mL) of deionized water to obtain a dispersion of covalent organic framework nanoprobes (i.e., TBA-COF nanosheets), recorded as the dispersion of TBA-COF nanosheets, with a concentration of 33 mg / mL.

[0025] The method for detecting methylated DNA sequences by the system of this embodiment comprises the following steps: S1. Prepare the DNA sequence to be tested by the DNA sample processing unit: S1-1. First, use the DNA methylation conversion kit (Thermo Scientific TM ) was used to convert the DNA samples, as follows: 20 mL of the sample containing methylated DNA (10 ng / mL, experimental group, sequence see Table 1) and the sample containing unmethylated DNA (10 ng / mL, control group) were added to two PCR tubes, and then 120 mL of Modification Reagent Solution was added. After heat denaturation at 98 °C for 10 min, the two DNAs were incubated at 60 °C for 150 min to complete the conversion. The converted DNA was then purified by column chromatography. After the above treatment, the unmethylated cytosine in the DNA sequence can be converted into uracil, while the methylated cytosine remains unchanged.

[0026] S1-2. Using the converted and purified DNA as a template to perform an asymmetric PCR process to obtain single-stranded DNA. The specific method is as follows: The total volume of the asymmetric PCR reaction solution was 50 mL, including 2 mL template DNA, 3 mL forward primer (100 mM, sequence see Table 1), 1.5 µL reverse primer (10 mM), 25 mL Phusion U PCR master mix (Thermo Scientific TM ) and 18.5 mL of sterile enzyme-free water. The asymmetric PCR program was set as follows: 98 °C denaturation for 30 s, 98 °C denaturation for 10 s, 57 °C annealing for 30 s, 72 °C extension for 15 s, 35 cycles, and a final extension at 72 °C for 5 min. The PCR product was purified by column chromatography and the Qubit quantification kit (Invitrogen) was used. TM ) to detect the concentration and perform capillary electrophoresis test.

[0027] S1-3. Afterwards, the PCR product is fluorescently labeled to obtain the DNA sequence to be tested. The specific method is as follows: the PCR product is mixed with an equal concentration of 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, i.e., Texas Red, see Table 1) at a volume ratio of 1:1, and then 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 allow the two to hybridize, and finally obtain the DNA sequence to be tested. If the original sample used in step S1-1 is a sample to be tested containing methylated DNA (i.e., the experimental group), then after bisulfite treatment, asymmetric PCR process and fluorescent labeling, the obtained DNA sample is recorded as the DNA of the experimental group to be tested; if the original sample used in step S1-1 is a sample to be tested containing non-methylated DNA (i.e., the control group), then after bisulfite treatment, asymmetric PCR process and fluorescent labeling, the obtained DNA sample is recorded as the DNA of the control group to be tested.

[0028] S2. Detect the fluorescence emission spectrum of the DNA sequence to be detected by the detection unit: The dispersion of TBA-COF nanosheets (50 mL, 33 mg / mL.) was mixed with the DNA of the experimental group to be tested (2.5 mL, 10 mM) and the DNA of the control group to be tested (2.5 mL, 10 mM), and diluted to 500 mL with TE buffer solution. The final concentration of TBA-COF nanosheets was 3.3 mg / mL, and the final concentration of the DNA sequence to be tested was 50 nM. After incubation for 20 min, the fluorescence emission spectrum of the solution was detected using a fluorescence spectrometer with 590 nm as the excitation wavelength. The fluorescence intensity at 612 nm was obtained, and the fluorescence intensity value was correlated with the DNA methylation status in the original sample used in step S1-1.

[0029] Table 1 DNA sequences involved in Example 1

[0030] Experimental results: Reference Figure 1 , which is the chemical structure of TBA-COF; after ultrasonic exfoliation of TBA-COF powder in anhydrous ethanol, the SEM image of TBA-COF nanosheets obtained is as follows Figure 2 As shown, the layered structure of the nanosheets can be seen from the SEM image, and the ultra-thin thickness and ultra-small pores of the nanosheets can be seen from the TEM image. Figure 3 ).

[0031] Bisulfite can convert unmethylated cytosine (C) in DNA sequences into uracil (U), while methylated cytosine (C) remains unchanged. Figure 4 As shown in the figure, the original methylated DNA (experimental group) is converted into a sequence rich in cytosine (C), while the non-methylated DNA (control group) is converted into a sequence rich in thymine (T). The covalent organic framework nanoprobe of the present invention has a strong adsorption force on DNA sequences rich in thymine (T), and can effectively quench the fluorescence connected to the DNA sequence after adsorption and binding, while the adsorption force on DNA sequences rich in cytosine is weaker. Therefore, compared with the fluorescent molecules connected to the DNA sequence of the experimental group to be tested, the fluorescent molecules connected to the DNA sequence of the control group to be tested will be quenched to a greater extent by COF, as shown in the figure. Figure 5 As shown, the fluorescence intensity of the experimental group was significantly higher than that of the control group. Example 2

[0032] Effect of the ratio of methylated DNA to unmethylated DNA on fluorescence intensity: The DNA of the experimental group to be tested treated by the DNA sample processing unit in Example 1 was mixed with the DNA of the control group to be tested in a certain ratio, that is, the DNA of the experimental group to be tested / the DNA of the control group to be tested = 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 according to the method of Example 1, and the fluorescence emission spectrum was detected using a fluorescence spectrometer. The results are as follows Figure 6 As shown in the figure, with the increase of the proportion of DNA in the experimental group to be tested, that is, methylated DNA, the content of thymine (T) gradually decreased, the adsorption capacity of TBA-COF nanosheets decreased, and the fluorescence intensity at 612 nm (maximum emission wavelength) increased linearly. The linear equation is: y=171.01x+169.62, R 2=0.9842. The linear equation can be used to calculate the proportion of methylated DNA according to the fluorescence intensity, thereby achieving qualitative and quantitative detection of methylated DNA. Example 3

[0033] The fluorescent labeling method of the PCR product in the DNA sample processing unit in Example 1 is replaced by: using terminal deoxynucleotidyl transferase to label the end of the PCR product with Texas red-dCTP, and using column chromatography to purify the DNA to remove unreacted Texas red-dCTP. The remaining steps are the same as in Example 1.

[0034] The experimental results are as follows Figure 7 As shown, the COF of this embodiment can still detect methylated DNA. Example 4

[0035] The reaction temperature in the preparation process of the covalent organic framework nanoprobe in Example 1 was changed to 80°C, the reaction time was changed to 96 hours, the concentration of TBA-COF nanosheets dispersed in water was changed to 40 mg / mL, and the rest of the process was the same as Example 1.

[0036] After testing, the COF of this embodiment can still detect methylated DNA. Example 5

[0037] The reaction temperature in the preparation process of the covalent organic framework nanoprobe in Example 1 was changed to 120° C., the reaction time was changed to 24 hours, the concentration of TBA-COF nanosheets dispersed in water was changed to 30 mg / mL, and the rest of the process was the same as Example 1.

[0038] After testing, the COF of this embodiment can still detect methylated DNA. Example 6

[0039] The fluorescent molecule used in the fluorescent labeling process of the DNA sample processing unit is changed to Cy3, and the rest of the process is the same as that of Example 1.

[0040] The experimental results are as follows Figure 8 As shown, the COF of this embodiment can still detect methylated DNA.

[0041] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to specific details.

Claims

1. A methylated DNA sequence detection system based on covalent organic framework materials, characterized in that: include: Covalent organic framework nanoprobes, which are prepared by Schiff base reaction using organic small molecules containing triphenylamine structure as monomer raw materials, and have different adsorption capacities for different bases; A DNA sample processing unit, which obtains a DNA sequence to be tested by performing bisulfite treatment, asymmetric PCR amplification, and fluorescent labeling on 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 tested is combined with 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 tested.

2. The methylated DNA sequence detection system based on covalent organic framework materials according to claim 1, characterized in that: The monomer raw material includes at least one of tris(4-aminophenyl)amine and tris(4-benzoyl)amine.

3. The methylated DNA sequence detection system based on covalent organic framework material according to claim 2, characterized in that: The covalent organic framework nanoprobe is prepared by the following method: 1) Tris(4-aminophenyl)amine and tris(4-benzoyl)amine as monomer raw materials are added into a solvent and ultrasonically treated until clear; 2) adding acetic acid solution to the solution in step 1), mixing well and then degassing; 3) After degassing, seal and heat to react. After the reaction is completed, collect the product, wash and dry; 4) adding the powder product obtained in step 3) into anhydrous ethanol for ultrasonic exfoliation to obtain an ethanol dispersion of the covalent organic framework nanoprobe; 5) The product of step 4) is allowed to settle, and the obtained upper suspension is re-dispersed in water after centrifugation to obtain the dispersion of the covalent organic framework nanoprobe.

4. The methylated DNA sequence detection system based on covalent organic framework material according to claim 3, characterized in that: In step 3), the heating reaction temperature is 80-120° C. and the reaction time is 24-96 hours.

5. The methylated DNA sequence detection system based on covalent organic framework material according to claim 4, characterized in that: In the dispersion of the covalent organic framework nanoprobe, the concentration of the covalent organic framework nanoprobe is 30-40 mg / mL.

6. The methylated DNA sequence detection system based on covalent organic framework material according to claim 5, characterized in that: The covalent organic framework nanoprobe is prepared by the following method: 1) A mixture of 10-40 mg of tris(4-aminophenyl)amine and 10-40 mg of tris(4-benzoyl)amine as raw monomers is added to 3-12 mL of a solvent consisting of o-dichlorobenzene and ethanol in a volume ratio of 4:1, and ultrasonically treated for 2-10 minutes to obtain a clear solution; 2) adding 120-480 mL of 1.5-6 mol / L acetic acid solution to the product of step 1), mixing well, freezing in liquid nitrogen at 77K, and degassing by 2-6 freeze-thaw cycles; 3) After degassing, seal the mixture 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; 4) taking 0.5-2 mg of the powder product obtained in step 3) and adding it to 15-60 mL of anhydrous ethanol, and ultrasonically exfoliating at 50-220 W for 3-12 h to obtain an ethanol dispersion of the covalent organic framework nanoprobe; 5) After the ethanol dispersion of the covalent organic framework nanoprobe in step 4) is allowed to settle at room temperature for 12 h, the bottom precipitate is discarded, the upper suspension is centrifuged at 7000-15000 rpm for 3-15 min, the upper ethanol is discarded, and the obtained covalent organic framework nanoprobe is re-dispersed in the same volume of deionized water to obtain the dispersion of the covalent organic framework nanoprobe.

7. The methylated DNA sequence detection system based on covalent organic framework materials according to claim 1, characterized in that: In the DNA sample processing unit, the method for fluorescently labeling the PCR product is: labeling the complementary sequence with a fluorescent substance or terminal labeling with terminal deoxynucleotidyl transferase.

8. The methylated DNA sequence detection system based on covalent organic framework materials according to claim 1, characterized in that: In the DNA sample processing unit, the fluorescent material used in the fluorescent labeling process is quantum dots or at least one of the following fluorescent molecules or their derivatives: Texas Red fluorescent dye, cyanine series fluorescent dye, rhodamine fluorescent dye, Alexa series fluorescent dye, Atto series fluorescent dye.

9. An application of the methylated DNA sequence detection system based on a covalent organic framework material as claimed in any one of claims 1 to 8 in detecting methylated DNA sequences, wherein the application is for the purpose of non-disease diagnosis and treatment.

10. A method for detecting methylated DNA sequences using the methylated DNA sequence detection system based on a covalent organic framework material as claimed in any one of claims 1 to 8, characterized in that: The method comprises the following steps: S1. The sample DNA is first converted using bisulfite by a DNA sample processing unit, and then asymmetric PCR amplification is performed, and then the PCR product is fluorescently labeled using a fluorescent substance to obtain a DNA sequence to be tested; S2. Mix the dispersion of the covalent organic framework nanoprobe with the DNA sequence to be tested and incubate them, use a fluorescence spectrometer to detect the fluorescence emission spectrum of the incubated product, obtain the fluorescence intensity of the emission peak position of the fluorescent substance labeled in the DNA sequence to be tested, and judge the proportion of methylated DNA in the sample to be tested according to the value of the fluorescence intensity; wherein the value of the fluorescence intensity is positively correlated with the proportion of methylated DNA in the sample to be tested.

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