Application of covalent organic framework material based on triphenylamine unit in DNA sequence recognition

Abstract

The invention discloses application of a covalent organic framework material based on a triphenylamine unit in DNA (deoxyribonucleic acid) sequence recognition, and provides COFs (covalent organic frameworks) with ultra-thin thickness and an ordered conjugated structure aiming at the problems that the existing COFs need to be additionally doped in DNA sequence recognition, a detected target substance is single, a preparation process is complex and the like. A triphenylamine unit is used as a monomer raw material, and the triphenylamine unit-based nano-sheet is prepared through a Schiff base reaction, can be a single-layer or multi-layer nano-sheet, has an ultrathin thickness and an ordered conjugated structure, has high selectivity to DNA of different sequences, and is simple in preparation process and easy to operate. During application, other materials do not need to be loaded, and recognition of DNA of different sequences can be achieved only through COFs. In addition, the recognition process is simple, different DNA sequences can be distinguished only by measuring the fluorescence quenching degree, the application capability is improved, the preparation process is simplified, and wide research and application prospects are achieved.

Description

Technical Field

[0001] The invention relates to the field of nanomaterials, and in particular to a covalent organic framework material based on triphenylamine units, a preparation method thereof and an application thereof in DNA sequence recognition. 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, COFs' huge specific surface area and abundant active sites can interact with nucleic acids through non-covalent or covalent forces, showing great application potential in DNA sequence recognition and detection.

[0003] Prior art (doi: 10.1039 / D4BM01018C) reported a novel rare earth doped covalent organic framework (Ln-COFs) fluorescent probe for rapid detection of 7-methylguanine (m7Gua), a biomarker reflecting the degree of DNA methylation, which is of great significance for early cancer screening and diagnosis. The probe uses pyridine-2,6-dicarboxylic acid (DPA) as an energy donor and recognition site, Eu 3+ Ions are used as signal molecules, and the two are integrated into a stable COFs matrix with high porosity and available binding sites, achieving highly selective and fast response detection of m7Gua. However, the above COFs require additional rare earth doping, and the target substance detected is single, which cannot be applied to a wider range of DNA sequence recognition.

[0004] Another research team has developed a fluorescent and colorimetric visualization nanoprobe (CN113493820A), including: a two-dimensional material and a probe DNA; the two-dimensional material is a two-dimensional material that can adsorb the probe DNA, or the two-dimensional material can be used as a substrate to synthesize a two-dimensional material-metal nanoparticle complex for catalytic colorimetric detection; the probe DNA can be complementary paired with a disease-related target gene; the binding force between the probe DNA and the target gene is greater than the binding force between the probe DNA and the two-dimensional material. The above method achieves the recognition of specific DNA sequences, but the above nanoprobe requires COFs to synthesize a two-dimensional material-metal nanoparticle complex, which has a complicated preparation process and limitations in use.

[0005] Therefore, developing COFs with broader DNA sequence recognition functions has important research and application significance. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a triphenylamine unit-based covalent organic framework material for use in DNA sequence recognition in view of the deficiencies in the above-mentioned prior art. The present invention provides a COFs material with ultra-thin thickness and ordered conjugated structure, denoted as TBA-COF, in view of the problems that the existing COFs need to be additionally doped, the detection target substance is single, and the preparation process is complicated in DNA sequence recognition. The material is prepared by Schiff base reaction using triphenylamine unit as a monomer raw material, and can be a single-layer or multi-layer nanosheet, which has an ultra-thin thickness and an ordered conjugated structure, has a high degree of selectivity for DNA of different sequences, and the preparation process is simple and easy to operate. When applied, there is no need to load other materials, and only TBA-COF can be used to achieve recognition of DNA of different sequences. In addition, its recognition process is simple, and different DNA sequences can be distinguished by measuring the degree of fluorescence quenching, which improves the application ability, simplifies the preparation process, and has broad research and application prospects.

[0007] To achieve the above-mentioned purpose, the technical scheme adopted by the present invention is as follows: In the first aspect of the present invention, a covalent organic framework material based on triphenylamine units is provided for use in DNA sequence recognition, and the preparation method of the covalent organic framework material comprises the following steps: a compound having a chemical structure shown in the following formula I is added as a monomer raw material to a solvent, and ultrasonically treated until clarified; then an acetic acid solution is added, mixed evenly and then degassed; after degassed, the mixture is sealed and heated to carry out a Schiff base reaction, and after the reaction is completed, the product is collected, washed and dried, and the obtained product is added to anhydrous ethanol, ultrasonically peeled, and the obtained covalent organic framework material is redispersed in water to obtain a dispersion of the covalent organic framework material based on triphenylamine units; the specific method of applying the covalent organic framework material based on triphenylamine units to DNA sequence recognition is as follows: the dispersion of the covalent organic framework material based on triphenylamine units is mixed with DNA to be identified of different sequences labeled with fluorescent molecules, diluted with a buffer solution and incubated, and the fluorescence spectrum of the solution is detected, the sequences of the DNA to be identified are at least 2, and the application is for the purpose of non-disease diagnosis and treatment; ; Ⅰ; Wherein, R is an amino group or an aldehyde group.

[0008] Preferably, the monomer raw material is at least one of tris(4-aminophenyl)amine and tris(4-benzoyl)amine.

[0009] Preferably, the monomer raw material further comprises one of 1,3,5-tris(4-aminophenyl)benzene and 1,3,5-tris(p-formylphenyl)benzene.

[0010] Preferably, the solvent is o-dichlorobenzene and ethanol or a mixture of both.

[0011] Preferably, the method for preparing the covalent organic framework material comprises the following steps: One or two of tri(4-aminophenyl)amine and tri(4-benzoyl)amine are mixed with one of 1,3,5-tri(4-aminophenyl)benzene and 1, 3, 5-tri(p-formylphenyl)benzene, added to a solvent, and ultrasonically treated until clarified; then an acetic acid solution is added, mixed evenly, and degassed through several freeze-thaw cycles; after degassed, the mixture is sealed and reacted at 80-120° C. for 24-96 hours. After the reaction is completed, the product is collected, washed with tetrahydrofuran, and dried. Anhydrous ethanol is added to the obtained product, ultrasonically stripped, and redispersed in water to obtain a dispersion of the covalent organic framework material based on triphenylamine units.

[0012] Preferably, the method for preparing the covalent organic framework material comprises the following steps: 10-40 mg of one or both of tris(4-aminophenyl)amine and tris(4-benzoyl)amine and 10-40 mg of one of 1,3,5-tris(4-aminophenyl)benzene and 1,3,5-tris(p-formylphenyl)benzene were mixed, 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, and then 120-480 mL of 1.5-6 mol / L acetic acid solution was added, mixed evenly, and heated at 77 The method comprises the steps of freezing the mixture in liquid nitrogen of 400 μm and degassing the mixture through 2-6 freeze-thaw cycles; sealing the mixture after degassing, reacting the mixture at 80-120°C for 24-96 hours, collecting the product after the reaction is completed, washing the product with tetrahydrofuran, and drying the product; taking 0.5-2 mg of the obtained powder product and adding the mixture to 15-60 mL of anhydrous ethanol, and ultrasonically peeling the mixture at 50-220 W for 3-12 hours to obtain an ethanol dispersion of the covalent organic framework material based on triphenylamine units; standing the ethanol dispersion of the covalent organic framework material at room temperature for 12 hours, discarding the bottom precipitate, centrifuging the upper suspension at 7000-15000 rpm for 3-15 minutes, discarding the upper ethanol, and redispersing the obtained covalent organic framework material in deionized water to obtain a dispersion of the covalent organic framework nanoprobe based on triphenylamine units.

[0013] Preferably, the concentration of the covalent organic framework material in the dispersion of the covalent organic framework nanoprobes is 30-40 mg / mL.

[0014] Preferably, the application method is: The dispersion of the covalent organic framework material based on triphenylamine units is diluted with TE buffer solution to obtain a working solution, which is added to a solution of a DNA sample to be identified that is labeled with a fluorescent substance and incubated, and the fluorescence intensity is detected, which is recorded as F. The dispersion of the covalent organic framework material based on triphenylamine units in the above step is replaced with the same volume of TE buffer solution to obtain the fluorescence intensity F. 0 , through F 0 / F value for DNA sequence identification.

[0015] Preferably, the fluorescent substance is quantum dots or at least one of the following fluorescent molecules or their derivatives: Texas Red fluorescent dye, cyanine series fluorescent dye, rhodamine series fluorescent dye, Alexa series fluorescent dye, Atto series fluorescent dye.

[0016] The second aspect of the present invention provides a DNA sequence recognition kit, which includes the covalent organic framework material based on triphenylamine units as described above.

[0017] The beneficial effects of the present invention are: 1. The present invention provides an application of a covalent organic framework material based on triphenylamine units in DNA sequence recognition. The covalent organic framework material (abbreviated as TBA-COF) of the present invention uses molecules having triphenylamine units as monomer raw materials. Due to the non-planarity of the structure of the triphenylamine unit itself, the synthesized TBA-COF sheet has a certain three-dimensional structure, and the interlayer pp force is weak. Therefore, when used, it is conducive to peeling it into ultra-thin nanosheets, increasing the effective binding area between TBA-COF and a DNA sequence to be detected, magnifying the difference in the force between TBA-COF and different DNA sequences, and facilitating the distinction of DNAs of different sequences.

[0018] 2. The triphenylamine unit-based TBA-COF provided by the present invention has an ordered conjugated structure. Once the fluorescently labeled DNA is adsorbed on the surface of TBA-COF, its fluorescence can be efficiently quenched, which is conducive to detecting the adsorption capacity of TBA-COF for DNA of different sequences, thereby improving the application capacity; the TBA-COF has different adsorption capacities for different bases, specifically, the adsorption capacity for thymine (T) is strong, and the adsorption capacity for adenine (A) and cytosine (C) is weakened in turn. According to the selective adsorption characteristics, it can realize the recognition of DNA sequences.

[0019] 3. The preparation method and application process of the present invention are simple and easy to operate; the product of the present invention does not need to be loaded with rare earth metal ions, metal nanoparticles, graphene, DNA complementary sequences, aptamers and other materials, and only uses TBA-COF to achieve recognition of DNA of different sequences, thereby simplifying the preparation process; in addition, when applied, different DNA sequences can be distinguished only by measuring the degree of fluorescence quenching, thereby improving the application capability and having broad research and application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is a schematic diagram of the route for synthesizing TBA-COF in Example 1; Figure 2 is a TEM image of the TBA-COF nanosheet of Example 1; Figure 3 is an AFM image of the TBA-COF nanosheet of Example 1; Figure 4 is the XRD pattern of the TBA-COF nanosheets of Example 1; Figure 5 are the initial fluorescence emission spectra of the three DNA sequences in Example 1 (at a concentration of 50 nM) and the fluorescence emission spectra after incubation with TBA-COF nanosheets (at a concentration of 3.3 mg / mL) for 20 min, with an excitation wavelength of 590 nm; Figure 6 is the F of the three DNA sequences in Example 1 0 / F value comparison results, where F 0 is the fluorescence intensity of the three DNA sequences (50 nM) at 612 nm, and F is the fluorescence intensity of the three DNA sequences at 612 nm after incubation with TBA-COF nanosheets (concentration of 3.3 mg / mL) for 20 min; Figure 7 is the F of the three DNA sequences in Example 2 0 / F value comparison results, where F 0 is the fluorescence intensity of the three DNA sequences (50 nM) at 612 nm, and F is the fluorescence intensity of the three DNA sequences at 612 nm after incubation with TBA-COF nanosheets (concentration of 4 mg / mL) for 20 min; Figure 8 is the F of the three DNA sequences in Example 4 0 / F value comparison results, where F 0 is the fluorescence intensity of the three DNA sequences (50 nM) at 612 nm, and F is the fluorescence intensity of the three DNA sequences at 612 nm after incubation with COF-1 nanosheets (concentration of 3.3 mg / mL) for 20 min; Fig. 9 is the F of the three DNA sequences in Example 6 0 / F value comparison results, where F 0 is the fluorescence intensity of the three DNA sequences (50 nM) at 565 nm, F is the fluorescence intensity of the three DNA sequences at 565 nm after incubation with TBA-COF nanosheets (concentration of 3.3 mg / mL) for 20 min, and the excitation wavelength is 550 nm; Fig.10 are the initial fluorescence emission spectra of the three DNA sequences (concentration of 50 nM) in the comparative example and the fluorescence emission spectra after incubation with COF-2 nanosheets for 20 min, with an excitation wavelength of 590 nm; Fig.11 is the F of the three DNA sequences in the comparative example 0 / F value comparison results, where F 0 is the fluorescence intensity of the three DNA sequences (50 nM) at 612 nm, and F is the fluorescence intensity of the three DNA sequences at 612 nm after incubation with COF-2 nanosheets for 20 min. DETAILED DESCRIPTION

[0021] 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.

[0022] It should be understood that 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.

[0023] 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

[0024] (1) A method for preparing a covalent organic framework material based on triphenylamine units, comprising the following steps: 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) peeling was performed for 6 h to obtain an ethanol dispersion of a covalent organic framework material based on triphenylamine units. After standing and settling at room temperature for 12 h, the bottom precipitate was discarded, the upper nanosheet suspension was collected and centrifuged at 14000 rpm for 10 min, the upper ethanol was discarded, and the nanosheets (i.e., covalent organic framework materials based on triphenylamine units) were resuspended in the same volume (30 mL) of deionized water to obtain a dispersion of covalent organic framework materials based on triphenylamine units, recorded as TBA-COF nanosheet dispersion, with a concentration of 33 mg / mL.

[0025] (2) Application of TBA-COF nanosheet dispersion to the recognition of three different DNA sequences: The three DNA sequences are shown in Table 1 below: Table 1 ; The TBA-COF nanosheet dispersion (50 mL, 33 mg / mL) was mixed with the three DNA sequences labeled with Texas Red (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 DNA sequences labeled with Texas Red was 50 nM. After incubation for 20 min, the fluorescence spectrum of the solution was detected with an excitation wavelength of 590 nm, and the fluorescence intensity at 612 nm (i.e., the fluorescence intensity after quenching) was obtained, which was recorded as F. At the same time, the fluorescence spectra of the three DNA sequences (50 nM) without TBA-COF sheets were detected, and the fluorescence intensity at 612 nm (i.e., the initial fluorescence intensity) was obtained, which was recorded as F 0 .

[0026] (3) Experimental results: The structures of tri(4-aminophenyl)amine, tri(4-benzoyl)amine and the product TBA-COF are as follows Figure 1After ultrasonic peeling in anhydrous ethanol, the ultrathin thickness and ultrasmall pores of the nanosheets can be seen from the TEM image ( Figure 2 ). The AFM image shows that the thickness of the TBA-COF nanosheet is about 3 nm ( Figure 3 ). XRD experimental pattern of TBA-COF nanosheets ( Figure 4 , experimental) and simulation results ( Figure 4 , simulation) are in good agreement, and o Sharp diffraction peaks are shown near the surface, which proves the ordered crystal structure of TBA-COF.

[0027] By measuring the fluorescence spectra of different solution systems at an excitation wavelength of 590 nm, it can be seen that the ethanol dispersion of TBA-COF nanosheets has an effect on AAAAAA (A 6 ), TTTTTT (T 6 ) and CCCCCC (C 6 ) The fluorescence quenching degree of the three DNA sequences is different ( Figure 5 ). In order to facilitate the parallel comparison of the fluorescence quenching degree of the three DNA sequences by TBA-COF nanosheets, the fluorescence intensity ratio of the three DNA sequences before and after quenching was calculated: F 0 / F, and used as a comparison, where F 0 is the initial fluorescence intensity of the three DNA sequences (50 nM) at 612 nm, F is the fluorescence intensity of the three DNA sequences at 612 nm after incubation with TBA-COF nanosheet dispersion (concentration of 3.3 mg / mL) for 20 min. Figure 6 The results shown clearly show that T 6 The fluorescence quenching degree of sequence A is the largest, followed by 6 Sequence, C 6 The fluorescence quenching degree of the sequence is minimal, which proves that TBA-COF has a recognition effect on different DNA sequences. Example 2

[0028] The heating reaction temperature 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 preparation process and the application process in DNA sequence recognition were the same as in Example 1.

[0029] Figure 7 The test results show that the TBA-COF of this embodiment can still achieve the three different DNA sequences AAAAAA (A 6 ), TTTTTT (T 6 ) and CCCCCC (C 6 ) identification. Example 3

[0030] The heating reaction temperature 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 preparation process and the application process in DNA sequence recognition were the same as in Example 1.

[0031] After testing, the TBA-COF of this embodiment can still achieve three different DNA sequences AAAAAA (A 6 ), TTTTTT(T 6 ) and CCCCCC (C 6 ) identification. Example 4

[0032] The monomer raw materials were replaced with 20 mg tris(4-aminophenyl)amine and 26.9 mg 1, 3, 5-tris(p-formylphenyl)benzene to obtain COF-1. The rest of the preparation process and the application process in DNA sequence recognition were the same as those in Example 1.

[0033] Figure 8 As shown in the test results, COF-1 of this embodiment can still achieve the three different DNA sequences AAAAAA (A 6 ), TTTTTT (T 6 ) and CCCCCC (C 6 ) identification. Example 5

[0034] The monomer raw materials were replaced with 24 mg 1,3,5-tris(4-aminophenyl)benzene and 20 mg tris(4-benzoyl)amine. The rest of the preparation process and the application process in DNA sequence recognition were the same as those in Example 1.

[0035] After testing, the COF of this embodiment can still achieve three different DNA sequences AAAAAA (A 6 ), TTTTTT (T 6 ) and CCCCCC (C 6 ) identification. Example 6

[0036] The fluorescent molecule connected to the DNA sequence was changed to Cy3, and the rest of the process was the same as in Example 1.

[0037] Fig. 9 The test results show that the TBA-COF of this embodiment can still achieve the three different DNA sequences AAAAAA (A 6 ), TTTTTT (T 6 ) and CCCCCC (C 6 ) identification. Comparative Example

[0038] The monomer raw materials were replaced with 24 mg 1,3,5-tris(4-aminophenyl)benzene and 26.9 mg 1,3,5-tris(p-formylphenyl)benzene to obtain COF-2. The rest of the preparation process and the application process in DNA sequence recognition were the same as those in Example 1.

[0039] Fig.10 and Fig.11 As shown in the test results, the COF-2 material in the comparative example has good 6 ), TTTTTT (T 6 ) and CCCCCC (C 6 ) is obviously not as good as Examples 1 to 6 of the present application, and the above three DNA sequences cannot be effectively distinguished through the above application process. The reason is that the two monomers of COF-2, 1,3,5-tris(4-aminophenyl)benzene and 1,3,5-tris(p-formylphenyl)benzene, are planar structures, resulting in a strong pp effect between the layers of COF-2, making it difficult to be peeled off. Therefore, the exposed effective sites of COF-2 nanosheets are far less than those of TBA-COF and COF-1, and cannot fully contact with the DNA sequence, and cannot effectively distinguish the DNA sequence.

[0040] 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. Application of a covalent organic framework material based on triphenylamine units in DNA sequence recognition, characterized in that: The preparation method of the covalent organic framework material comprises the following steps: adding a compound having a chemical structure shown in the following formula I as a monomer raw material to a solvent, and ultrasonically treating the mixture until it is clear; then adding an acetic acid solution, mixing the mixture evenly, and then degassing the mixture; sealing the mixture after degassing, and heating the mixture to perform a Schiff base reaction; after the reaction is completed, collecting the product, washing it, and drying it; adding anhydrous ethanol to the obtained product, and ultrasonically peeling the obtained covalent organic framework material; and re-dispersing the obtained covalent organic framework material in water to obtain a dispersion of the covalent organic framework material based on triphenylamine units; and the specific method of applying the covalent organic framework material based on triphenylamine units to DNA sequence recognition is as follows: mixing the dispersion of the covalent organic framework material based on triphenylamine units with DNA to be recognized of different sequences labeled with fluorescent molecules, diluting the mixture with a buffer solution, and incubating the mixture, and then detecting the fluorescence spectrum of the solution; the sequences of the DNA to be recognized are at least two, and the application is for the purpose of non-disease diagnosis and treatment; ； Ⅰ； Wherein, R is an amino group or an aldehyde group.

2. The use of the covalent organic framework material based on triphenylamine units in DNA sequence recognition according to claim 1, characterized in that: The monomer raw material is at least one of tris(4-aminophenyl)amine and tris(4-benzoyl)amine.

3. The use of the covalent organic framework material based on triphenylamine units in DNA sequence recognition according to claim 2, characterized in that: The monomer raw material also includes one of 1,3,5-tris(4-aminophenyl)benzene and 1,3,5-tris(p-formylphenyl)benzene.

4. The use of the covalent organic framework material based on triphenylamine units in DNA sequence recognition according to claim 3, characterized in that: The solvent is o-dichlorobenzene and ethanol or a mixture of both.

5. The use of the covalent organic framework material based on triphenylamine units in DNA sequence recognition according to claim 4, characterized in that: The preparation method of the covalent organic framework material comprises the following steps: One or two of tri(4-aminophenyl)amine and tri(4-benzoyl)amine are mixed with one of 1,3,5-tri(4-aminophenyl)benzene and 1,3,5-tri(p-formylphenyl)benzene, added to a solvent, and ultrasonically treated until clarified; then an acetic acid solution is added, mixed evenly, and degassed through several freeze-thaw cycles; after degassed, the mixture is sealed and reacted at 80-120°C for 24-96 hours. After the reaction is completed, the product is collected, washed with tetrahydrofuran, and dried. Anhydrous ethanol is added to the obtained product, ultrasonically stripped, and redispersed in water to obtain a dispersion of the covalent organic framework material based on triphenylamine units.

6. The use of the covalent organic framework material based on triphenylamine units in DNA sequence recognition according to claim 5, characterized in that: The preparation method of the covalent organic framework material comprises the following steps: 10-40 mg of one or both of tris(4-aminophenyl)amine and tris(4-benzoyl)amine and 10-40 mg of one of 1,3,5-tris(4-aminophenyl)benzene and 1,3,5-tris(p-formylphenyl)benzene were mixed, 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, and then 120-480 mL of 1.5-6 mol / L acetic acid solution was added, mixed evenly, and heated at 77 The method comprises the steps of freezing the obtained product in liquid nitrogen of K and degassing the product by 2-6 freeze-thaw cycles; sealing the product after degassing, reacting the product at 80-120°C for 24-96 hours, collecting the product after the reaction, washing the product with tetrahydrofuran and drying the product; taking 0.5-2 mg of the obtained powder product and adding the product to 15-60 mL of anhydrous ethanol, and ultrasonically peeling the product at 50-220 W for 3-12 hours to obtain an ethanol dispersion of the covalent organic framework material based on triphenylamine units; standing the ethanol dispersion of the covalent organic framework material at room temperature for 12 hours, discarding the bottom precipitate, centrifuging the upper suspension at 7000-15000 rpm for 3-15 minutes, discarding the upper ethanol, and redispersing the obtained covalent organic framework material in deionized water to obtain a dispersion of the covalent organic framework nanoprobe based on triphenylamine units.

7. The use of the covalent organic framework material based on triphenylamine units in DNA sequence recognition according to claim 6, characterized in that: The concentration of the covalent organic framework material in the dispersion of the covalent organic framework nanoprobe is 30-40 mg / mL.

8. Use of the covalent organic framework material based on triphenylamine units in DNA sequence recognition according to any one of claims 1 to 7, characterized in that: The application method is: The dispersion of the covalent organic framework material based on triphenylamine units is diluted with TE buffer solution to obtain a working solution, which is added to a DNA sample solution to be identified that is labeled with a fluorescent substance and incubated, and the fluorescence intensity is detected and recorded as F. The dispersion of the covalent organic framework material based on triphenylamine units in the above step is replaced with the same volume of TE buffer to obtain the fluorescence intensity F0, and the DNA sequence is identified by the value of F0 / F.

9. The use according to claim 8, characterized in that: The fluorescent substance 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.

10. A DNA sequence recognition kit, characterized in that: It comprises a covalent organic framework material based on triphenylamine units as claimed in any one of claims 1 to 9.

Images