Application of triphenylamine-based covalent organic frameworks in DNA sequence recognition

Abstract

The present invention discloses a covalent organic framework material based on triphenylamine units for use in DNA sequence recognition. The present invention addresses the problems of existing COFs requiring additional doping, a single target substance for detection, and a complex preparation process in DNA sequence recognition. A COF with ultra-thin thickness and an ordered conjugated structure is provided. The triphenylamine unit is used as a monomer raw material and is prepared by a Schiff base reaction. The COF can be a single-layer or multi-layer nanosheet with an ultra-thin thickness and an ordered conjugated structure. The COF has a high 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 COFs can be used to recognize DNA of different sequences. In addition, the recognition process is simple, and different DNA sequences can be distinguished by measuring the degree of fluorescence quenching, thereby improving the application capability and simplifying the preparation process. The COF has broad research and application prospects.

Description

Technical Field

[0001] The present 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 class of porous crystalline polymers formed by covalently linked organic molecules. Due to their highly ordered pore structure, tunable pore size, and surface chemical properties, they have been extensively studied in the field of biosensing. In particular, COFs' large surface area and abundant active sites allow them to interact with nucleic acids through non-covalent or covalent interactions, demonstrating great potential for 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, and Eu 3+ ions as signal molecules, and integrating them into a stable COFs matrix with high porosity and available binding sites, achieved highly selective and fast-response detection of m7Gua. However, the aforementioned COFs require additional rare earth doping and have a single target substance, making them unsuitable for broader DNA sequence recognition.

[0004] Another research team has developed a fluorescent and colorimetrically visualized nanoprobe (CN113493820A), comprising a two-dimensional material and a probe DNA. The two-dimensional material can adsorb the probe DNA, or it can serve as a substrate for synthesizing a two-dimensional material-metal nanoparticle complex for catalytic colorimetric detection. The probe DNA can complementarily pair with a disease-related target gene, and 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. This method achieves recognition of specific DNA sequences, but the nanoprobe requires synthesizing a two-dimensional material-metal nanoparticle complex from COFs, resulting in a complex preparation process and limitations in its 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 covalent organic framework material based on triphenylamine units for use in DNA sequence recognition in response to the deficiencies in the above-mentioned prior art. The present invention addresses the problems that existing COFs require additional doping, a single target substance for detection, and a complex preparation process in DNA sequence recognition, and provides a COFs material with ultra-thin thickness and an ordered conjugated structure, denoted as TBA-COF. The material uses triphenylamine units as monomer raw materials and is prepared by Schiff base reaction. It can be a single-layer or multi-layer nanosheet with an ultra-thin thickness and an ordered conjugated structure. It has a high 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 is used to achieve recognition of DNA of different sequences. In addition, its recognition process is simple, and different DNA sequences can be distinguished by simply measuring the degree of fluorescence quenching, which improves the application capability, simplifies the preparation process, and has broad research and application prospects.

[0007] To achieve the above objectives, the present invention adopts the following technical solutions: In a first aspect, the present invention provides an application of a covalent organic framework material based on triphenylamine units in DNA sequence recognition, wherein the preparation method of the covalent organic framework material comprises the following steps: adding a compound having the chemical structure shown in Formula I below as a monomer raw material to a solvent, and ultrasonically treating the mixture until clarified; then adding an acetic acid solution, mixing uniformly, and degassing; sealing the mixture after degassing, heating the mixture to perform a Schiff base reaction, collecting the product after the reaction, washing, and drying the product; adding anhydrous ethanol to the obtained product, ultrasonically stripping the product, and redispersing the obtained covalent organic framework material in water to obtain a dispersion of the covalent organic framework material based on triphenylamine units; the specific method for applying the covalent organic framework material based on triphenylamine units in DNA sequence recognition is as follows: mixing the dispersion of the covalent organic framework material based on triphenylamine units with DNA to be identified of different sequences labeled with fluorescent molecules, diluting the mixture with a buffer solution, incubating the mixture, and detecting the fluorescence spectrum of the solution, wherein the sequences of the DNA to be identified are at least two, and the application is for the diagnosis and treatment of non-diseases;

[0008] ;

[0009] Ⅰ;

[0010] Wherein, R is an amino group or an aldehyde group.

[0011] Preferably, the monomer raw material is at least one of tris(4-aminophenyl)amine and tris(4-benzoyl)amine.

[0012] Preferably, the monomer raw material further comprises one of 1,3,5-tris(4-aminophenyl)benzene and 1,3,5-tris(p-formylphenyl)benzene.

[0013] Preferably, the solvent is o-dichlorobenzene and ethanol or a mixture of both.

[0014] Preferably, the method for preparing the covalent organic framework material comprises the following steps:

[0015] One or two of tris(4-aminophenyl)amine and tris(4-benzoyl)amine are mixed with one of 1,3,5-tris(4-aminophenyl)benzene and 1,3,5-tris(p-formylphenyl)benzene, added to a solvent, and ultrasonically treated until clear; 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. The obtained product is added to anhydrous ethanol, ultrasonically exfoliated, and redispersed in water to obtain a dispersion of the covalent organic framework material based on triphenylamine units.

[0016] Preferably, the method for preparing the covalent organic framework material comprises the following steps:

[0017] 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 and added to 3-12 mL of a solvent consisting of o-dichlorobenzene and ethanol in a volume ratio of 4:1. The mixture was ultrasonically treated for 2-10 minutes to obtain a clear solution. 120-480 mL of 1.5-6 mol / L acetic acid solution was then added and mixed thoroughly. The mixture was then heated at 77 The mixture was frozen in liquid nitrogen at 4000 K and degassed by 2-6 freeze-thaw cycles; after degassed, the mixture was sealed and reacted at 80-120°C for 24-96 hours. After the reaction, the product was collected, washed with tetrahydrofuran, and dried; 0.5-2 mg of the obtained powder product was added to 15-60 mL of anhydrous ethanol, and ultrasonically stripped at 50-220 W for 3-12 hours to obtain an ethanol dispersion of the covalent organic framework material based on triphenylamine units; the ethanol dispersion of the covalent organic framework material was allowed to settle at room temperature for 12 hours, the bottom precipitate was discarded, the upper suspension was centrifuged at 7000-15000 rpm for 3-15 minutes, the upper ethanol was discarded, and the obtained covalent organic framework material was redispersed in deionized water to obtain a dispersion of the covalent organic framework nanoprobe based on triphenylamine units.

[0018] Preferably, the concentration of the covalent organic framework material in the dispersion of the covalent organic framework nanoprobes is 30-40 mg / mL.

[0019] Preferably, the application method is:

[0020] 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. 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.

[0021] 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 fluorescent dye, Alexa series fluorescent dye, Atto series fluorescent dye.

[0022] In a second aspect, the present invention provides a DNA sequence recognition kit, which comprises the covalent organic framework material based on triphenylamine units as described above.

[0023] The beneficial effects of the present invention are:

[0024] 1. The present invention provides a covalent organic framework material based on triphenylamine units for use in DNA sequence recognition. The covalent organic framework material (abbreviated as TBA-COF) of the present invention uses molecules containing triphenylamine units as monomer raw materials. Due to the non-planar structure of the triphenylamine units themselves, the synthesized TBA-COF sheets have a certain three-dimensional structure and weak interlayer p-p forces. Therefore, when used, they are easily exfoliated into ultrathin nanosheets, increasing the effective binding area between TBA-COF and the DNA sequence to be detected, amplifying the difference in the interaction force between TBA-COF and different DNA sequences, and facilitating the differentiation of DNA sequences of different sequences.

[0025] 2. The triphenylamine-based TBA-COF provided by the present invention has an ordered conjugated structure. Once fluorescently labeled DNA is adsorbed on the TBA-COF surface, its fluorescence is efficiently quenched, facilitating the detection of TBA-COF's adsorption capacity for DNA of different sequences, thereby improving its application capabilities. The TBA-COF has different adsorption capacities for different bases. Specifically, it has a strong adsorption capacity for thymine (T) and a decreasing adsorption capacity for adenine (A) and cytosine (C). This selective adsorption property enables DNA sequence recognition.

[0026] 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 require loading materials such as rare earth metal ions, metal nanoparticles, graphene, DNA complementary sequences, and aptamers. It can recognize DNA of different sequences using only TBA-COF, thus simplifying the preparation process. In addition, during application, different DNA sequences can be distinguished simply by measuring the degree of fluorescence quenching, which improves the application capability and has broad research and application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Schematic diagram of the synthesis route of TBA-COF in Example 1;

[0028] Figure 2 is a TEM image of the TBA-COF nanosheets of Example 1;

[0029] Figure 3 is an AFM image of the TBA-COF nanosheets of Example 1;

[0030] Figure 4 is the XRD pattern of the TBA-COF nanosheets of Example 1;

[0031] Figure 5 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;

[0032] Figure 6 is the comparison result of the F0 / F values ​​of the three DNA sequences in Example 1, where F0 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;

[0033] Figure 7 is the comparison result of the F0 / F values ​​of the three DNA sequences in Example 2, where F0 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;

[0034] Figure 8 is the comparison result of the F0 / F values ​​of the three DNA sequences in Example 4, where F0 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;

[0035] Figure 9 is the comparison result of the F0 / F values ​​of the three DNA sequences in Example 6, where F0 is the fluorescence intensity of the three DNA sequences (50 nM) at 565 nm, and 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, with an excitation wavelength of 550 nm;

[0036] Figure 10 The initial fluorescence emission spectra of the three DNA sequences (at a 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;

[0037] Figure 11 This is the comparison result of the F0 / F values ​​of the three DNA sequences in the comparative example, where F0 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

[0038] The present invention is further described in detail below with reference to the embodiments so that those skilled in the art can implement the invention with reference to the description.

[0039] It should be understood that terms such as “having”, “including” and “comprising” used herein do not preclude the existence or addition of one or more other elements or combinations thereof.

[0040] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Materials and reagents used in the following examples are commercially available unless otherwise specified. In the following examples, where specific conditions are not specified, the experiments were conducted under conventional conditions or those recommended by the manufacturer. Reagents and instruments used, where the manufacturer is not specified, are commercially available conventional products. Example 1

[0041] (1) A method for preparing a covalent organic framework material based on triphenylamine units, comprising the following steps:

[0042] To a reaction tube, 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 (4:1 by volume) were added. After sonication for 5 minutes, a clear solution was obtained, to which 240 mL of 3 mol / L acetic acid solution was added. After mixing, the reaction tube was frozen in liquid nitrogen at 77 K and degassed by three freeze-thaw cycles. After degassing, the tube was sealed and heated at 120°C for 72 hours. After the reaction, the product was collected, washed with tetrahydrofuran, and vacuum-dried to constant weight to obtain a powder product (denoted as TBA-COF powder).

[0043] 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 material based on triphenylamine units) were resuspended in the same volume (30 mL) of deionized water to obtain a dispersion of covalent organic framework material based on triphenylamine units, recorded as TBA-COF nanosheet dispersion, with a concentration of 33 mg / mL.

[0044] (2) Application of TBA-COF nanosheet dispersion to the recognition of three different DNA sequences:

[0045] The three DNA sequences are shown in Table 1 below:

[0046] Table 1

[0047] ;

[0048] A TBA-COF nanosheet dispersion (50 mL, 33 mg / mL) was mixed with each of the three Texas Red-labeled DNA sequences (2.5 mL, 10 mM) and diluted to 500 mL with TE buffer. The final concentration of the TBA-COF nanosheets was 3.3 mg / mL, and the final concentration of the Texas Red-labeled DNA sequence was 50 nM. After incubation for 20 minutes, the fluorescence spectrum of the solution was measured at an excitation wavelength of 590 nm. The fluorescence intensity at 612 nm (i.e., the fluorescence intensity after quenching) was obtained and recorded as F. Simultaneously, the fluorescence spectrum of the three DNA sequences (50 nM) in the absence of TBA-COF nanosheets was measured, and the fluorescence intensity at 612 nm (i.e., the initial fluorescence intensity) was obtained and recorded as F0.

[0049] (3) Experimental results:

[0050] The structures of tris(4-aminophenyl)amine, tris(4-benzoyl)amine and the product TBA-COF are as follows Figure 1 After ultrasonic peeling in anhydrous ethanol, the ultrathin thickness and ultrasmall pores of the nanosheets can be seen from the TEM image ( Figure 2 ). AFM images show that the thickness of TBA-COF nanosheets 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 of the ionized carbonyl group, demonstrating the ordered crystal structure of TBA-COF.

[0051] 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 different degrees of fluorescence quenching for the three DNA sequences AAAAAA (A6), TTTTTT (T6) and CCCCCC (C6) ( 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: F0 / F, and used for comparison, where F0 is the initial 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 nanosheet dispersion (concentration of 3.3 mg / mL) for 20 minutes. Figure 6 The results clearly show that the fluorescence quenching degree of the T6 sequence is the greatest, followed by the A6 sequence, and the C6 sequence has the least fluorescence quenching degree, which confirms that TBA-COF has a recognition effect on different DNA sequences. Example 2

[0052] The heating reaction temperature in Example 1 was changed to 80° C., the reaction time was changed to 96 hours, and the concentration of TBA-COF nanosheets dispersed in water was changed to 40 mg / mL. The rest of the preparation process and the application process in DNA sequence recognition were the same as in Example 1.

[0053] Figure 7 From the test results, it can be seen that the TBA-COF of this embodiment can still recognize three different DNA sequences: AAAAAA (A6), TTTTTT (T6) and CCCCCC (C6). Example 3

[0054] The heating reaction temperature in Example 1 was changed to 120° C., the reaction time was changed to 24 hours, and the concentration of TBA-COF nanosheets dispersed in water was changed to 30 mg / mL. The rest of the preparation process and the application process in DNA sequence recognition were the same as in Example 1.

[0055] After testing, the TBA-COF of this example can still recognize three different DNA sequences: AAAAAA (A6), TTTTTT (T6) and CCCCCC (C6). Example 4

[0056] The monomer raw materials were replaced with 20 mg of tris(4-aminophenyl)amine and 26.9 mg of 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 in Example 1.

[0057] Figure 8 From the test results, it can be seen that COF-1 of this embodiment can still recognize three different DNA sequences: AAAAAA (A6), TTTTTT (T6) and CCCCCC (C6). Example 5

[0058] The monomer raw materials were replaced with 24 mg of 1,3,5-tris(4-aminophenyl)benzene and 20 mg of tris(4-benzoyl)amine. The rest of the preparation process and the application process in DNA sequence recognition were the same as in Example 1.

[0059] After testing, the COF of this embodiment can still recognize three different DNA sequences: AAAAAA (A6), TTTTTT (T6) and CCCCCC (C6). Example 6

[0060] 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.

[0061] Figure 9 From the test results, it can be seen that the TBA-COF of this embodiment can still recognize three different DNA sequences: AAAAAA (A6), TTTTTT (T6) and CCCCCC (C6). Comparative Example

[0062] The monomer raw materials were replaced with 24 mg of 1,3,5-tris(4-aminophenyl)benzene and 26.9 mg of 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 in Example 1.

[0063] Figure 10 and Figure 11 As shown in the test results, the difference in the fluorescence quenching degree of the COF-2 material of the comparative example for three different DNA sequences AAAAAA (A6), TTTTTT (T6) and CCCCCC (C6) is significantly worse than that of Examples 1 to 6 of the present application, and it is impossible to achieve effective distinction of the above three DNA sequences 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 COF-2 layers, making it difficult to be peeled off. Therefore, the exposed effective sites of the COF-2 nanosheets are far less than those of TBA-COF and COF-1, and they cannot fully contact the DNA sequences, and cannot effectively distinguish the DNA sequences.

[0064] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the description and implementation methods. 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 base recognition, characterized in that: The preparation method of the covalent organic framework material comprises the following steps: adding a monomer raw material to a solvent, and ultrasonically treating it until it is clear; then adding an acetic acid solution, mixing it evenly, and degassing it; after degassing, sealing it, heating it to carry out a Schiff base reaction, collecting the product after the reaction is completed, washing it, and drying it; adding anhydrous ethanol to the obtained product, ultrasonically peeling it, and redispersing the obtained covalent organic framework material in water to obtain a dispersion of the covalent organic framework material nanosheet based on triphenylamine units; the specific method of applying the covalent organic framework material based on triphenylamine units to base recognition is as follows: mixing the dispersion of the covalent organic framework material nanosheet based on triphenylamine units with different bases to be recognized marked with fluorescent molecules, diluting it with a buffer solution, incubating it, and detecting the fluorescence spectrum of the solution, wherein the bases to be recognized are at least two, and the application is for the purpose of non-disease diagnosis and treatment; The monomer raw material is one of the following combinations: a mixture of tris(4-aminophenyl)amine and tris(4-benzoyl)amine, or a mixture of tris(4-aminophenyl)amine and 1,3,5-tris(p-formylphenyl)benzene, or a mixture of 1,3,5-tris(4-aminophenyl)benzene and tris(4-benzoyl)amine; When used, the covalent organic framework material has different adsorption capacities for different bases, thereby enabling recognition of the bases to be identified. The adsorption capacity of the covalent organic framework material for thymine, adenine and cytosine decreases in sequence.

2. The use of the covalent organic framework material based on triphenylamine units in base recognition according to claim 1, characterized in that: The solvent is o-dichlorobenzene and ethanol or a mixture of both.

3. The use of the covalent organic framework material based on triphenylamine units in base recognition according to claim 2, characterized in that: The preparation method of the covalent organic framework material comprises the following steps: The monomer raw material is added to a solvent and ultrasonically treated until it is clear; 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. The obtained product is added to anhydrous ethanol, ultrasonically exfoliated, and redispersed in water to obtain a dispersion of the triphenylamine unit-based covalent organic framework material nanosheets.

4. The use of the covalent organic framework material based on triphenylamine units in base recognition according to claim 3, characterized in that: The preparation method of the covalent organic framework material comprises the following steps: The monomer raw material 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, and then 120-480 mL of an acetic acid solution with a concentration of 1.5-6 mol / L is added. After mixing evenly, the solution is frozen in liquid nitrogen at 77 K and degassed by 2-6 freeze-thaw cycles; after degassed, the solution 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; 0.5-2 mg of the obtained powder product is added to 15-60 mL of anhydrous ethanol, and ultrasonically stripped at 50-220W for 3-12 hours to obtain an ethanol dispersion of the covalent organic framework material based on the triphenylamine unit; after the ethanol dispersion of the covalent organic framework material is allowed to stand at room temperature for 12 hours, the bottom precipitate is discarded, and the upper suspension is centrifuged at 7000-15000 rpm for 3-15 min, discard the upper ethanol, and redisperse the obtained covalent organic framework material in deionized water to obtain a dispersion of the covalent organic framework material nanosheets based on triphenylamine units.

5. The use of the covalent organic framework material based on triphenylamine units in base recognition according to claim 4, characterized in that: The concentration of the covalent organic framework material in the dispersion of the covalent organic framework nanomaterial nanosheets is 30-40 mg / mL.

6. Use of the covalent organic framework material based on triphenylamine units in base recognition according to any one of claims 1 to 5, characterized in that: The application method is: The dispersion of triphenylamine-based covalent organic framework material nanosheets is diluted with TE buffer solution to obtain a working solution, which is added to a sample solution of a base to be identified that is labeled with a fluorescent substance and incubated. The fluorescence intensity is detected and recorded as F. The dispersion of triphenylamine-based covalent organic framework material nanosheets in the above step is replaced with the same volume of TE buffer to obtain the fluorescence intensity F0, and the base is identified by the value of F0 / F.

7. The use according to claim 6, 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.

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