COFs (covalent organic frameworks)-based nano material, preparation method and hybridization chain reaction p53 gene fluorescence-SERS (Surface Enhanced Raman Scattering) dual-mode detection method

Abstract

The invention relates to the technical field of detection, in particular to a COFs (covalent organic frameworks)-based nano material, a preparation method and a hybridization chain reaction p53 gene fluorescence-SERS (Surface Enhanced Raman Scattering) dual-mode detection method. The nano material is prepared from the following raw materials: Au nano particles and COFs (Covalent Organic Frameworks); and the material is prepared in a self-assembly manner. The p53 gene fluorescence-SERS dual-mode detection method based on the nano material and the hybridization chain reaction, provided by the invention, has the following beneficial effects that (1) the hybridization chain reaction is an isothermal enzyme-free nucleic acid signal amplification technology, a signal far stronger than that of a non-hybridization chain reaction can be initiated, a signal change range is widened, and the p53 gene detection sensitivity is improved; (2) the fluorescence-SERS dual-path signal output combines respective advantages and makes up respective deficiencies, the fluorescence-SERS dual-path signal output complements each other, and the reliability and accuracy of analysis and detection are improved; and (3) by properly designing and changing the H-Cy5 sequence, the method can be theoretically expanded to detection of other cancer markers and shows good universality.

Description

Technical Field

[0001] The present invention relates to the field of detection technologies, and in particular, to a fluorescence-SERS dual-mode detection method for p53 gene based on COF-based nanomaterials, a preparation method, and hybridization chain reaction. Background Art

[0002] Cancer is a disease caused by the loss of normal growth regulation and excessive proliferation of body cells. It is the leading cause of death globally and one of the diseases with the highest fatality rate. Ensuring accurate cancer diagnosis is crucial for improving patient prognosis and reducing cancer mortality. Cancer diagnosis methods usually include in vivo imaging analysis, in vitro histopathological analysis, and cancer biomarker detection. Among them, the quantitative determination of cancer-specific biomarkers is of great significance for tumor discovery and typing, cancer progression assessment, and treatment effect evaluation, and also plays a key role in disease management and patient quality of life. However, the concentrations of many cancer biomarkers are extremely low under physiological or pathological conditions and are easily interfered by complex biological matrices. Therefore, the sensitive and accurate analysis of trace cancer biomarkers still poses a challenge.

[0003] Nucleic acid signal amplification technology has been widely used to improve the detection sensitivity of ultra-trace cancer markers (such as DNA, RNA, proteins, etc.). Polymerase chain reaction is one of the earliest proposed in vitro nucleic acid amplification technologies, which can amplify target nucleic acids with ultra-high efficiency and even achieve the detection of cancer markers at the single-molecule level. However, the implementation of polymerase chain reaction technology depends on strict temperature cycling control of the equipment, which is time-consuming and costly, limiting its application in the field of bioanalysis detection. To overcome the deficiencies of polymerase chain reaction technology, a more general isothermal nucleic acid signal amplification technology has emerged. According to different key factors for realizing signal amplification, it can be divided into enzyme-involved nucleic acid signal amplification technology and enzyme-free nucleic acid signal amplification technology. The former, such as nicking enzyme-assisted signal amplification, exonuclease-assisted signal amplification, rolling circle amplification, and strand displacement amplification, have been successfully applied in improving the detection sensitivity of cancer markers. However, they have some defects to a certain extent, such as profound reaction mechanisms, longer reaction times, enzyme activity being easily affected by the environment, highly relying on the characteristics of enzymes, making their reaction conditions relatively harsh and the reaction system complex, which hinders their application in complex biological samples. Enzyme-free nucleic acid signal amplification technologies mainly include catalytic hairpin assembly strategy and hybridization chain reaction strategy, which occur depending on toehold-mediated strand displacement reaction. The core principle is the competition hybridization between different nucleic acid molecules and complementary strands to obtain a more thermodynamically stable double strand. This process is a kinetically controlled reaction and does not require any enzyme participation. The concept of hybridization chain reaction was first proposed by Dirks and Pierce in 2004. It generally consists of a single-stranded DNA and two metastable hairpin DNAs. The single-stranded DNA serves as an initiator to trigger the cascade hybridization event between the two hairpin DNAs, inducing the generation of a nicked double helix long-chain DNA with dozens to hundreds of repeating units until the hairpins are exhausted. Hybridization chain reaction is simple to operate, efficient, has mild reaction conditions, and low cost. More importantly, hybridization chain reaction can achieve a signal amplification effect with a ratio of initiator to signal of 1:n, and is expected to significantly improve the detection sensitivity when used for cancer marker detection.

[0004] In addition to sensitivity, accuracy is also a key factor in evaluating the detection performance of cancer markers, and the dual-mode sensing strategy provides a new idea for solving the problems of improving detection reliability and accuracy. Compared with sensors based on single-signal output, dual-mode sensors provide two optional signal sensing modes simultaneously, which can select the suitable mode according to different actual application scenarios and have stronger detection flexibility. In addition, dual-mode sensors have two signals from independent transmission paths, which can be mutually verified to ensure the reliability and accuracy of the detection results. So far, a variety of dual-mode sensing strategies for cancer marker detection have been reported, such as electrochemical-electrochemiluminescence type, photoelectrochemical-colorimetric type, fluorescence-colorimetric type, surface-enhanced Raman scattering-colorimetric type, and fluorescence-surface-enhanced Raman scattering (SERS) type, etc. Among them, the fluorescence-SERS dual-mode sensor combines the advantages of convenient and fast fluorescence detection, easy signal reading, good reproducibility, good stability, and fingerprint recognition, multiplexing, and high sensitivity of SERS detection, which has attracted great attention. The fluorescence-SERS dual-mode sensing strategy is expected to obtain multi-scale information for precision medicine cancer diagnosis.

[0005] Covalent organic frameworks (COFs) are a new type of crystalline material with periodically ordered molecular arrangement formed by the topological directional covalent connection of different types of building blocks. COFs have excellent properties such as simple design, clear composition, diverse main chain structures, large specific surface area, adjustable pore size, high stability, simple functionalization, easy surface modification, and strong loading capacity, and have achieved remarkable development in the fields of gas storage, chromatographic separation, catalysis, environmental remediation, and biosensing. It is worth noting that, on the one hand, the long-range ordered structure of COFs endows them with strong broad-spectrum light absorption ability, which is expected to be used as a receptor for capturing and quenching dye fluorescence; on the other hand, the large specific surface area of COFs makes them an ideal carrier for anchoring metal nanoparticles. While preventing the aggregation of MNPs, it also makes them approach each other to form high-density "hot spots" and improve SERS performance. Based on the above excellent properties, COFs can be easily involved in the design of fluorescence-SERS dual-mode probes for bioanalysis.

[0006] Introducing COFs into the hybridization chain reaction-assisted fluorescence-SERS dual-mode sensor will bring new opportunities for the sensitive and accurate detection of cancer biomarkers. Summary of the Invention

[0007] To solve the above technical problems, the present invention provides a COF-based nanomaterial, and the raw materials for preparing the nanomaterial include: Au nanoparticles, COFs; and it is prepared by a self-assembly method.

[0008] As an embodiment of the present invention, the COFs are prepared by the reaction of trifunctional aromatic aldehyde monomers and linear diamine monomers.

[0009] As an embodiment of the present invention, the trifunctional aromatic aldehyde monomer is selected from one of 1,3,5-tris(4-formylphenyl)benzene, trimellitic anhydride benzene, tris(2-formylvinyl)benzene, tris(4-formylphenylethynyl)benzene, tris(formylpyridyl)benzene.

[0010] As an embodiment of the present invention, the linear diamine monomer is selected from one of p-phenylenediamine, biphenyl diamine, ethylenediamine, styrene diamine, pyrene diamine, naphthalene diamine, diaminotriazine, p-aminobenzoic acid, 2,5-diamino-p-xylene.

[0011] As an embodiment of the present invention, a method for preparing a kind of COFs-based nanomaterial, the steps are as follows: (1) Preparation of Au NPs: Add chloroauric acid solution and ultrapure water into a 100 mL three-necked flask connected with a reflux condenser, and heat the solution to boiling; subsequently, quickly add trisodium citrate solution into the flask, and heat the mixture under reflux for 25 - 35 min until the sol color turns wine red to obtain Au NPs; finally, naturally cool the prepared Au NPs sol to room temperature, dispense it with an ep tube, and store it in a 4°C refrigerator for later use; (2) Preparation of COFs: First, dissolve the trifunctional aromatic aldehyde monomer and the linear diamine monomer in acetonitrile; subsequently, add acetic acid, and magnetically stir at room temperature for 24 h; finally, wash the obtained product with acetonitrile, tetrahydrofuran and absolute ethanol twice respectively, and dry it in a vacuum drying oven to obtain COFs; (3) Preparation of COFs@PEI@Au NPs: First, ultrasonically disperse COFs in a mixed solution of water and absolute ethanol; then, add the surface-functionalized carrier solution, ultrasonically treat it and then magnetically stir; finally, wash the obtained product with ultrapure water and dry it in vacuum to obtain the surface-functionalized carrier-modified COFs; (4) Preparation of the nanomaterial by electrostatic self-assembly method: Add AuNPs sol into the surface-functionalized carrier-modified COFs dispersion, magnetically stir and then ultrasonically treat; wash the obtained product with ultrapure water, redisperse it in 5 mL of ultrapure water to obtain the nanomaterial storage solution, and store it in a 4°C refrigerator for later use.

[0012] As an embodiment of the present invention, the surface-functionalized carrier is selected from one of polyethyleneimine, polylysine, chitosan, PAMAM dendrimer, poly(dimethyldiallylammonium chloride), polyquaternium-10 / PQ-10, poly(vinyl alcohol)-poly(methacrylate) copolymer.

[0013] As an embodiment of the present invention, a method for preparing a COFs@PEI@Au NPs composite, the method comprising the following steps: (1) Preparation of Au NPs: First, add chloroauric acid solution (0.1 M, 0.1 mL) and ultrapure water (40 mL) into a 100 mL three-necked flask equipped with a reflux condenser, and heat the solution to boiling; subsequently, quickly add trisodium citrate solution (0.01 g / mL, 0.6 mL) into the flask, and heat the mixture under reflux for 25 - 35 min until the sol color turns wine red to obtain Au NPs; finally, naturally cool the prepared Au NPs sol to room temperature, aliquot it with an ep tube, and store it in a 4°C refrigerator for later use.

[0014] (2) Preparation of COFs: First, dissolve TPB (0.0141 g) and TP (0.008 g) in 20 mL of acetonitrile; subsequently, add acetic acid (0.5, 1, 2, 3, 4 mL), and magnetically stir at room temperature for 24 h; finally, wash the obtained product twice with acetonitrile, tetrahydrofuran, and absolute ethanol respectively, and dry it in a vacuum drying oven for 12 h to obtain COFs.

[0015] Preferably, the preferred usage amount of the acetic acid is 1 mL.

[0016] (3) Preparation of COFs@PEI@Au NPs: First, ultrasonically disperse COFs (12 mg) in a mixture of 20 mL of water and 10 mL of absolute ethanol; then, add PEI solution (15 mg / mL, 2 mL), ultrasonically treat for 30 min and then magnetically stir for 30 min; finally, wash the obtained product three times with ultrapure water, and dry it in a vacuum drying oven for 12 h to obtain COFs@PEI.

[0017] Subsequently, prepare COFs@PEI@Au NPs by electrostatic self-assembly method. First, add Au NPs sol (12 mL) to the COFs@PEI dispersion (1.6 mg / mL, 5 mL), magnetically stir for 10 min and then ultrasonically treat for 20 min; then wash the obtained product three times with ultrapure water, and redisperse it in 5 mL of ultrapure water to obtain a COFs@PEI@Au NPs storage solution, and store it in a 4°C refrigerator for later use.

[0018] The preparation process of COFs@PEI@Au NPs with different Au NPs loadings is the same as above, the difference being that the usage amount of Au NPs (0, 4, 8, 12, 16, 20 mL) is different during the preparation process. Preferably, the usage amount of the Au NPs is 12 mL.

[0019] As an embodiment of the present invention, the application of the COFs-based nanomaterial is applied to the fluorescence-SERS dual-mode detection of p53 gene in hybridization chain reaction.

[0020] As an embodiment of the present invention, the steps of the detection method for the fluorescence-SERS dual-mode of p53 gene in hybridization chain reaction are as follows: (1) Add H1-Cy5, H2-Cy5 and p53 gene standard solutions with different concentrations into Tris-HCl buffer solution; (2) After incubating at 37 °C for a period of time, add the dispersion of the nanomaterial into the system and continue to incubate at a certain temperature for a period of time; (3) Measure the fluorescence intensity at 667 nm and the SERS intensity at 555 cm −1 respectively for each p53 gene concentration using a fluorescence spectrophotometer and a Raman spectrometer.

[0021] A fluorescence-SERS dual-mode detection method for p53 gene based on COFs@PEI@Au NPs and hybridization chain reaction, comprising the following steps: (1) Add H1-Cy5 (0, 25, 50, 75 or 100 nM, 1 μL), H2-Cy5 (0, 25, 50, 75 or 100 nM, 1 μL) and p53 gene standard solutions with different concentrations (10 μL) into Tris-HCl buffer solution (20 mM, 75.5 μL, pH 6.5, 7, 7.5, 8, 8.5, 9 or 9.5, containing 10 mM MgCl2); (2) After incubating at 37 °C for 0, 10, 20, 30, 40, 50, 60, 80, 100 or 120 min, add the COFs@PEI@Au NPs dispersion (50, 100, 200, 300, 400, 600, 800, 1000 or 1200 μg / mL, 12.5 μL) into the system and continue to incubate at 5, 15, 25, 30 or 45 °C for 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or 60 min; (3) Measure the fluorescence intensity at 667 nm (λ ex = 610, 620, 630, 635, 640 or 650 nm) and the SERS intensity at 555 cm −1 respectively for each p53 gene concentration using a fluorescence spectrophotometer and a Raman spectrometer (λ ex = 532, 633 or 780 nm).

[0022] As an embodiment of the present invention, the concentration ratio of H1-Cy5 to H2-Cy5 is 1:1; H1-Cy5 is 50 nM; H2-Cy5 is 50 nM; Tris-HCl buffer has a pH of 8.0; incubation is carried out at 37 °C for 40 min; the COFs@PEI@Au NPs dispersion is 200 μg / mL; incubation is continued at 25 °C for 25 min; fluorescence λ ex = 630 nm; SERS λ ex = 780 nm.

[0023] A fluorescence-SERS dual-mode detection method for p53 gene based on COFs@PEI@Au NPs and hybridization chain reaction, wherein: The sequence of H1-Cy5 is 5’-CACTGGAAGACTCTTAACGGAGTCTTCCAGTGTGATGA-Cy5-3’ The sequence of H2-Cy5 is 5’-Cy5-CGTTAAGAGTCTTCCAGTGTCATCACACTGGAAGACTC-3’ The sequence of p53 gene is 5’-TCATCACACTGGAAGACTC-3’.

[0024] Among them, H1-Cy5 and H2-Cy5 are DNA hairpin probe 1 and DNA hairpin probe 2 respectively; when they do not encounter the p53 gene target, they are in a hairpin self-closed state; once the p53 gene sequence is introduced, H1 is opened, triggering H2, and then in turn opening H1, thus initiating a chain self-assembly reaction.

[0025] As an embodiment of the present invention, the COF-based nanomaterial is applied to the field of medical testing technology.

[0026] As an embodiment of the present invention, the COF-based nanomaterial is applied to the field of spectroscopy technology.

[0027] Adopting the above technical solution, the present invention has the following beneficial effects: Compared with the prior art, the present invention provides a fluorescence-SERS dual-mode detection method for p53 gene based on nanomaterials and hybridization chain reaction, which has the following beneficial effects: (1) Hybridization chain reaction is an isothermal enzyme-free nucleic acid signal amplification technology, which can trigger signals and signal change ranges far stronger than those of non-hybridization chain reaction, improving the detection sensitivity of p53 gene; (2) The fluorescence-SERS dual-path signal output combines the respective advantages, while making up for the respective deficiencies, complementing each other, and improving the reliability and accuracy of analysis and detection; (3) By appropriately designing and changing the H-Cy5 sequence, this method can theoretically be extended to the detection of other cancer markers, showing good generality. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings required for the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0029] Figure 1 Schematic diagram of the preparation of COFs@PEI@Au NPs in Example 1.

[0030] Figure 2 Characterization of the Au NPs synthesized in Example 1. Among them, Figure A is a transmission electron microscopy image, Figure B is a hydrodynamic diameter distribution diagram, and Figure C is an ultraviolet-visible absorption spectrum.

[0031] Figure 3 Characterization of the COFs synthesized in Example 1. Among them, Figure A is a transmission electron microscopy image (the inset is a scanning electron microscopy image), Figure B is a hydrodynamic diameter distribution diagram, Figure C is an X-ray diffraction pattern, Figure D is an infrared spectrum of COFs and its synthetic components, and Figures E and F are the X-ray photoelectron total energy spectrum and the high-resolution X-ray photoelectron spectrum of N (1s), respectively.

[0032] Figure 4 Characterization of the COFs@PEI@Au NPs synthesized in Example 1. Among them, Figure A is a high-angle annular dark-field scanning transmission electron microscopy image of COFs@PEI@Au NPs and its mapping images of energy-dispersive X-ray spectroscopy of C, N, O, and Au elements, Figure B is a distribution diagram of the hydrodynamic diameters of COFs@PEI@Au NPs, COFs@PEI, and COFs, Figure C is the zeta potential of COFs@PEI@Au NPs, Au NPs, COFs@PEI, and COFs, Figure D is the small-angle (left) and wide-angle (right) powder X-ray diffraction patterns of COFs@PEI@Au NPs, COFs@PEI, and COFs, and Figures E and F are the N2 adsorption / desorption isotherm diagram and pore size distribution diagram of COFs@PEI@Au NPs, respectively.

[0033] Figure 5Comparison of signal changes caused by hybridization chain reaction and non - hybridization chain reaction. Among them, Figure A is the fluorescence emission spectrogram in the hybridization chain reaction (left) and non - hybridization chain reaction (right) systems. Figure B is the Raman spectrogram in the hybridization chain reaction (left) and non - hybridization chain reaction (right) systems. Figure C is the agarose gel electrophoresis diagram of each component in the hybridization chain reaction system under various combinations. Figure D is a schematic diagram for comparing the hybridization chain reaction and non - hybridization chain reaction, and Figure E is a comparison of the corresponding fluorescence and SERS signal changes.

[0034] Figure 6 Optimization comparison diagram of the best fluorescence excitation wavelength and the best SERS excitation wavelength.

[0035] Figure 7 Optimization comparison diagram of the dosage of Au NPs during the preparation of COFs@PEI@Au NPs and optimization comparison diagram of the detection conditions for p53 gene.

[0036] Figure 8 Schematic diagram of using combined COFs@PEI@Au NPs and hybridization chain reaction for fluorescence - SERS dual - mode detection of p53 gene.

[0037] Figure 9 Performance diagram of using combined COFs@PEI@Au NPs and hybridization chain reaction for fluorescence - SERS dual - mode detection of p53 gene.

[0038] Figure 10 Performance diagram of using combined COFs@PEI@Au NPs and non - hybridization chain reaction for fluorescence - SERS dual - mode detection of p53 gene.

[0039] Figure 11 Specificity of using combined COFs@PEI@Au NPs and hybridization chain reaction for p53 gene detection.

[0040] Figure 12 Uniformity of the detection platform.

[0041] Figure 13 Reproducibility of the detection platform. Specific implementation method

[0042] The following examples further illustrate the content of the present invention, but should not be construed as a limitation to the present invention. Without departing from the spirit and essence of the present invention, modifications and substitutions made to the methods, steps or conditions of the present invention all fall within the scope of the present invention. If not specifically specified, the technical means used in the examples are conventional means well known to those skilled in the art. Example

[0043] This embodiment provides a method for preparing COFs@PEI@Au NPs composites, which includes the following steps: (1) Preparation of Au NPs: First, add chloroauric acid solution (0.1 M, 0.1 mL) and ultrapure water (40 mL) into a 100 mL three-necked flask equipped with a reflux condenser, and heat the solution to boiling; then, quickly add trisodium citrate solution (0.01 g / mL, 0.6 mL) into the flask, and heat the mixture under reflux for about 30 min until the sol color turns wine red to obtain Au NPs; finally, naturally cool the prepared Au NPs sol to room temperature, aliquot it with ep tubes, and store it in a refrigerator at 4°C for later use.

[0044] (2) Preparation of COFs: First, dissolve TPB (0.0141 g) and TP (0.008 g) in 20 mL of acetonitrile; then, add acetic acid (1 mL), and stir magnetically at room temperature for 24 h; finally, wash the obtained product twice with acetonitrile, tetrahydrofuran, and absolute ethanol respectively, and dry it in a vacuum drying oven for 12 h to obtain COFs.

[0045] (3) Preparation of COFs@PEI@Au NPs: First, disperse COFs (12 mg) ultrasonically in a mixture of 20 mL of water and 10 mL of absolute ethanol; then, add PEI solution (15 mg / mL, 2 mL), ultrasonically treat for 30 min and then stir magnetically for 30 min; finally, wash the obtained product three times with ultrapure water, and dry it in a vacuum drying oven for 12 h to obtain COFs@PEI.

[0046] Subsequently, prepare COFs@PEI@Au NPs by electrostatic self-assembly method. First, add Au NPs sol (12 mL) to the COFs@PEI dispersion (1.6 mg / mL, 5 mL), stir magnetically for 10 min and then ultrasonically treat for 20 min; then wash the obtained product three times with ultrapure water, and redisperse it in 5 mL of ultrapure water to obtain the COFs@PEI@Au NPs storage solution, and store it in a refrigerator at 4°C for later use.

[0047] Optimization of the dosage of Au NPs during the preparation of COFs@PEI@Au NPs Preparation of COFs@PEI@Au NPs with different Au NPs loadings: Except for the different usage amounts of Au NPs (0, 4, 8, 12, 16, 20 mL) during the preparation process, the remaining preparation methods are the same as in Example 1.

[0048] Comparison of the sensing performance of COFs@PEI@Au NPs with different Au NP loadings: The following analysis steps were carried out on the above 6 kinds of COFs@PEI@Au NPs with different Au NP loadings respectively: (1) Add H1-Cy5 (50 nM, 1 μL), H2-Cy5 (50 nM, 1 μL) and p53 gene standard solution (250 pM, 10 μL) into Tris-HCl buffer (20 mM, 75.5 μL, pH 8.0, containing 10 mM MgCl2); (2) After incubating at 37 °C for 40 min, add the COFs@PEI@Au NPs dispersion (200 μg / mL, 12.5 μL) into the system and continue to incubate at 25 °C for 25 min; (3) Measure the fluorescence intensity at 667 nm (λ ex = 635 nm) using a fluorescence spectrophotometer. At the same time, a blank experiment was designed. Except for not adding the p53 gene standard solution, other conditions were the same as above. The amount of Au NPs used in the preparation of COFs@PEI@Au NPs was optimized according to the fluorescence recovery efficiency.

[0049] Figure 7 Figure A is an optimized comparison chart of the amount of Au NPs used in the preparation of COFs@PEI@Au NPs. From Figure 7 Figure A, it can be seen that when the amount of Au NPs used is 12 mL, the best fluorescence recovery efficiency can be obtained. Therefore, the amount of Au NPs used is preferably 12 mL.

[0050] Example 1-1 The difference between this example and Example 1 is that in step (2), the amount of acetic acid is 0.5 mL.

[0051] Example 1-2 The difference between this example and Example 1 is that in step (2), the amount of acetic acid is 2 mL.

[0052] Example 1-3 The difference between this example and Example 1 is that in step (2), the amount of acetic acid is 3 mL.

[0053] Example 1-4 The difference between this example and Example 1 is that in step (2), the amount of acetic acid is 4 mL.

[0054] Example 1-5 The difference between this example and Example 1 is that in step (3), the amount of Au NP sol is 0 mL.

[0055] Example 1-6 The difference between this example and Example 1 is that in step (3), the amount of Au NP sol is 4 mL.

[0056] Examples 1 - 7 The difference between this example and Example 1 lies in that in step (3), the amount of Au NPs sol is 8 mL.

[0057] Examples 1 - 8 The difference between this example and Example 1 lies in that in step (3), the amount of Au NPs sol is 16 mL.

[0058] Examples 1 - 9 The difference between this example and Example 1 lies in that in step (3), the amount of Au NPs sol is 20 mL.

[0059] Among them, Figure 1 is the schematic diagram for the preparation of COFs@PEI@Au NPs in Example 1.

[0060] Figure 2 is the characterization of the Au NPs synthesized in Example 1. Among them, Figure A is the transmission electron microscopy image, Figure B is the hydrodynamic diameter distribution diagram, and Figure C is the ultraviolet - visible absorption spectrum. It can be seen from Figure 2 that the synthesized Au NPs are in a relatively uniform circular shape, with good monodispersity, the particle size is about 29.0 nm, the hydrodynamic diameter is about 43.6 nm, and it has a characteristic plasma peak at 529 nm with a full width at half maximum of about 104 nm.

[0061] Figure 3 is the characterization of the COFs synthesized in Example 1. Among them, Figure A is the transmission electron microscopy image (the inset is the scanning electron microscopy image), Figure B is the hydrodynamic diameter distribution diagram, Figure C is the X - ray diffraction pattern, Figure D is the infrared spectrum of COFs and its synthetic components, and Figures E and F are the X - ray photoelectron total energy spectrum and the high - resolution X - ray photoelectron spectrum of N(1s), respectively. It can be seen from Figure 3 that the average particle size of the synthesized COFs is 481.6 ± 30.0 nm, with good crystallinity, and the - NH2 in the linker TPB and the - CH = O in the linker TP are successfully condensed to form imine.

[0062] Figure 4Characterization of COFs@PEI@Au NPs synthesized in Example 1. Among them, Figure A is the high-angle annular dark-field scanning transmission electron microscopy image of COFs@PEI@Au NPs and the mapping images of energy-dispersive X-ray spectroscopy of C, N, O, and Au elements, Figure B is the hydrodynamic diameter distribution diagram of COFs@PEI@Au NPs, COFs@PEI, and COFs, Figure C is the zeta potential of COFs@PEI@Au NPs, Au NPs, COFs@PEI, and COFs, Figure D is the small-angle (left) and wide-angle (right) powder X-ray diffraction patterns of COFs@PEI@Au NPs, COFs@PEI, and COFs, and Figures E and F are the N2 adsorption / desorption isotherm diagram and pore size distribution diagram of COFs@PEI@Au NPs respectively. It can be seen from Figure 4 the figure that COFs@PEI@Au NPs are positively charged on the surface, a high density of Au NPs is loaded on the surface of COFs@PEI, and the loading of Au NPs does not affect the morphology and crystal structure of COFs@PEI. COFs@PEI@Au NPs have a mesoporous structure, a large specific surface area, and good porosity.

[0063] Comparison of signal changes caused by hybridization chain reaction and non-hybridization chain reaction Preparation of COFs@PEI@Au NPs: The preparation method of COFs@PEI@Au NPs in this example is the same as that in Example 1.

[0064] Signal changes caused by hybridization chain reaction: (1) Add H1-Cy5 (50 nM, 1 μL), H2-Cy5 (50 nM, 1 μL), and p53 gene standard solution (10 μL) to Tris-HCl buffer (20 mM, 75.5 μL, pH 8.0, containing 10 mM MgCl2); (2) After incubating at 37 °C for 40 min, add the COFs@PEI@Au NPs dispersion (200 μg / mL, 12.5 μL) to the system and continue to incubate at 25 °C for 25 min; (3) Measure the fluorescence intensity at 667 nm (λ ex = 635 nm) and the SERS intensity at 555 cm −1 in the reaction system using a fluorescence spectrophotometer and a Raman spectrometer respectively (λ ex = 780 nm). At the same time, a blank experiment was designed, and other conditions were the same as above except that the p53 gene standard solution was not added.

[0065] Signal changes caused by non-hybridization chain reaction: (1) H1-Cy5 (50 nM, 1 μL) and p53 gene standard solution (10 μL) were added to Tris-HCl buffer (20 mM, 76.5 μL, pH 8.0, containing 10 mM MgCl2); (2) After incubation at 37°C for 40 min, COFs@PEI@Au NPs dispersion (200 μg / mL, 12.5 μL) was added to the system and incubated at 25°C for another 25 min; (3) The fluorescence intensity (λ ex = 635 nm) and 555 cm −1 SERS intensity at (λ ex = 780 nm). A blank experiment was also designed. Except for not adding the p53 gene standard solution, all other conditions were the same as above.

[0066] Comparison of signal changes caused by hybridization chain reaction and non-hybridization chain reaction: Calculate the ratio of signal changes caused by hybridization chain reaction to non-hybridization chain reaction.

[0067] Figure 5 Figure 1 is a comparison of signal changes caused by hybridization chain reaction and non-hybridization chain reaction. Figure A is the fluorescence emission spectrum of hybridization chain reaction (left) and non-hybridization chain reaction (right) systems. Figure B is the Raman spectrum of hybridization chain reaction (left) and non-hybridization chain reaction (right) systems. Figure C is the agarose gel electrophoresis of various components in the hybridization chain reaction system under various combinations. Figure D is a schematic diagram comparing hybridization chain reaction and non-hybridization chain reaction, and Figure E is a comparison of the corresponding fluorescence and SERS signal changes. Figure 5 It can be seen that the designed hybridization chain reaction system indeed undergoes p53-induced cascade self-assembly of H1-Cy5 and H2-Cy5, forming a high-molecular-weight, long double-stranded DNA. The fluorescence and SERS signal changes caused by the 1:n long double-stranded DNA structure induced by the hybridization chain reaction are much stronger than those caused by the 1:1 short double-stranded DNA structure induced by the non-hybridization chain reaction, with enhancements of approximately 9.93 times (fluorescence mode) and 8.01 times (SERS mode).

[0068] Optimization of optimal fluorescence excitation wavelength and optimal SERS excitation wavelength Preparation of COFs@PEI@Au NPs: The preparation method of COFs@PEI@Au NPs in this example is the same as that in Example 1.

[0069] Optimization of the best fluorescence excitation wavelength and the best SERS excitation wavelength: (1) Add H1-Cy5 (50 nM, 1 μL), H2-Cy5 (50 nM, 1 μL), and p53 gene standard solution (10 μL) into Tris-HCl buffer (20 mM, 75.5 μL, pH 8.0, containing 10 mM MgCl2); (2) After incubating at 37 °C for 40 min, add the COFs@PEI@Au NPs dispersion (200 μg / mL, 12.5 μL) into the system, and continue to incubate at 25 °C for 25 min; (3) Measure the fluorescence spectra ((λ ex = 610, 620, 630, 635, 640 or 650 nm) and Raman spectra (λ ex = 532, 633 or 780 nm) of the reaction system using a fluorescence spectrophotometer and a Raman spectrometer respectively.

[0070] Figure 6 It is a comparison chart for the optimization of the best fluorescence excitation wavelength and the best SERS excitation wavelength. As can be seen from Figure 6 A, in the range where the excitation wavelength changes from 610 nm to 650 nm, the emission spectrum of Cy5 in the mixed system of COFs@PEI@Au NPs, H-Cy5 and p53 gene shows non-excitation light dependence, and the fluorescence intensity gradually increases. However, the noise interference of the fluorescence spectrophotometer also increases accordingly. In contrast, 635 nm, which can excite relatively high fluorescence intensity and relatively less noise interference, is selected as the excitation wavelength for subsequent fluorescence experiments. As can be seen from Figure 6 B, under the excitation of 532 nm laser, basically no SERS signal of Cy5 can be collected in the mixed system of COFs@PEI@Au NPs and H-Cy5; under the excitation of 633 nm laser, although strong SERS signals of Cy5 are collected in the mixed system of COFs@PEI@Au NPs and H-Cy5, there is also huge fluorescence interference at the same time, and the SERS signal will be submerged by the high and wide fluorescence background; under the excitation of 780 nm laser, clear and relatively high SERS signals of Cy5 are collected in the mixed system of COFs@PEI@Au NPs and H-Cy5, and the fluorescence interference is relatively small. Therefore, 780 nm is selected as the best excitation wavelength for subsequent SERS experiments.

[0071] Optimization of experimental conditions Preparation of COFs@PEI@Au NPs: The preparation method of COFs@PEI@Au NPs in this example is the same as that in Example 1.

[0072] Optimization of experimental conditions: (1) Add H1-Cy5 (0, 25, 50, 75, or 100 nM, 1 μL), H2-Cy5 (0, 25, 50, 75, or 100 nM, 1 μL), and p53 gene standard solution (250 pM, 10 μL) into Tris-HCl buffer (20 mM, 75.5 μL, pH 6.5, 7, 7.5, 8, 8.5, 9, or 9.5, containing 10 mM MgCl2); (2) After incubating at 37 °C for 0, 10, 20, 30, 40, 50, 60, 80, 100, or 120 min, add COFs@PEI@Au NPs dispersion (50, 100, 200, 300, 400, 600, 800, 1000, or 1200 μg / mL, 12.5 μL) into the system, and continue to incubate at 5, 15, 25, 30, or 45 °C for 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or 60 min; (3) Measure the fluorescence intensity at 667 nm (λ ex = 635 nm) using a fluorescence spectrophotometer. At the same time, design a blank experiment, with other conditions the same as above except without adding the p53 gene standard solution. Optimize each experimental condition according to the fluorescence recovery efficiency.

[0073] Figure 7 B-7G is a comparison graph for optimizing the detection conditions of the p53 gene. As can be seen from Figure 7 B-7G, the experimental conditions such as (B) the concentration of COFs@PEI@Au NPs; (C) the concentration ratio of H1-Cy5 to H2-Cy5; (D) the concentrations of H1-Cy5 and H2-Cy5; (E) the hybridization chain reaction time; (F) the pH of the system; and (G) the adsorption time and adsorption temperature are optimized to be: (B) 200 μg / mL; (C) 1:1; (D) 50 nM; (E) 40 min; (F) 8.0; and (G) 25 min and 25 °C respectively.

[0074] Combined use of COFs@PEI@Au NPs and hybridization chain reaction for fluorescence-SERS dual-mode detection of p53 gene Preparation of COFs@PEI@Au NPs: The preparation method of COFs@PEI@Au NPs in this example is the same as that in Example 1.

[0075] Combined COFs@PEI@Au NPs and hybridization chain reaction for fluorescence-SERS dual-mode detection of p53 gene: (1) Add H1-Cy5 (50 nM, 1 μL), H2-Cy5 (50 nM, 1 μL) and different concentrations of p53 gene standard solution (10 μL) into Tris-HCl buffer (20 mM, 75.5 μL, pH 8.0, containing 10 mM MgCl2); (2) After incubating at 37 °C for 40 min, add COFs@PEI@Au NPs dispersion (200 μg / mL, 12.5 μL) into the system and continue to incubate at 25 °C for 25 min; (3) Measure the fluorescence intensity at 667 nm (λ ex = 635 nm) and the SERS intensity at 555 cm −1 using a fluorescence spectrophotometer and a Raman spectrometer respectively for each p53 gene concentration (λ ex = 780 nm). Establish a quantitative model based on the relationship between the fluorescence intensity and the SERS intensity with the change of p53 gene concentration for p53 gene concentration prediction.

[0076] Figure 8 Schematic diagram of combined COFs@PEI@Au NPs and hybridization chain reaction for fluorescence-SERS dual-mode detection of p53 gene.

[0077] Figure 9 Performance graph of combined COFs@PEI@Au NPs and hybridization chain reaction for fluorescence-SERS dual-mode detection of p53 gene. It can be seen from Figure 9 that in the fluorescence mode, the linear range for detecting p53 gene by the detection platform is 5 - 250 pM, and the detection limit is 2.65 pM; in the SERS mode, the linear range for detecting p53 gene by the detection platform is 10 - 800 pM, and the detection limit is 6.72 pM.

[0078] Combined COFs@PEI@Au NPs and non-hybridization chain reaction for fluorescence-SERS dual-mode detection of p53 gene Preparation of COFs@PEI@Au NPs: The preparation method of COFs@PEI@Au NPs in this example is the same as that in Example 1.

[0079] Combined COFs@PEI@Au NPs and non-hybrid chain reaction for fluorescence-SERS dual-mode detection of p53 gene: (1) Add H1-Cy5 (50 nM, 1 μL) and different concentrations of p53 gene standard solution (10 μL) into Tris-HCl buffer (20 mM, 76.5 μL, pH 8.0, containing 10 mM MgCl2); (2) After incubating at 37 °C for 40 min, add COFs@PEI@Au NPs dispersion (200 μg / mL, 12.5 μL) into the system and continue to incubate at 25 °C for 25 min; (3) Measure the fluorescence intensity at 667 nm (λ ex = 635 nm) and the SERS intensity at 555 cm −1 using a fluorescence spectrophotometer and a Raman spectrometer respectively for each p53 gene concentration (λ ex = 780 nm). Establish a quantitative model based on the relationship between the fluorescence intensity and the SERS intensity with the change of p53 gene concentration for p53 gene concentration prediction.

[0080] Figure 10 Performance graph for combined COFs@PEI@Au NPs and non-hybrid chain reaction for fluorescence-SERS dual-mode detection of p53 gene. As can be seen from Figure 10 , in the fluorescence mode, the linear range for detecting p53 gene by the detection platform is 50 - 2500 pM, and the detection limit is 28.9 pM; in the SERS mode, the linear range for detecting p53 gene by the detection platform is 200 - 10000 pM, and the detection limit is 91.4 pM. Comparing Figure 9 and Figure 10 results, the detection limit of the hybridization chain reaction-assisted COFs@PEI@Au NPs sensor is ~10.9 times lower (fluorescence analysis) and ~13.6 times lower (SERS analysis) than that of the hybridization chain reaction-assisted COFs@PEI@Au NPs sensor.

[0081] Specificity of combined COFs@PEI@Au NPs and hybridization chain reaction for p53 gene detection Preparation of COFs@PEI@Au NPs: The preparation method of COFs@PEI@Au NPs in this example is the same as that in Example 1.

[0082] Specificity of the combined COFs@PEI@Au NPs and hybridization chain reaction for p53 gene detection: (1) Add H1-Cy5 (50 nM, 1 μL), H2-Cy5 (50 nM, 1 μL) and p53 gene standard solution (10 μL) or interfering substances (two single-base mismatched DNAs (MD1-1 and MD1-2), two double-base mismatched DNAs (MD2-1 and MD2-2), one triple-base mismatched DNA (MD3), one quadruple-base mismatched DNA (MD4) and two full-base mismatched DNAs (MDa-1 and MDa-2), 10 μL) into Tris-HCl buffer (20 mM, 75.5 μL, pH 8.0, containing 10 mM MgCl2); (2) After incubating at 37 °C for 40 min, add the COFs@PEI@Au NPs dispersion (200 μg / mL, 12.5 μL) to the system and continue to incubate at 25 °C for 25 min; (3) Measure the fluorescence intensity at 667 nm (λ ex = 635 nm) and the SERS intensity at 555 cm −1 for each group of experiments using a fluorescence spectrophotometer and a Raman spectrometer respectively (λ ex = 780 nm).

[0083] Figure 11 This is the specificity of the combined COFs@PEI@Au NPs and hybridization chain reaction for p53 gene detection. As can be seen from Figure 11 it, the detection platform constructed in the present invention has good specificity for the fluorescence-SERS dual-mode detection of p53 gene.

[0084] Homogeneity of the detection platform Preparation of COFs@PEI@Au NPs: The preparation method of COFs@PEI@Au NPs in this example is the same as that in Example 1.

[0085] Homogeneity of the detection platform: (1) Add H1-Cy5 (50 nM, 1 μL), H2-Cy5 (50 nM, 1 μL) and p53 gene standard solution (10 μL) into Tris-HCl buffer (20 mM, 75.5 μL, pH 8.0, containing 10 mM MgCl2); (2) After incubating at 37 °C for 40 min, add the COFs@PEI@Au NPs dispersion (200 μg / mL, 12.5 μL) to the system and continue to incubate at 25 °C for 25 min; (3) Measure the fluorescence intensity at 667 nm (λ ex = 635 nm) and 555 cm−1 SERS intensity at ex (λ

[0086] Figure 12 To detect the uniformity of the detection platform. From Figure 12 it can be seen that the fluorescence signals at 667 nm and the SERS signals at 555 cm −1 are basically the same, and the relative standard deviations are 3.69% and 4.94% respectively, indicating that the H-Cy5 and C@P@A mixed detection system has good uniformity.

[0087] Reproducibility of the detection platform Preparation of COFs@PEI@Au NPs: The preparation method of COFs@PEI@Au NPs in this example is the same as that in Example 1.

[0088] Reproducibility of the detection platform: The following analysis steps were carried out separately with 5 batches of parallel-prepared COFs@PEI@Au NPs: (1) Add H1-Cy5 (50 nM, 1 μL), H2-Cy5 (50 nM, 1 μL) and p53 gene standard solution (10 μL) to Tris-HCl buffer (20 mM, 75.5 μL, pH 8.0, containing 10 mM MgCl2); (2) After incubating at 37 °C for 40 min, add the COFs@PEI@Au NPs dispersion (200 μg / mL, 12.5 μL) to the system and continue to incubate at 25 °C for 25 min; (3) Use a fluorescence spectrophotometer and a Raman spectrometer to measure the fluorescence intensity at 667 nm (λ ex = 635 nm) and the SERS intensity at 555 cm −1 (λ ex = 780 nm).

[0089] Figure 13 To detect the reproducibility of the detection platform. From Figure 13 it can be seen that the relative standard deviations of detecting the p53 gene in the fluorescence mode and the SERS mode are calculated to be 4.16% and 3.29% respectively, proving that the detection platform has acceptable reproducibility. Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A COF-based nanomaterial, characterized in that, The raw materials for preparing the nanomaterials include: Au nanoparticles, COFs; and they are prepared by a self-assembly method.

2. The nanomaterial based on COFs according to claim 1, wherein The COFs are prepared by reacting a trifunctional aromatic aldehyde monomer with a linear diamine monomer.

3. The nano-material based on COFs according to claim 2, characterized in that, The trifunctional aromatic aldehyde monomer is selected from one of 1,3,5-tris(4-formylphenyl)benzene, trimesic aldehyde, tris(2-formylvinyl)benzene, tris(4-formylphenylethynyl)benzene, tris(formylpyridyl)benzene.

4. The nanomaterial based on COFs according to claim 2, characterized in that, The linear diamine monomer is selected from one of p-phenylenediamine, biphenyl diamine, ethylenediamine, styrene diamine, pyrene diamine, naphthalene diamine, diaminotriazine, p-aminobenzoic acid, 2,5-diamino-p-xylene.

5. A method for preparing a COF-based nanomaterial according to any one of claims 1 to 4, characterized in that The steps are as follows: (1) Preparation of Au NPs: Add chloroauric acid solution and ultrapure water into a 100 mL three-necked flask connected with a reflux condenser, and heat the solution to boiling; then, quickly add trisodium citrate solution into the flask, and heat the mixture under reflux for 25 - 35 min until the sol color turns wine red to obtain Au NPs; finally, naturally cool the prepared Au NPs sol to room temperature, dispense it with an ep tube, and store it in a 4 °C refrigerator for later use; (2) Preparation of COFs: First, dissolve the trifunctional aromatic aldehyde monomer and the linear diamine monomer in acetonitrile; then, add acetic acid and stir magnetically at room temperature for 24 h; finally, wash the obtained product twice with acetonitrile, tetrahydrofuran, and absolute ethanol respectively, and dry it in a vacuum drying oven to obtain COFs; (3) Preparation of COFs@PEI@Au NPs: First, ultrasonically disperse COFs in a mixed solution of water and absolute ethanol; then, add the surface-functionalized carrier solution, perform ultrasonic treatment and then magnetic stirring; finally, wash the obtained product with ultrapure water and dry it in vacuum to obtain surface-functionalized carrier-modified COFs; (4) Preparation of nanomaterials by electrostatic self-assembly method: Add Au NPs sol to the surface-functionalized carrier-modified COFs dispersion, stir magnetically and then perform ultrasonic treatment; Wash the obtained product with ultrapure water, redisperse it in 5 mL of ultrapure water to obtain a nanomaterial storage solution, and store it in a 4 °C refrigerator for later use.

6. The preparation method of a COF-based nanomaterial according to claim 5, characterized in that, The surface-functionalized carrier is selected from one of polyethyleneimine, polylysine, chitosan, PAMAM dendrimer, polydimethyldiallylammonium chloride, polyquaternium-10 / PQ-10, polyvinyl alcohol-poly(methacrylate) copolymer.

7. Use of a COF-based nanomaterial according to any one of claims 1 to 4, characterized in that, Applied to the fluorescence-SERS dual-mode detection of p53 gene in hybridization chain reaction.

8. The application of a COF-based nanomaterial according to claim 7, wherein, The detection method steps of the fluorescence-SERS dual-mode of the p53 gene in the hybridization chain reaction are as follows: (1) Add H1-Cy5, H2-Cy5, and different concentration p53 gene standard solutions into Tris-HCl buffer solution; (2) After incubating at 37 °C for a period of time, add the dispersion of nanomaterials into the system, and continue to incubate at a certain temperature for a period of time; (3)Measure the fluorescence intensity at 667 nm and the SERS intensity at 555 cm −1 for each p53 gene concentration using a fluorescence spectrophotometer and a Raman spectrometer, respectively.

9. A COF-based nanomaterial according to any one of claims 1 to 4, characterized in that, Applied to the field of medical testing technology.

10. A COF-based nanomaterial according to any one of claims 1 to 4, characterized in that, Applied to the field of spectroscopy technology.

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