Diagnosis and treatment integrated difunctional material as well as preparation method and application thereof

Abstract

The invention provides a diagnosis and treatment integrated bifunctional material, and belongs to the technical field of fluorescent nanomaterials, the diagnosis and treatment integrated bifunctional material is named as COF-Bpy, and the structural formula of the COF-Bpy comprises a structural unit shown in the formula I. The invention further provides a preparation method of the diagnosis and treatment integrated bifunctional material. The material has the functions of fluorescence detection and adsorption, can be used for biological fluorescence imaging, can also be used for detection and adsorption of in-vivo or in-vitro copper ions, and plays a role of integration of diagnosis and treatment. The invention further provides a preparation method and application of the diagnosis and treatment integrated difunctional material.

Description

Technical Field

[0001] The present invention belongs to the technical field of fluorescent nanomaterials, and in particular relates to a dual-functional material integrating diagnosis and treatment, and a preparation method and application thereof. Background Art

[0002] Recently, the dual-functional platform of fluorescence sensing and adsorption has attracted wide attention. Covalent organic framework materials (COFs), as a new type of porous nanomaterials, have good crystalline porous structure and ordered π-π structure, which can be used to develop luminescent materials with adsorption function. Some research groups have reported a wide variety of fluorescent covalent organic frameworks for sensing and adsorption of different targets. For example, Liu et al. constructed a glutathione-functionalized covalent organic framework for fluorescent sensing and adsorption of cationic dyes; Deng et al. developed a fluorescent covalent organic framework that can selectively detect and adsorb myricetin in rattan tea; Wang et al. synthesized a phenylboronic acid-functionalized covalent organic framework for sensing and adsorption of rifamycin antibiotics; Chen et al. synthesized a two-dimensional fluorescent covalent organic framework nanosheet to achieve the detection and removal of p-nitrophenol; Geng et al. reported a covalent organic framework with dual functions of iodine adsorption and sensing. Fluorescent COFs have become promising candidates in the field of fluorescence sensing and adsorption because of their rich pores and satisfactory specific surface area, which can provide sufficient binding sites to interact with the target.

[0003] However, conventional COFs have poor fluorescence properties and low quantum yields, and do not have the potential for application in fluorescence detection. They usually only have an adsorption function and are used for the adsorption and removal of targets. They have a single function and a narrow range of applications. This has caused difficulties in the development of sensing and adsorption dual-functional materials based on COFs materials. In order to solve this problem, people have made many efforts. For example, fluorescein, rare earth elements and carbon dots are combined with COFs to obtain strongly emitting COFs composite materials, which perfectly combine the fluorescence sensing function of the luminophore with the adsorption function of COFs. Although this strategy can give COFs the ability to emit light, its reaction is more complicated and the synthesis cycle is longer. During the reaction, the pore structure of COFs may collapse, the crystallinity may decrease, and the adsorption effect of COFs may be affected. Summary of the invention

[0004] In order to solve the problems of poor fluorescence performance and single function of conventional COFs, the present invention provides an integrated diagnosis and treatment dual-functional material, which has the functions of fluorescence detection and adsorption, can be used for biological fluorescence imaging, and can also be used for the detection and adsorption of copper ions in vivo or in vitro, playing the role of "diagnosis and treatment in one".

[0005] The present invention also provides a preparation method and application of the diagnosis and treatment integrated dual-function material.

[0006] The present invention is achieved through the following technical solutions:

[0007] The present invention provides a diagnosis and treatment integrated dual-function material, the diagnosis and treatment integrated dual-function material is named COF-Bpy, and the structural formula of the COF-Bpy contains the structural unit shown in Formula I:

[0008]

[0009] Based on the same inventive concept, the present invention provides a method for preparing a dual-functional material integrating diagnosis and treatment, the preparation method comprising:

[0010] Adding 5,5'-diamino-2,2'-bipyridine, 2,6-diformylpyridine and a catalyst into an organic solvent to obtain a reaction solution;

[0011] The reaction solution is allowed to stand for reaction, and the obtained product is washed and dried to obtain a COF-Bpy material.

[0012] Optionally, the step of adding 5,5'-diamino-2,2'-bipyridine, 2,6-diformylpyridine and a catalyst into an organic solvent to obtain a reaction solution specifically comprises:

[0013] Adding 5,5'-diamino-2,2'-bipyridine and 2,6-diformylpyridine into acetone, and then performing ultrasonic mixing to obtain a mixed solution;

[0014] Add glacial acetic acid to the mixed solution, and then perform ultrasonic mixing to obtain a reaction solution.

[0015] Furthermore, in the reaction solution, the molar ratio of 5,5'-diamino-2,2'-bipyridine to 2,6-diformylpyridine is 1:1;

[0016] In the reaction solution, the molar concentration of 5,5'-diamino-2,2'-bipyridine is 6 to 10 mmol·L -1 ;

[0017] In the reaction solution, the molar concentration of the catalyst is 0.19 to 0.45 mol·L -1 .

[0018] Further, the reaction solution is allowed to stand for reaction, and the obtained product is washed and dried to obtain a COF-Bpy material, which specifically includes:

[0019] The reaction solution was allowed to react at room temperature for 72±3 hours, and the obtained product was washed with tetrahydrofuran and then dried to obtain a COF-Bpy material.

[0020] Based on the same inventive concept, the present invention provides an application of an integrated diagnosis and treatment dual-functional material in copper ion detection and adsorption.

[0021] Based on the same inventive concept, the present invention provides an application of an integrated diagnosis and treatment dual-function material in bioluminescence imaging.

[0022] Based on the same inventive concept, the present invention provides an application of an integrated diagnosis and treatment dual-function material in the preparation of an integrated diagnosis and treatment agent or a bioluminescent imaging agent.

[0023] Based on the same inventive concept, the present invention provides a reagent for detecting and adsorbing copper ions, wherein the reagent comprises the above-mentioned integrated diagnosis and treatment dual-functional material.

[0024] Based on the same inventive concept, the present invention provides a bioluminescent imaging agent, which comprises the above-mentioned dual-functional material integrating diagnosis and treatment.

[0025] Based on the same inventive concept, the present invention also provides a drug for alleviating copper poisoning, wherein the active ingredient of the drug comprises the above-mentioned integrated diagnosis and treatment dual-function material.

[0026] Based on the same inventive concept, the present invention also provides an application of an integrated diagnosis and treatment dual-functional material as or in the preparation of information encryption ink or cell pH probe.

[0027] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0028] 1. The present invention discloses an integrated diagnosis and treatment bifunctional material. 5,5'-diamino-2,2'-bipyridine and 2,6-diformylpyridine are reacted at room temperature to produce a Schiff base reaction to synthesize a bipyridine group-rich fluorescent COFs, named COF-Bpy. COF-Bpy has the functions of fluorescence detection and adsorption. It has an AIE (aggregation-induced emission) effect, can emit green fluorescence, and exhibits a satisfactory quantum yield (24.72%). Since COF-Bpy provides a large number of bipyridine N,N"-complexation sites, it is easy to coordinate with copper ions and undergo protonation / deprotonation. The copper ions in the sample can be removed by fluorescence quenching and the change of the pH of the monitoring system can be monitored. Therefore, COF-Bpy can be used to construct a fluorescence sensing-adsorption bifunctional platform for the detection and adsorption of copper ions in vitro and in vivo, playing the role of "integrated diagnosis and treatment", and can also be used for bioluminescence imaging and information encryption.

[0029] 2. The present invention discloses an integrated diagnosis and treatment dual-functional material. By rationally designing the skeleton structure and spatial structure of COFs, special AI Egens (5,5'-diamino-2,2'-bipyridine) are directly introduced to participate in the synthesis. Under relatively simple reaction conditions, AI E fluorescent COFs that simultaneously satisfy the DIA (destruction of π-π interlayer accumulation), IMLP (interference in molecular layer flattening) and RIR (restriction of intramolecular rotation) effects are synthesized. Combined with the porous characteristics of COF-Bpy itself, it can be used to construct a fluorescence detection-adsorption dual-functional platform based on COFs. The COF-Bpy of the present invention can sensitively and specifically detect trace copper ions in water through the fluorescence quenching effect, and effectively enrich and remove copper ions. Moreover, COF-Bpy can also detect copper ions in cells and nematodes and improve the poisoning reaction of cells and nematodes caused by copper accumulation through adsorption. It has the potential to be used as an "integrated diagnosis and treatment" drug for the treatment of copper overdose.

[0030] 3. The present invention discloses a method for preparing a dual-functional material for integrated diagnosis and treatment. The method uses 5,5'-diamino-2,2'-bipyridine and 2,6-diformylpyridine to undergo a Schiff base reaction at room temperature to synthesize COF-Bpy which has both fluorescence detection and adsorption functions. The preparation method has simple reaction steps, mild reaction conditions, and low reaction energy consumption. Compared with the existing fluorescent COFs synthesis process, it has the advantages of low cost, low risk, and environmental friendliness. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0032] Figure 1 The simulated structure of COF-Bpy: Figure A is a top view of COF-Bpy; Figure B is a side view of COF-Bpy; Figure C is a topological schematic diagram of the twisted arrangement of six-layer COF-Bpy units.

[0033] Figure 2 A is a schematic diagram of the vertical backbone structure of COF-Bpy.

[0034] Figure 2 B is a comparison of the spatial conformations of C3+C2 type COF and C2+C2 type COF.

[0035] Figure 3 This is the synthetic route of COF-Bpy.

[0036] Figure 4PXRD patterns of COF-Bpy prepared under different conditions: Figure A shows different solvents; Figure B shows different volumes of solvents (6, 7, 8, 9 and 10 mL); Figure C shows different volumes of catalysts (0.3, 0.4, 0.5, 0.6 and 0.7 mL); Figure D shows different reaction times (24, 48 and 72 h).

[0037] Figure 5 A is the infrared spectra of the precursor and COF-Bpy.

[0038] Figure 5 B is the solid state of COF-Bpy 13 C NMR spectrum.

[0039] Figure 5 C is the XPS elemental analysis spectrum of COF-Bpy.

[0040] Figure 5 D is the C1s region spectrum of COF-Bpy.

[0041] Figure 5 E is the N1s region spectrum of COF-Bpy.

[0042] Figure 5 F is the O1s region spectrum of COF-Bpy.

[0043] Figure 5 G is the SEM image of COF-Bpy.

[0044] Figure 5 H is the TEM image of COF-Bpy.

[0045] Figure 5 I is the N2 adsorption-desorption isotherm of COF-Bpy.

[0046] Figure 5 J is the thermogravimetric analysis curve of COF-Bpy.

[0047] Figure 5 K is the change in water contact angle of COF-Bpy within 10 s.

[0048] Figure 6 For different DMSO components (f w ) in THF / DMSO solution: The upper left inset shows the photoluminescence emission intensity of COF-Bpy at different f w Ultraviolet image.

[0049] Figure 7 A is a schematic diagram of the complexation site between copper ions and COF-Bpy.

[0050] Figure 7B is the selective detection of copper ions by COF-Bpy.

[0051] Figure 7 C is the effect of different concentrations of copper ions on the fluorescence spectrum of COF-Bpy.

[0052] Figure 7 D is the linear relationship between the fluorescence intensity of COF-Bpy and the concentration of copper ions.

[0053] Figure 8 A is the adsorption isotherm model diagram of COF-Bpy for copper ions.

[0054] Figure 8 B is the adsorption kinetic model diagram of COF-Bpy for copper ions.

[0055] Fig. 9 The results of copper ion detection in HCCC cells by COF-Bpy: Figure A is the inverted fluorescence imaging of different concentrations of copper ions (range 10nM-100nM) in COF-Bpy detection cells; Figure B is the relative fluorescence imaging intensity (I / I0) of COF-Bpy under different concentrations of copper ions (range 10nM-100nM); Figure C is the inverted fluorescence imaging of different concentrations of copper ions (range 0.1μM-1.0μM) in COF-Bpy detection cells; Figure D is the relative fluorescence imaging intensity (I / I0) of COF-Bpy under different concentrations of copper ions (range 0.1μM-1.0μM); Figure E is the inverted fluorescence imaging of different concentrations of copper ions (range 1μM-10μM) in COF-Bpy detection cells; Figure F is the relative fluorescence imaging intensity (I / I0) of COF-Bpy under different concentrations of copper ions (range 1μM-10μM).

[0056] Fig.10 The results of COF-Bpy detection of copper ions in nematodes: Figure A is the positive fluorescence imaging of COF-Bpy detecting different concentrations of copper ions in nematodes (range 0-2mM); Figure B is the linear relationship between the fluorescence imaging intensity of nematodes and the copper ion concentration.

[0057] Fig.11 A is the effect of COF-Bpy on the viability of copper-poisoned cells.

[0058] Fig.11 B is the effect of COF-Bpy on the survival rate of copper-poisoned nematodes.

[0059] Fig.12 A is the fluorescence spectrum of COF-Bpy at different pH values: the inset in the upper right corner is the UV image of COF-Bpy at different pH values.

[0060] Fig.12B shows that the effect of different pH (1-14) on the fluorescence intensity of COF-Bpy is linear.

[0061] Fig.12 C is the reversible pH response of COF-Bpy.

[0062] Fig.12 D is an information encryption image drawn using COF-Bpy ink.

[0063] Fig.13 A is the inverted fluorescence imaging of HCCC cells by COF-Bpy at different pH values: the scale bar is 100 μm.

[0064] Fig.13 B is the linear relationship between COF-Bpy fluorescence imaging intensity and pH value: the inset in the lower left corner is the fluorescence imaging intensity statistics. DETAILED DESCRIPTION

[0065] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

[0066] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

[0067] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0068] The overall idea of ​​the present invention is as follows:

[0069] To date, the most widely used COFs are usually synthesized by the Schiff base reaction of aldehyde precursors and amino precursors to form imine bonds. Although conventional imine COFs have high crystallinity and can quickly achieve target enrichment, most of them do not have obvious fluorescence due to aggregation-induced quenching (ACQ) caused by strong π-π interactions between COFs layers. This limitation is particularly evident in the application of analyzing targets in vivo based on fluorescence imaging methods. When fluorescent COFs accumulate in vivo, the ACQ property will cause fluorescence quenching, thereby greatly reducing the detection sensitivity.

[0070] The present invention improves the construction strategy of COFs and increases the fluorescence emission intensity of COFs by introducing the aggregation-induced emission (AIE) mechanism into the structure of COFs.

[0071] The inventors believe that since the flattening of the COFs molecular layer promotes the formation of a tight π-π stacking structure between the layers, the energy of the excited state is lost through non-radiative transitions, resulting in the ACQ effect and weak fluorescence. Therefore, in order to achieve the AIE of fluorescent COFs, it is necessary to destroy the π-π interlayer accumulation (DIA) and interfere with the flattening of the molecular layer (IMLP). In addition, since the objective existence of molecular motion also makes COFs more prone to non-radiative transition energy loss, which is not conducive to the construction of fluorescent COFs, it is also necessary to limit the intramolecular rotation (RIR) motion of COFs. Based on this, the inventors believe that promoting DIA, IMLP and RIR effects is a key strategy for manufacturing AIE fluorescent COFs with high fluorescence properties.

[0072] In order to solve the problem of poor fluorescence performance and single function of conventional COFs, the present invention provides a method for preparing a dual-functional material for integrated diagnosis and treatment. The method uses 5,5'-diamino-2,2'-bipyridine and 2,6-diformylpyridine to undergo Schiff base reaction at room temperature to synthesize fluorescent COFs rich in bipyridine groups, named COF-Bpy. COF-Bpy has the functions of fluorescence detection and adsorption at the same time. It has an AIE effect and can emit green fluorescence, showing a satisfactory quantum yield (24.72%). Since COF-Bpy provides a large number of bipyridine N,N"-complexation sites, it is easy to coordinate with copper ions, produce fluorescence quenching and remove copper ions in the sample. Therefore, COF-Bpy can be used to construct a fluorescence sensing-adsorption dual-functional platform for the detection and adsorption of copper ions in vitro and in vivo, play the role of "integrated diagnosis and treatment", and can also be used for bioluminescence imaging.

[0073] The present invention rationally designs the skeleton structure and spatial structure of COFs, directly introduces special AIEgens (5,5'-diamino-2,2'-bipyridine) to participate in the synthesis, and synthesizes high-emission fluorescent COFs under relatively simple conditions. In view of the formation of a vertically arranged skeleton structure and a six-membered ring chair-folded spatial structure, the DIA, IMLP and RIR triple effects are promoted, showing the properties of AIE. Combined with the porous characteristics of COF-Bpy itself, it can be used to construct a fluorescence detection-adsorption dual-functional platform based on COFs.

[0074] The COF-Bpy synthesized in the present invention, in a dispersed state (in a well-dispersed solvent), the COF-Bpy skeleton part belonging to bipyridine can rotate freely until it is perpendicular to the pyridine ring ( Figure 1A). In the aggregated state (in a poorly dispersed solvent), the free rotation of the bipyridine skeleton is restricted, thus showing the characteristics of AIE. Moreover, this behavior further leads to the formation of a distorted chair-like spatial structure of COF-Bpy, resulting in non-planar molecular layers, increased interlayer spacing, and weakened interlayer π-π interactions ( Figure 1 B. Figure 1 C). The AIE performance of COF-Bpy has been extensively enhanced by DIA, IMLP and RIR. Unlike the prior art, the present invention does not introduce some side chains or hydrogen bonds to enhance the fluorescence performance of COFs, but adopts a simpler and cheaper method to directly introduce special AIEgens to form a unique vertical skeleton and twisted spatial structure. The present invention combines the spatial solidification of COFs with the skeleton solidification to achieve "one stone, three birds" of DIA, IMLP and RIR, and improves the optical performance of COF-Bpy (strong green light and satisfactory fluorescence quantum yield). In addition, due to the active nitrogen sites in the bipyridine unit, COF-Bpy may have strong metal ion coordination and pH monitoring capabilities, and its wide application potential such as copper ion detection (by fluorescence sensing) and copper poisoning mitigation ability (by adsorption) has attracted our attention and has been discussed in detail.

[0075] After testing, the invented COF-Bpy can sensitively and specifically detect trace copper ions in water through the fluorescence quenching effect, and effectively enrich and remove copper ions. In addition, COF-Bpy can also detect copper ions in cells and nematodes and improve the poisoning reaction of cells and nematodes caused by copper accumulation through adsorption. It has the potential to be used as a "diagnosis and treatment" drug for the treatment of copper overdose. In addition, COF-Bpy can achieve detection in a wide range of pH (1-14) through the protonation and deprotonation of the bipyridine nitrogen site. Based on this property, it can be developed as an ink for information encryption.

[0076] Specifically, the present invention provides a method for preparing a dual-functional material for diagnosis and treatment, comprising:

[0077] S1. adding 5,5'-diamino-2,2'-bipyridine, 2,6-diformylpyridine and a catalyst to an organic solvent to obtain a reaction solution;

[0078] S2. The reaction solution is allowed to stand for reaction, and the obtained product is washed and dried to obtain a COF-Bpy material.

[0079] Step S1 specifically includes:

[0080] Adding 5,5'-diamino-2,2'-bipyridine and 2,6-diformylpyridine into acetone, and then performing ultrasonic mixing to obtain a mixed solution;

[0081] Add glacial acetic acid to the mixed solution, and then perform ultrasonic mixing to obtain a reaction solution.

[0082] In the present invention, 5,5'-diamino-2,2'-bipyridine and 2,6-diformylpyridine are first added to acetone and mixed, and the catalyst is then added to make the catalyst contact evenly with the mixed monomers to avoid excessive local concentration to generate other products.

[0083] In the present invention, the advantage of using 5,5'-diamino-2,2'-bipyridine and 2,6-diformylpyridine as raw materials to prepare COF-Bpy is that the synthesized C2+C2 configuration COFs are easy to produce skeleton solidification and spatial solidification mechanisms, promote the generation of AIE, and improve the fluorescence performance. The skeleton solidification is caused by 2,6-diformylpyridine as a non-rotating monomer that can keep the six-membered ring skeleton structure stable, while 5,5'-diamino-2,2'-bipyridine as a freely rotatable monomer can rotate to be perpendicular to 2,6-diformylpyridine, forming a special vertical skeleton structure ( Figure 2 A), thereby increasing the interlayer spacing and promoting the DIA effect. The spatial solidification is caused by the fact that, unlike the conventional C3+C2 COFs (C3+C2 COFs can form an infinitely extended six-membered ring honeycomb COFs layer), the C2+C2 COFs present an independent six-membered ring arrangement ( Figure 2 B). Therefore, each COFs ring can undergo twisted chair folding, further promoting the DIA and IMLP effects. When the COF layers are arranged together, this special twisted structure is conducive to the occurrence of RIR. Finally, by screening suitable reactive monomers and rationally designing the skeleton and spatial arrangement of the COFs structure, we obtained highly emitting fluorescent AIE-COFs.

[0084] In step S1, in the reaction solution, the molar ratio of 5,5'-diamino-2,2'-bipyridine to 2,6-diformylpyridine is 1:1;

[0085] In the reaction solution, the molar concentration of 5,5'-diamino-2,2'-bipyridine is 6 to 10 mmol·L -1 ;

[0086] In the reaction solution, the molar concentration of the catalyst is 0.19 to 0.45 mol·L -1 .

[0087] In the present invention, the molar ratio of 5,5'-diamino-2,2'-bipyridine and 2,6-diformylpyridine is 1:1. 5,5'-diamino-2,2'-bipyridine contains two -NH2, and 2,6-diformylpyridine contains two -C=O. The reaction at a molar ratio of 1:1 is conducive to the complete reaction between -NH2 and -C=O. A ratio lower or higher than this range will result in incomplete reaction.

[0088] In the present invention, the molar concentration of 5,5'-diamino-2,2'-bipyridine is 6 to 10 mmol·L -1 The appropriate monomer concentration can make the reaction proceed at an appropriate speed. If the concentration is too low, the reaction speed will be too slow, the reaction time will be increased, and the yield will be reduced; if the concentration is too high, the reaction speed will be too fast, which will result in poor product crystal form.

[0089] In the present invention, the molar concentration of the catalyst is 0.19 to 0.45 mol·L -1 The advantage is that it can adjust the acidity of the reaction system and speed up the reaction. If the catalyst concentration is too low, the reaction will be too slow, the reaction time will be long, and the yield will be low. If the concentration is too high, the reaction will be too fast, which will be unfavorable for the crystallization of the product.

[0090] The following will describe in detail an integrated diagnosis and treatment dual-functional material, its preparation method and application in combination with examples and experimental data.

[0091] Example 1

[0092] This embodiment provides a method for preparing and optimizing a dual-functional material for integrated diagnosis and treatment.

[0093] The crystal structure of COFs is one of the most important factors in the synthesis of COFs materials. At present, the most widely used COFs solvent thermal synthesis process can obtain products with high crystallinity, but this method requires the use of a large amount of toxic reagents, high temperature and high energy consumption, and produces glass waste. This process not only has high operating costs and high risks, but also does not meet the principles of green chemistry and the goal of sustainable development. Therefore, in order to obtain low-energy consumption, easily available crystalline COFs materials, this embodiment optimizes the synthesis parameters (reaction solvent type, solvent dosage, catalyst dosage, reaction time) of the reaction on the basis of room temperature synthesis to obtain stable crystalline fluorescent COFs.

[0094] 1. The reagents used are as follows:

[0095] 5,5'-diamino-2,2'-bipyridine (purity>97%), 2,6-diformylpyridine (purity>98%), acetone, acetonitrile, dioxane, N,N-dimethylformamide, mesitylene, n-butanol, o-dichlorobenzene, glacial acetic acid, tetrahydrofuran.

[0096] 2. Specifically, the preparation method of COF-Bpy material is as follows:

[0097] 5,5'-diamino-2,2'-bipyridine and 2,6-diformylpyridine are ultrasonically mixed in an organic solvent; glacial acetic acid is then added as a catalyst, and ultrasonically mixed to obtain a reaction solution; the reaction solution is allowed to react at room temperature for a certain period of time, washed with tetrahydrofuran, and dried to obtain an AIE fluorescent COFs material COF-Bpy (such as Figure 3 )

[0098] 3. Optimization of synthesis parameters:

[0099] 3.1 Appropriate solvent polarity, combination and ratio are crucial to achieve a balance between COFs skeleton growth and crystallization. Therefore, this example first studies the effects of solvents acetonitrile, acetone, dioxane, N,N-dimethylformamide, different ratios of mesitylene / dioxane and n-butanol / o-dichlorobenzene on the crystallinity of COF-Bpy, and the crystallinity of COF-Bpy is measured by powder X-ray diffractometer. Figure 4 A shows that the best solvent is acetone.

[0100] 3.2 This example explores the amount of acetone used. Figure 4 B shows that the optimal amount of acetone is 9 mL (5,5'-diamino-2,2'-bipyridine concentration is 6.7 mmol·L -1 ).

[0101] 3.3 Since the crystallinity of imine 2D-COFs is affected by the catalyst concentration during the reaction, the amount of catalyst used was optimized in this example. Figure 4 C shows that when the amount of glacial acetic acid is 0.4 mL (the concentration of glacial acetic acid is 0.26 mol·L -1 ), the crystallinity of the product is optimal.

[0102] 3.4 Since the synthesis of COFs is an "aging process", sufficient time is required to achieve "self-adjustment". This example explores the synthesis time. Figure 4 D shows that the crystallinity of COF-Bpy increases with the increase of reaction time, and an obvious peak is formed at 72 h. Considering the time cost in the synthesis process, 72 h is considered to be the optimal reaction time.

[0103] 4. The optimal preparation method of COF-Bpy material in this embodiment is as follows:

[0104] 2,6-diformylpyridine (8.11 mg, 0.06 mmol) and 5,5'-diamino-2,2'-bipyridine (11.17 mg, 0.06 mmol) were weighed and placed in 9 mL of acetone and ultrasonicated for 10 min to ensure thorough mixing. Subsequently, 0.4 mL of glacial acetic acid (6 mol·L -1 ) was slowly added to the mixture, and ultrasonic treatment was continued for 5 min. The reaction system was placed at room temperature for 72 h. Finally, the resulting yellow precipitate was washed three times with tetrahydrofuran, and the precipitate was collected by centrifugation (7800 rpm, 5 min) and placed in a vacuum drying oven at 60 ° C for 12 h to obtain COF-Bpy.

[0105] Finally, light yellow powder COF-Bpy was synthesized with a yield of 83.79%.

[0106] Example 2

[0107] In this example, the COF-Bpy material prepared in Example 1 is characterized.

[0108] 1. Infrared analysis (FT-IR)

[0109] FT-IR spectrum shows ( Figure 5 A), aldehyde group C=O (1720 cm -1 ) absorption peak, aldehyde CH (2860cm -1 ) and NH stretching characteristic peaks of 5,5'-diamino-2,2'-bipyridine (Bpy) (3331-3199 cm -1 ) disappeared. On the contrary, the 1629cm -1 The peak of , corresponding to C = N stretching vibration. These results indicate that a Schiff base reaction occurred between 5,5'-diamino-2,2'-bipyridine and 2,6-diformylpyridine, resulting in an imine bond and the successful synthesis of COF-Bpy.

[0110] 2. Nuclear magnetic resonance analysis (NMR) NMR spectrum shows ( Figure 5 B), the response at 155.24ppm is classified as a -C*=N-characteristic absorption peak. The peaks at 148.25ppm and 139.89ppm correspond to -C*-N=C- and -N=CC*-, respectively. The peak at 133.88ppm is associated with the C atom para to the N atom on the pyridine ring. The peaks at 119.40 / 115.98ppm correspond to the C atom connecting the pyridine ring and the terminal -C*-NH2 at the edge. There is a small peak at 191.94ppm attributed to the incompletely reacted -C*=O at the edge of the structure.

[0111] 3. Elemental analysis (XPS)

[0112] XPS spectrum of COF-Bpy ( Figure 5 CF) shows the presence of element C (77.34%), element N (16.51%) and element O (6.14%). The C 1s spectrum can be divided into four peaks, corresponding to pyridine C (284.60 eV), CC (284.00 eV), C=N (285.40 eV) and CN (287.20 eV). The N 1s spectrum can be fitted into three peaks, including pyridine N (398.50 eV), N=C (398.96 eV) and NC (399.40 eV). The formation of N=C further indicates that the condensation of COF-Bpy is successful. The O1s spectrum of COF-Bpy mainly comes from the incompletely reacted O=C in 2,6-diformylpyridine (531.60 eV), oxygen in the air (533.30 eV) and moisture (531.90 eV).

[0113] 4. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) analysis

[0114] like Figure 5 As shown in Figure GH, the microstructure and surface morphology of COF-Bpy were observed by SEM and TEM. The SEM images showed that COF-Bpy exhibited a microstructure of stacked flakes, and the surface showed a microscopic granular texture, showing a certain degree of roughness. The TEM images further showed that the COF-Bpy nanosheets exhibited a layered stacked flake crystal morphology.

[0115] 5. Specific surface area analysis (BET)

[0116] like Figure 5 As shown in Figure 1, the specific surface area of ​​COF-Bpy was analyzed by N2 adsorption-desorption isotherm, and the specific surface area was calculated to be 106.96 m 2 ·g -1 .

[0117] 6. Thermogravimetric analysis (TGA)

[0118] like Figure 5 As shown in Figure J, the thermal stability of COF-Bpy was evaluated by thermogravimetric analysis (TGA). It can be found that the thermal decomposition of COF-Bpy can be divided into two processes. When the temperature reaches 350°C, the weight loss rate is about 14.30%, which is mainly attributed to the evaporation of residual solvent and water in COF-Bpy. When the temperature rises to 900°C, the weight of COF-Bpy continues to decrease by about 29.05%. This process is attributed to the destruction of the carbon skeleton of COF-Bpy, and the final residual mass is 56.65%. These results show that COF-Bpy has good thermal stability.

[0119] 7. Water contact angle analysis

[0120] The hydrophilicity / hydrophobicity of COF-Bpy was studied using AFES contact angle meter. Figure 5 As shown in Figure 4, the water contact angle (WCA) of COF-Bpy decreased from 53.72° to 46.64° within 10 s, showing a hydrophilic trend. This indicates that COF-Bpy is suitable for aqueous systems and can be adapted to a wider range of applications.

[0121] Example 3

[0122] Equal amounts of COF-Bpy were dispersed in solutions of different DMSO components (DMSO / THF system, where DMSO was a poor solvent for COF-Bpy and THF was a good solvent for COF-Bpy) at a concentration of 100 μg mL -1 The fluorescence intensity of COF-Bpy with DMSO components ranging from 0% to 99% was measured. It was found that the fluorescence intensity increased with the increase of DMSO components ( Figure 6 ), which is due to the aggregation of COF-Bpy with the increase of poor solvent components, confirming the AIE effect of COF-Bpy.

[0123] Example 4

[0124] In this example, the COF-Bpy material prepared in Example 1 was tested for its copper ion fluorescence detection performance and adsorption performance.

[0125] 1. Fluorescence performance test

[0126] In order to study the selectivity of COF-Bpy in detecting copper ions, 300 μL of ion solution (1 mM) was added to 2700 μL of COF-Bpy (25 μg mL -1 ) solution; after blowing three times with a pipette, the fluorescence intensity of different mixtures was tested at an excitation wavelength of 390 nm. To evaluate the sensitivity of COF-Bpy to copper ions, copper ion solutions with concentrations of 10 pM-10 μM were prepared, and 300 μL of copper ion solution was added to 2700 μL COF-Bpy (25 μg mL -1 ) solution; after thorough mixing, the fluorescence intensity before and after adding the copper ion solution was tested at an excitation wavelength of 390 nm. Since copper ions can selectively quench the fluorescence of COF-Bpy by complexing with the bipyridine sites ( Figure 7 AB), its fluorescence intensity shows a good linear relationship with copper ions in an extremely low concentration range (10pM-4μM) ( Figure 7 CD), indicating that COF-Bpy can sensitively and specifically detect trace amounts of copper ions in water.

[0127] 2. Adsorption performance test

[0128] (1) Isotherm adsorption curve

[0129] In this example, the adsorption performance of COF-Bpy was tested at room temperature and neutral pH. 2 mg COF-Bpy was added to 5 mL copper ion solution (concentration range 0-70.4 μg mL -1 ). Oscillate at room temperature for 3 h to reach complete adsorption equilibrium. After filtering through a 0.10 μm membrane, the supernatant was analyzed by atomic absorption spectroscopy to obtain the adsorption isotherm of copper ions. The equilibrium adsorption capacity (Q) was calculated using the following formula.

[0130]

[0131] Where C0 (μg·mL -1 ) and C e (μg·mL -1 ) are the analyte concentrations before and after adsorption, respectively. Q is the equilibrium adsorption capacity (mg·g -1 ), V is the volume of added solution (mL), and m is the mass of adsorbent (mg).

[0132] The changes in the concentration of COF-Bpy after adsorbing different concentrations of copper ions, such as Figure 8 As shown in A, under the same adsorption time, when the copper ion concentration is at a low level, the adsorption amount increases with the copper ion concentration and then reaches equilibrium. -1 When COF-Bpy reaches adsorption saturation, the equilibrium adsorption capacity is 15.85 mg·g -1 This is because there are a large number of adsorption sites distributed on the porous structure of COF-Bpy, which can quickly complex with copper ions. As the concentration of copper ions increases, the adsorption sites gradually become saturated, so the change in adsorption capacity tends to equilibrium.

[0133] (2) Kinetic curve

[0134] In this example, the adsorption kinetics of COF-Bpy was tested at room temperature and neutral pH to study the change of the adsorption amount of copper ions after COF-Bpy adsorbed copper ions under the same concentration conditions. In the adsorption kinetics analysis, 2 mg of COF-Bpy was added to 5 mL of 12.8 μg mL -1 The supernatant was then ultrasonically treated for 1 min in a copper ion solution. After oscillating at room temperature for different times (0-55 min), it was filtered through a 0.10 μm filter membrane. The supernatant was analyzed by atomic absorption spectrometry.

[0135] like Figure 8As shown in Figure B, the adsorption amount increases rapidly from 0 to 10 minutes, increases slowly from 10 to 40 minutes, and reaches saturation at 40 minutes. In the stage of rapid increase in adsorption rate, copper ions can quickly complex with COF-Bpy because the adsorption sites are sufficient. As the adsorption sites decrease, the adsorption rate slows down and tends to equilibrium after reaching saturation. The above results show that COF-Bpy has potential application value in the enrichment and removal of copper ions.

[0136] Example 5

[0137] In this example, the COF-Bpy material prepared in Example 1 was applied to the detection of copper ions in HCCC cells and nematodes.

[0138] 1. COF-Bpy material is used for copper ion detection in cells:

[0139] HCCC cells were seeded in 96-well plates in DMEM containing 10% FBS and cultured in a CO2 incubator (37°C, 5% CO2) for 24 h. When copper ions were detected, 200 μg mL -1 The cells were treated with COF-Bpy for 30 min. Subsequently, different concentrations of sterile copper ion aqueous solution (10 nM-100 μM) were added, and fluorescence imaging was performed after incubation for another 30 min.

[0140] Fig. 9 AF shows that the fluorescence intensity of the cells gradually decreases with the addition of copper ions (10nM-10μM), showing a linear correlation within the corresponding range. When the intracellular copper ion level is very low (10-100nM), the fluorescence intensity of COF-Bpy shows a linear detection relationship with the copper ion concentration (I0 / I=3.5116x+1.0371, R 2 =0.9865). Meanwhile, the response of COF-Bpy to copper ions is in the range of 0.1-1 μM (I0 / I=0.7596x+1.3468, R 2 =0.9937) and 1-10 μM range (I0 / I = 0.1905x + 1.8597, R 2 =0.9953), indicating that COF-Bpy has the potential to accurately sense trace amounts of copper ions in cells.

[0141] 2. COF-Bpy material is used for copper ion detection in nematodes:

[0142] Use bleach solution (0.5 mol·L -1The wild-type strain N2 of Caenorhabditis elegans was synchronized with 1% NaOH and 1% NaClO, and cultured on nematode growth medium (NGM) agar plates with Escherichia coli OP50 as the food source to obtain L1 larvae. After reaching the L4 stage, the nematodes were transferred to FUDR-NGM plates (containing 5 mg·L -1 FUDR) inhibited offspring hatching. Subsequently, the nematodes were exposed to different concentrations of copper ions (0.25-2 mM) on FUDR-NGM plates and incubated for 48 h to ensure complete absorption of copper ions, thus establishing an acute copper poisoning model in nematodes. After incubation, 200 μg mL -1 The nematodes were treated with COF-Bpy for 60 min, and the fluorescence intensity of the nematodes was observed using an orthogonal fluorescence microscope. At least 30 nematodes were analyzed in each group, and the experiment was performed in triplicate.

[0143] Fig.10 AB showed that as the copper ion concentration in the nematode increased, the fluorescence intensity of COF-Bpy gradually decreased, and showed a good linear correlation in the copper ion concentration range of 0-2.0 mM (y = -1.1364x + 7.5376, R 2 =0.9916). In addition, COF-Bpy can sensitively detect the accumulation of copper ions above 1 mM in nematodes. This indicates that COF-Bpy can sensitively detect the content and changes of copper ions in organisms.

[0144] Example 6

[0145] In this example, the COF-Bpy material prepared in Example 1 is used to alleviate copper poisoning of cells and nematodes.

[0146] 1. HCCC cells were seeded in 96-well plates in DMEM containing 10% FBS and cultured in a CO2 incubator (37°C, 5% CO2) for 24 h. To induce rapid copper poisoning, 1 mM copper ion sterile aqueous solution was added to 5 wells to establish an acute copper poisoning cell model. Similarly, 1 mM copper ion sterile aqueous solution and different concentrations of COF-Bpy sterile aqueous solution (50-300 μg mL -1 ) were added to 5 wells respectively. The other 5 wells were used as the control group, and the same amount of sterilized water was added. After 24 h of co-incubation, the cell viability of each group was evaluated using the MTT method, and it was found that 200 μg mL -1 COF-Bpy can restore cell activity to normal levels ( Fig.11 A).

[0147] 2. The L4 nematodes obtained as described above were transferred to FUDR-NGM plates containing 10 mM copper ions and cultured for 48 h to induce an acute copper poisoning nematode model. Subsequently, the nematodes were transferred to plates containing 100-400 μg mL -1The nematodes were cultured in FUDR-NGM plates of COF-Bpy. The survival rates of the nematodes in the control group, model group (copper ion treatment) group and treatment group (COF-Bpy treatment) were observed under a microscope, where the nematodes that showed sensitive movement and maintained an S-shaped wavy body shape were considered alive, while those that did not move, had a stiff body, and looked white and shortened were considered dead. The survival rate (SR) and the mean lifespan extension rate (MLS) were calculated using the following formulas.

[0148]

[0149] Where T a is the number of surviving nematodes after treatment with C copper ions or COF-Bpy. b and n is the total number of nematodes, T i is the life span of the i-th nematode.

[0150] It was found that COF-Bpy effectively increased the survival rate of copper-poisoned nematodes (the average life span extension rate was 26.25-38.51%) and delayed the death of nematodes caused by copper poisoning ( Fig.11 B).

[0151] Example 7

[0152] In this example, the pH fluorescence detection performance of the AIE-COF material prepared in Example 1 was tested.

[0153] Since the bipyridine nitrogen site is easily protonated and deprotonated, COF-Bpy can be used as a pH probe for linear detection of a wide range of pH (1-14). COF-Bpy fluorescence turns on under acidic conditions and turns off under alkaline conditions. Based on this phenomenon, we developed alkaline fluorescent ink based on COF-Bpy for information encryption. After writing the pattern on a clean filter paper and air-drying it, the pattern cannot be seen under visible light or UV light. When the filter paper is immersed in acidic lemon juice, the pattern emitting green fluorescence can be clearly seen under UV light, and the information is successfully extracted. In order to further expand the application of COF-Bpy-based pH probes in cells, it was successfully used to monitor pH fluctuations in cells.

[0154] 1. Information encryption ink

[0155] Given that the structure of COF-Bpy is rich in bipyridyl nitrogen, it is an excellent protonation site. The fluorescence spectra of COF-Bpy were measured at different pH levels. Fig.12 AB shows that the fluorescence intensity of COF-Bpy increases significantly with the decrease of pH value, and shows a linear correlation in the pH range of 1-14. 2The value is 0.9940. Further studies have shown that COF-Bpy exhibits a reversible "on-off" fluorescence response through protonation and deprotonation processes. Fig.12 C showed that COF-Bpy undergoes three reversible transitions between protonated and deprotonated states under strongly acidic and alkaline conditions (pH 2.0 and 13.0). This unique property enables COF-Bpy to be formulated as a fluorescent ink suitable for hiding sensitive information that is only visible under specific conditions. Therefore, COF-Bpy was dispersed in an alkaline solution (concentration of 25 μg mL -1 , using a pH meter to adjust the pH to 12.0) to produce ink for information encryption. Fig.12 D shows that the pattern drawn with COF-Bpy ink looks inconspicuous under visible light or UV light. When exposed to acidic substances such as lemon juice, the hidden information becomes visible under 365nm UV light. Therefore, the pH-sensitive COF-Bpy developed in this study provides powerful capabilities for information encryption and a simple deciphering method was developed.

[0156] 2. Cell pH Probe

[0157] Cellular pH plays a vital role in cellular functions such as proliferation, apoptosis, and enzyme activity. Abnormal fluctuations in cellular pH can lead to cell dysfunction and various diseases, therefore, monitoring cellular pH is crucial in medical research. Given the wide range of pH monitoring capabilities of COF-Bpy, it can be used to image and detect pH levels in HCCC cells. HCCC cells were seeded in 96-well plates in DMEM containing 10% FBS and cultured in a CO2 incubator (37°C, 5% CO2) for 24 h. To monitor intracellular pH changes, 200 μg mL -1 The cells were treated with COF-Bpy for 30 min. After washing three times with PBS (pH 7.4), PBS with different pH values ​​(pH 2.0-11.99) was added to the cells and incubated for another 10 min before fluorescence imaging. Fig.13 A shows that as the cell pH value increased from 2.0 to 11.99, the fluorescence intensity of the cells treated with COF-Bpy gradually decreased, showing a negatively correlated linear relationship (y = -4.4356x + 76.356, R 2 =0.9935)( Fig.13 B). Notably, HCCC cells were rounded at pH 11.99, indicating an unhealthy state. These results confirm that COF-Bpy has excellent pH biosensing and imaging performance in a cellular environment.

[0158] In summary, COF-Bpy can detect copper ions in cells and nematodes, and can improve the poisoning reactions of cells and nematodes caused by copper accumulation through adsorption. It has the potential to be used as an "integrated diagnosis and treatment" drug for the treatment of copper overdose. It can also accurately monitor a wide range of pH fluctuations and can be used as an excellent pH probe and information encryption ink.

[0159] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements that are inherent to such process, method, article, or apparatus.

[0160] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0161] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A dual-functional material for diagnosis and treatment, characterized in that: The integrated diagnosis and treatment dual-function material is named COF-Bpy, and the structural formula of COF-Bpy includes the structural unit shown in Formula I:

2. The method for preparing a dual-functional material for diagnosis and treatment as claimed in claim 1, characterized in that: The preparation method comprises: Adding 5,5'-diamino-2,2'-bipyridine, 2,6-diformylpyridine and a catalyst into an organic solvent to obtain a reaction solution; The reaction solution is allowed to stand for reaction, and the obtained product is washed and dried to obtain a COF-Bpy material.

3. The method for preparing a dual-functional material for diagnosis and treatment integration according to claim 2, characterized in that: The step of adding 5,5'-diamino-2,2'-bipyridine, 2,6-diformylpyridine and a catalyst into an organic solvent to obtain a reaction solution specifically comprises: Adding 5,5'-diamino-2,2'-bipyridine and 2,6-diformylpyridine into acetone, and then performing ultrasonic mixing to obtain a mixed solution; Add glacial acetic acid to the mixed solution, and then perform ultrasonic mixing to obtain a reaction solution.

4. The method for preparing a dual-functional material for diagnosis and treatment integration according to claim 2 or 3, characterized in that: In the reaction solution, the molar ratio of 5,5'-diamino-2,2'-bipyridine to 2,6-diformylpyridine is 1:1; In the reaction solution, the molar concentration of 5,5'-diamino-2,2'-bipyridine is 6 to 10 mmol·L -1 ; In the reaction solution, the molar concentration of the catalyst is 0.19 to 0.45 mol·L -1 .

5. The method for preparing a dual-functional material for diagnosis and treatment integration according to claim 2, characterized in that: The reaction solution is allowed to stand for reaction, and the obtained product is washed and dried to obtain a COF-Bpy material, which specifically comprises: The reaction solution was allowed to react at room temperature for 72±3 hours, and the obtained product was washed with tetrahydrofuran and then dried to obtain a COF-Bpy material.

6. Application of the dual-functional material for diagnosis and treatment as described in claim 1 in copper ion detection and adsorption.

7. Application of the dual-functional material for diagnosis and treatment as claimed in claim 1 in bioluminescence imaging.

8. Use of the dual-functional material for diagnosis and treatment integration as described in claim 1 in the preparation of an integrated diagnosis and treatment agent or a bioluminescent imaging agent.

9. A reagent for detecting and adsorbing copper ions, characterized in that: The reagent comprises the dual-functional material for diagnosis and treatment as described in claim 1.

10. A bioluminescent imaging agent, characterized in that: The bioluminescent imaging agent comprises the diagnosis and treatment integrated dual-function material as described in claim 1.

11. A drug for alleviating copper poisoning, characterized in that: The active ingredient of the drug includes the dual-functional material for diagnosis and treatment as described in claim 1.

12. Use of the dual-functional material for diagnosis and treatment as claimed in claim 1 as or in the preparation of information encryption ink or cell pH probe.

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