Fluorescent imaging probes based on covalent organic frameworks, methods of preparation and use

Abstract

The application provides a fluorescent imaging probe based on a covalent organic framework, a preparation method and application. The fluorescent imaging probe is successfully prepared by the preparation method, the fluorescent intensity of the fluorescent imaging probe has a good linear relationship with the concentration of iron ions, the fluorescent imaging probe can be used for detecting the concentration of iron ions, and the fluorescent imaging probe has excellent biocompatibility and can be used for in vivo fluorescent imaging.

Description

Technical Field

[0001] This invention relates to the field of fluorescence detection technology, and in particular to a fluorescence imaging probe based on a covalent organic framework, its preparation method, and its application. Background Technology

[0002] Iron ions (Fe) 3+ Iron (Fe) is a transition metal that participates in various biological processes, such as oxygen transport, adenosine triphosphate (ATP) production, and trace element metabolism, and is crucial for human health. On the one hand, iron deficiency is associated with diseases such as anemia, cerebral thrombosis, peptic ulcers, and cancer. On the other hand, excessive iron intake can lead to iron poisoning, diabetes, cirrhosis, and neurodegenerative diseases. Furthermore, in industrial wastewater, excessive iron can cause changes in water color and taste, resulting in serious pollution. Therefore, detecting iron ions is essential for biological health and environmental assessment.

[0003] In recent years, electrochemical, ion chromatography, and anion exchange liquid chromatography-inductively coupled plasma mass spectrometry (ICP-MS) methods have been developed for the quantitative detection of iron ions and the exploration of their mechanisms. However, the high cost of instruments and cumbersome operation limit their widespread application. Compared with the above methods, fluorescence detection is one of the most efficient, convenient, and inexpensive methods. It refers to the significant change in the photophysical properties of the probe molecules before and after the fluorescent probe specifically binds to the analyte, thereby detecting the change in fluorescence signal and realizing the detection of different molecules or ions in organisms or the environment. Currently, more and more researchers are developing fluorescent probes for the detection of iron ions. However, these fluorescent probes still face some challenges, such as poor stability, low specificity, and high toxicity, making it impossible to achieve highly sensitive and specific detection of iron ions and in vivo fluorescence imaging. To overcome these challenges, it is urgent to develop a novel fluorescent probe with stable chemical structure and high biocompatibility, and to construct a simple, highly sensitive, and highly specific iron ion analysis platform to achieve in vivo fluorescence imaging.

[0004] Covalent organic frameworks (COFs) are a class of highly crystalline, porous organic polymers composed of light elements linked by covalent bonds. Due to their advantages such as high specific surface area, good stability, diverse topologies, and ease of functionalization, COFs have shown great potential in applications including gas storage, molecular adsorption / separation, drug delivery, and sensing. Furthermore, the π-stacking layers within COFs promote electron transfer, and their inherent porous structure provides sufficient nanoscale space for suitable analytes. Based on these advantages, various COF fluorescent probes have been developed for the detection of iron ions. However, currently reported COF fluorescent probes suffer from low quantum yield, poor biocompatibility, and unresolved issues regarding iron ion selectivity, preventing their application in the dynamic monitoring of iron ion levels in vivo.

[0005] Therefore, there is an urgent need to provide a fluorescent imaging probe based on a covalent organic framework, its preparation method, and its application to solve the problems existing in the prior art. Summary of the Invention

[0006] The main objective of this invention is to provide a fluorescent imaging probe based on a covalent organic framework, its preparation method, and its application, in order to solve the technical problems of low quantum yield, poor biocompatibility, and the need to improve iron ion selectivity in related technologies.

[0007] To achieve the above objectives, the present invention provides a fluorescent imaging probe based on a covalent organic framework, the structural formula of which is as follows: Wherein, Me represents methyl, n represents degree of polymerization, and the fluorescent imaging probe includes a covalent organic framework and polyvinyldioxythiophene.

[0008] Preferably, the degree of polymerization ranges from 1000 to 4000.

[0009] The beneficial effects of this invention are: (1) In the fluorescent imaging probe provided by the present invention, the thiophene unit in polyethylene dioxythiophene is rich in electrons, thus providing a high electron density environment for the covalent organic framework. Furthermore, the lone pair electrons of the S atoms on the thiophene can diffuse in the highly electron-rich conjugated system, thereby achieving effective electron transfer. Iron ions can provide unoccupied d orbitals and are ideal electron acceptors. Therefore, when the fluorescent imaging probe is mixed with iron ions, the reaction between the lone pair electrons of the S atoms on the thiophene site and the iron ions can lead to fluorescence quenching, achieving specificity and high sensitivity for the detection of iron ions.

[0010] (2) The fluorescence imaging probe provided by this invention has a higher quantum yield and higher fluorescence intensity compared with fluorescence probes in related technologies. By improving the rigidity of the covalent organic framework structure through polyvinyl dioxythiophene, increasing π-electron conjugation, reducing the loss of nonradiative transitions, and improving fluorescence efficiency; in addition, polyvinyl dioxythiophene provides a high electron density environment for the covalent organic framework, and the lone pair electrons of sulfur atoms diffuse in the highly electron-rich conjugated system, increasing the number of electron transitions, thereby improving fluorescence intensity.

[0011] (3) By controlling the appropriate degree of polymerization of polyethylene dioxythiophene, the present invention enables the formation of a strong spatial entanglement structure between polyethylene dioxythiophene and the covalent organic framework, making the obtained fluorescent imaging probe more stable, reducing experimental errors caused by the failure of the spatial structure in the fluorescent imaging probe, and improving the reliability of experimental results.

[0012] The present invention also provides a method for preparing the above-mentioned fluorescent imaging probe, comprising: 2,5-Dibromo-3,4-vinyldioxythiophene was dissolved in organic solvent A, mixed with covalent organic framework powder, stirred, and the organic solvent A was evaporated under vacuum to obtain a semi-finished product. The semi-finished product was purified to obtain a fluorescent imaging probe.

[0013] Preferably, the synthesis of the covalent organic framework includes: adding 1,3,5-tris(4-aminophenyl)benzene and 2,5-dimethoxyterephthalaldehyde to organic solvent B, sonicating, stirring, injecting acetic acid into the mixture, stirring, centrifuging, washing, and vacuum drying to obtain covalent organic framework powder.

[0014] Preferably, the organic solvent A includes one or more of acetone and diethyl ether; the organic solvent B includes one or more of acetonitrile and propionitrile.

[0015] Preferably, the mass ratio of the covalent organic framework powder to 2,5-dibromo-3,4-vinyldioxythiophene is 1:(1.4-2.2).

[0016] Preferably, the purification step includes: rinsing the semi-finished product; sealing under a protective gas and heating at 50-70°C for 2.5-3 days and at 75-90°C for 0.5-1.5 days; cooling, washing multiple times, and drying to obtain the fluorescent imaging probe.

[0017] The beneficial effects of this invention are: The fluorescent imaging probe prepared by the method provided in this invention has high sensitivity. The fluorescent imaging probe prepared by this method contains fewer impurities, thus reducing the impact of impurities on experimental results and improving the reliability of the results.

[0018] The present invention also provides an application of the above-described fluorescent imaging probe or the fluorescent imaging probe prepared by the above-described method in the detection of iron ions.

[0019] Preferably, the process of detecting iron ions includes: Obtain the linear equation between the fluorescence intensity of the fluorescence imaging probe solution and the iron ion concentration; The fluorescent imaging probe solution was mixed with the iron ion solution to be tested, the fluorescence intensity of the fluorescent imaging probe solution was measured, and the concentration of the iron ion solution to be tested was calculated by linear equation.

[0020] The beneficial effects of this invention are: by obtaining the linear equation between the fluorescence intensity of the fluorescence imaging probe solution and the iron ion concentration, and then measuring the fluorescence intensity, the concentration of the iron ion solution to be tested can be determined according to the linear equation. This not only enriches the means of measuring iron ion concentration, but also greatly simplifies the steps of concentration measurement and reduces the time cost in the concentration measurement process.

[0021] The present invention also provides an application of the above-described fluorescent imaging probe or the fluorescent imaging probe prepared by the above-described method in in vivo fluorescence imaging.

[0022] The beneficial effects of the present invention are as follows: The fluorescent imaging probe of the present invention for in vivo fluorescence imaging is prepared by mixing polyethylene dioxythiophene and covalent organic framework. Since polyethylene dioxythiophene and covalent organic framework have good biocompatibility, the fluorescent imaging probe also has good biocompatibility, which enables the fluorescent imaging probe provided by the present invention to be used for in vivo fluorescence imaging. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.

[0024] Figure 1 This is a flowchart of the preparation of a fluorescent imaging probe according to one embodiment of the present invention (Stirring means stirring, and Heating means heating). Figure 2 This is a bar chart comparing the fluorescence intensity of the fluorescence imaging probes obtained in Examples 1-3 and Comparative Examples 1-3 of the present invention; Figure 3 This is a transmission electron microscope image of (A) a covalent organic framework and (B) a fluorescence imaging probe in one embodiment of the present invention; Figure 4 Fourier transform infrared spectra of a covalent organic framework, polyethylene dioxythiophene, and a fluorescent imaging probe in one embodiment of the present invention; Figure 5 This is an X-ray diffraction pattern of a covalent organic framework, polyethylene dioxythiophene, and a fluorescent imaging probe in one embodiment of the present invention; Figure 6 This is the fluorescence excitation (Ex, left side) and emission (Em, right side) spectrum of a fluorescence imaging probe in one embodiment of the present invention; Figure 7 This is a standard curve diagram of iron ion detection by a fluorescent imaging probe in one embodiment of the present invention; Figure 8 This is a bar graph showing the cell viability of HeLa cells after 24 hours of staining with fluorescent imaging probes of different concentrations, according to one embodiment of the present invention. Figure 9This is a confocal fluorescence image of HeLa cells co-incubated with a fluorescence imaging probe and LysoTracker Red in one embodiment of the present invention; Figure 10 This is a fluorescence imaging probe toxicity test for zebrafish in one embodiment of the present invention; Figure 11 These are fluorescence images of zebrafish after incubation with a fluorescent imaging probe and iron ions, respectively, according to one embodiment of the present invention. Figure 12 This is an analytical graph showing the hemolysis rate test of fluorescent imaging probes of different concentrations in one embodiment of the present invention. Figure 13 This is a fluorescence image of a C57 mouse treated with a fluorescent imaging probe and iron ions in one embodiment of the present invention.

[0025] The realization of the objective, functional characteristics and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0026] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] It should be noted that all directional indicators (such as up, down, etc.) in the embodiments of the present invention are only used to explain the relative positional relationship and movement of each component in a certain specific posture (as shown in the figure). If the specific posture changes, the directional indicator will also change accordingly.

[0028] Furthermore, in this invention, descriptions involving "first," "second," etc., are for descriptive purposes only and should not be construed as indicating or implying their relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined with "first" or "second" may explicitly or implicitly include at least one of that feature.

[0029] Furthermore, the technical solutions of the various embodiments of the present invention can be combined with each other, but only if they are based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention.

[0030] This invention provides a fluorescent imaging probe based on a covalent organic framework, the structural formula of which is as follows: Wherein, Me represents methyl, n represents degree of polymerization, and the fluorescent imaging probe includes a covalent organic framework (COF) and polyvinyl dioxythiophene (PEDOT).

[0031] Polyvinyldioxythiophene (PVD) possesses a simple molecular structure, small band gap, and high conductivity. Covalent organic frameworks (COFs) are highly crystalline porous organic polymers composed of light elements linked by covalent bonds. Due to their advantages such as high specific surface area, good stability, variable topology, and ease of functionalization, COFs show great potential in applications such as gas storage, molecular adsorption / separation, drug delivery, and sensing. Furthermore, the π-stacking layers within COFs promote electron transfer, and their inherent porous structure provides sufficient nanoscale space for suitable analytes. This invention utilizes the porous structure within the COF to form a raw material for PDD (e.g., 2,5-dibromo-3,4-vinyldioxythiophene), forming PDD using the PDD structure as a polymerization site. This results in an entangled spatial structure of PDD and a COF, i.e., a fluorescent imaging probe. For details on the bonding method of PDD and COF, please refer to [link to relevant documentation]. Figure 1 .

[0032] In fluorescent imaging probes, the thiophene units in polyethylene dioxythiophene are rich in electrons, thus providing a high electron density environment for the covalent organic framework. Furthermore, the lone pair electrons of the S atoms on thiophene can diffuse in this highly electron-rich conjugated system, enabling efficient electron transfer. Iron ions, on the other hand, provide unoccupied d orbitals, making them ideal electron acceptors. Therefore, when the fluorescent imaging probe is mixed with iron ions, a reaction occurs between the lone pair electrons of the S atoms at the thiophene sites and the iron ions, leading to fluorescence quenching and achieving highly specific and sensitive detection of iron ions.

[0033] Compared to fluorescent probes in related technologies, this method exhibits higher quantum yield and fluorescence intensity. By using polyvinyl dioxythiophene to enhance the rigidity of the covalent organic framework structure, increasing π-electron conjugation, reducing losses from nonradiative transitions, and improving fluorescence efficiency, this method achieves higher fluorescence efficiency. Furthermore, polyvinyl dioxythiophene provides a high electron density environment for the covalent organic framework, and the lone pair electrons of sulfur atoms diffuse in the highly electron-rich conjugated system, increasing the number of electron transitions and thus enhancing fluorescence intensity.

[0034] The degree of polymerization ranges from 1000 to 4000. By controlling the appropriate degree of polymerization of polyethylene dioxythiophene, a strong spatial entanglement structure can be formed between polyethylene dioxythiophene and the covalent organic framework, making the obtained fluorescent imaging probe more stable, reducing experimental errors caused by the failure of the spatial structure in the fluorescent imaging probe, and improving the reliability of experimental results.

[0035] The present invention also provides a method for preparing the above-mentioned fluorescent imaging probe, comprising: 2,5-Dibromo-3,4-vinyldioxothiophene (DBrEDOT) was dissolved in organic solvent A, mixed with covalent organic framework powder, stirred, and the organic solvent A was evaporated under vacuum to obtain a semi-finished product. The semi-finished product was purified to obtain a fluorescent imaging probe.

[0036] In some embodiments, the synthesis of the covalent organic framework includes: adding 1,3,5-tris(4-aminophenyl)benzene (TAPB) and 2,5-dimethoxytetraphenyldialdehyde (DMTP) to organic solvent B, sonicating, stirring, injecting acetic acid into the mixture, stirring, centrifuging, washing, and vacuum drying to obtain covalent organic framework powder.

[0037] In some embodiments, organic solvent A includes one or more of acetone and diethyl ether; organic solvent B includes one or more of acetonitrile and propionitrile.

[0038] In some embodiments, the mass ratio of the covalent organic framework powder to 2,5-dibromo-3,4-vinyldioxythiophene is 1:(1.4-2.2), preferably 1:1.8. A suitable mass ratio can improve the conversion rate of both the covalent organic framework powder and 2,5-dibromo-3,4-vinyldioxythiophene, thereby reducing the impurity content in the resulting fluorescent imaging probe. This helps reduce the amount of rinsing agent used in subsequent purification steps, reducing preparation costs. Furthermore, the fluorescent imaging probe obtained with a suitable mass ratio has higher fluorescence intensity and can be better used for fluorescence imaging.

[0039] In some embodiments, the purification process includes: rinsing the semi-finished product with an rinsing agent (including one or more of n-hexane and n-heptane); sealing the rinsed semi-finished product under nitrogen as a protective gas, heating it at 60°C for 3 days and 85°C for 1 day, respectively; cooling it, and then washing it sequentially with acetone, sodium sulfite aqueous solution, and ultrapure water. Finally, the obtained powder is vacuum dried overnight to obtain a fluorescent imaging probe.

[0040] The fluorescent imaging probe prepared by the method provided in this invention has high sensitivity. The fluorescent imaging probe prepared by this method contains fewer impurities, thus reducing the impact of impurities on experimental results and improving the reliability of the results.

[0041] The present invention also provides an application of the above-described fluorescent imaging probe or the fluorescent imaging probe prepared by the above-described method in the detection of iron ions.

[0042] The process for detecting iron ions includes: Obtain the linear equation between the fluorescence intensity of the fluorescence imaging probe solution and the iron ion concentration; The fluorescent imaging probe solution is mixed with the iron ion solution to be tested, and the fluorescence intensity of the fluorescent imaging probe solution is measured. The concentration of the iron ion solution to be tested is calculated using a linear equation. By obtaining a linear equation between the fluorescence intensity of the fluorescent imaging probe solution and the iron ion concentration, and then measuring the fluorescence intensity, the concentration of the iron ion solution to be tested can be determined according to the linear equation. This not only enriches the methods for measuring iron ion concentration, but also greatly simplifies the concentration measurement process and reduces the time cost.

[0043] The present invention also provides an application of the above-described fluorescent imaging probe or the fluorescent imaging probe prepared by the above-described method in in vivo fluorescence imaging.

[0044] The fluorescent imaging probe is prepared by mixing polyethylene dioxythiophene and a covalent organic framework. Since polyethylene dioxythiophene and the covalent organic framework have good biocompatibility, the fluorescent imaging probe also has good biocompatibility, which enables the fluorescent imaging probe provided by the present invention to be used for in vivo fluorescence imaging.

[0045] Example 1: Preparation of Fluorescent Imaging Probes See Figure 1 1,3,5-Tris(4-aminophenyl)benzene (20 mg) and 2,5-dimethoxyterephthalaldehyde (20 mg) were dissolved in acetonitrile (40 mL), and sonicated for 30 seconds. Then, acetic acid (1 mL) was injected into the mixture under stirring, and the mixture was stirred vigorously at room temperature. Subsequently, the precipitate was collected by centrifugation, washed several times with anhydrous ethanol, and dried under vacuum at 50 °C to obtain a covalent organic framework powder.

[0046] 36 mg of 2,5-dibromo-3,4-vinyldioxothiophene was dissolved in 1.5 mL of acetone and mixed with 20 mg of covalent organic framework powder. The mixture was stirred at 25 °C, and the acetone was evaporated under vacuum. The semi-finished product was purified to obtain a fluorescent imaging probe. The purification steps included washing the powder with 5 mL of n-hexane. The washed powder was sealed under nitrogen as a protective gas and heated at 60 °C for 3 days and 85 °C for 1 day, respectively. After cooling, the powder was washed sequentially with 10 mL of acetone, 20 mL of 1.0 M sodium sulfite aqueous solution, and 20 mL of ultrapure water. Finally, the obtained powder was vacuum dried overnight to obtain the fluorescent imaging probe.

[0047] Example 2: The difference from Example 1 is that the amount of 2,5-dibromo-3,4-vinyldioxythiophene used is 28 mg, and the amount of covalent organic framework powder used is 20 mg.

[0048] Example 3: The difference from Example 1 is that the amount of 2,5-dibromo-3,4-vinyldioxythiophene used is 44 mg, and the amount of covalent organic framework powder used is 20 mg.

[0049] Comparative Example 1: The difference from Example 1 is that the amount of 2,5-dibromo-3,4-vinyldioxothiophene used is 20 mg, and the amount of covalent organic framework powder used is 20 mg.

[0050] Comparative Example 2: The difference from Example 1 is that the amount of 2,5-dibromo-3,4-vinyldioxothiophene used is 52 mg, and the amount of covalent organic framework powder used is 20 mg.

[0051] Comparative Example 3: The difference from Example 1 is that the amount of 2,5-dibromo-3,4-vinyldioxothiophene used is 60 mg, and the amount of covalent organic framework powder used is 20 mg.

[0052] The fluorescence intensity of the fluorescence imaging probes obtained in Examples 1-3 and Comparative Examples 1-3 can be found in [reference]. Figure 2 It can be seen that the fluorescence intensity of the fluorescence imaging probes in Examples 1-3 is higher than that of the fluorescence imaging probes in Comparative Examples 1-3, and they are better suited for fluorescence imaging. Among them, the fluorescence intensity of the fluorescence imaging probe obtained in Example 1 is the highest, and it is the most preferred embodiment.

[0053] Example 4: Characterization of the fluorescent imaging probe prepared in Example 1 The particle size, molecular structure, and crystallinity of the fluorescent imaging probe prepared in Example 1 were measured by transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FT-IR), and X-ray diffraction, respectively. TEM images of the fluorescent imaging probe and the covalent organic framework are shown below. Figure 3 As shown, the fluorescent imaging probe has a shape and size consistent with the covalent organic framework, being spherical with a particle size of approximately 850 nm. However, the spherical shape has a shadowed edge due to the encapsulation of poly(dioxythiophene). The Fourier transform infrared spectrum is as follows... Figure 4 As shown, the fluorescence imaging probe spectrum reveals characteristic absorption peaks for the covalent organic framework and polyvinyldioxythiophene, indicating that the covalent organic framework was retained during solid-state polymerization. (1617 cm⁻¹) -1 1353cm -1 and 1117cm -1 The absorption peak at 1070 cm⁻¹ corresponds to asymmetric C=N, C=C stretching, and interring CC stretching. Furthermore, the absorption peak at 1070 cm⁻¹... -1 976 cm -1 and 629 cm -1The absorption peaks correspond to the bending vibration of COC and the tensile vibration of CSC, respectively, indicating that polyethylene dioxythiophene has been successfully polymerized within the pores of the covalent organic framework. The X-ray diffraction results are shown in the figure below. Figure 5 As shown, the relative intensity of a single diffraction peak of the fluorescent imaging probe prepared in Example 1 is reduced compared to that of a covalent organic framework. This is because the polyvinyl dioxythiophene present in the pores of the covalent organic framework is a disordered guest.

[0054] Example 5: Determination of the absorption-emission spectrum of the fluorescent imaging probe prepared in Example 1 The fluorescent imaging probe synthesized in Example 1 was prepared to a concentration of 0.24 g·L⁻¹ using anhydrous ethanol solvent. -1 The solution was used. Fluorescence emission spectra were collected using a fluorescence visible spectrophotometer within the wavelength range of 200-800 nm under excitation at 365 nm. The results are as follows: Figure 6 As shown, the excitation peak of the fluorescent imaging probe in ethanol solution is located at 365 nm, and the emission peak is located at 475 nm. The excitation-emission spectrum of the fluorescent imaging probe in anhydrous ethanol solution is shown (the left part is the excitation spectrum, and the right part is the emission spectrum). This illustrates the excitation-emission peak positions of the fluorescent imaging probe.

[0055] Example 6: Detection of iron ions using the fluorescent imaging probe prepared in Example 1 The 0.24 g·L⁻¹ prepared in Example 1 -1 Different concentrations (selected according to requirements) of iron ion solutions were added to the fluorescent imaging probe solution. Changes in the fluorescence emission spectrum were collected using a fluorescence visible spectrophotometer under 365 nm excitation. The data were processed using Origin software to obtain... Figure 7 The linear equation shown illustrates that the fluorescence intensity of the fluorescent imaging probe prepared in Example 1 exhibits a good linear relationship with the iron ion concentration; when the detection range is 0-1 mM, the linear equation is: y = 1.0804 + 0.00512x, R 2 =0.994.

[0056] Example 7: Fluorescence imaging of the fluorescent imaging probe prepared in Example 1 in cells 1. Cell culture: HeLa cell lines were cultured in an incubator at 37°C and 5% CO2 concentration in a high-glucose DMEM medium containing a mixture of 10% fetal bovine serum and 1% penicillin and streptomycin.

[0057] 2. Cytotoxicity: The cytotoxicity of the fluorescent imaging probe prepared in Example 1 was evaluated using the CCK8 assay. Specifically, HeLa cells were cultured in 96-well plates containing 200 μL of DMEM cell culture medium with 10% fetal bovine serum at 37°C under humid conditions for 24 h with 5% CO2, at an initial density of 5000 cells / well. Subsequently, the culture medium was removed, and different concentrations (including 0.0 g·L⁻¹) were added. -1 0.1 g·L -1 0.2 g·L -1 0.3 g·L -1 0.4 g·L -1 and 0.5 g·L -1 The fluorescent imaging probe and culture medium were incubated for 24 h. Then, 20 μL of CCK8 reagent was added to each well, and the mixture was incubated again at 37 °C and 5% CO2 for 0.5 h. The absorbance of each well was then recorded. The results are as follows: Figure 8 As shown, 0.5 g·L -1 The fluorescent imaging probe did not affect the normal growth of HeLa cells within 24 hours, indicating that the fluorescent imaging probe has good biocompatibility. 3. Cell fluorescence imaging: HeLa cells were used as test cells and cultured in glass-bottomed dishes for cell imaging. The culture dishes contained 2 mL of culture medium, including 10% fetal bovine serum and 90% DMEM, and were cultured at 37°C and 5% CO2 humidity. Subsequently, the culture medium was discarded, and the cells were then imaged using a fluorescent imaging probe (0.24 g·L⁻¹). -1 Add 2 mL of fresh culture medium to the petri dish.

[0058] After culturing for 24 hours, the cells were washed three times with PBS to remove any residual fluorescent imaging probes. Then, they were cultured in iron-containing medium for 2 hours. The cells were washed three times with PBS, and HeLa cell lysosomes were counterstained with the lysosomal red fluorescent probe (LysoTracker RedDND-99) for 1 hour. Residual LysoTracker Red DND-99 was then removed with PBS. Subsequently, the fluorescence of HeLa cells was observed using a 40× confocal microscope. The results are as follows: Figure 9 The fluorescence image shown in (A) is collected in the red channel (λ). ex In the λ = 561 nm range, the fluorescence image of (B) was collected in the blue channel (λ). ex The fluorescence image of (D) was collected in the blue channel (λ = 405 nm). ex The fluorescence image of (E) was collected in the red channel (λ = 405 nm). ex=561nm). Image (C) is a merged image of (A) and (B), and image (F) is a merged image of (D) and (E). (A), (B), and (C) show the effect of using only 0.24 g·L⁻¹. -1 HeLa cells incubated with a fluorescent imaging probe. (D), (E), and (F) show HeLa cells with 0.24 g·L⁻¹ -1 The fluorescent imaging probe was incubated with 500 μM iron ions. (Scale bar: 50 μm). It was clearly observed that the fluorescent imaging probe prepared in Example 1 and the lysosomal probe LysoTracker Red ddn-99 achieved excellent co-localization, indicating that the fluorescent imaging probe prepared in Example 1 is internalized in the cell lysosomes. Furthermore, the intracellular fluorescence decreased after the addition of iron ions, indicating that the fluorescent imaging probe prepared in Example 1 can respond to intracellular iron ions.

[0059] Example 8: Fluorescence imaging of the fluorescent imaging probe prepared in Example 1 in zebrafish 1. Zebrafish incubation: The fish eggs are incubated at a constant temperature of 26℃.

[0060] 2. Zebrafish toxicity: Fluorescent imaging probe solutions of different concentrations were prepared and added to six-well plates. Twenty zebrafish were incubated for one day for each concentration group, and the survival rate of the zebrafish was recorded. Results are as follows: Figure 10 As shown, the fluorescent imaging probe prepared in Example 1 has low toxicity to zebrafish, especially at a concentration of 0.5 g·L⁻¹. -1 At that time, it will not affect the zebrafish's survival ability within 24 hours.

[0061] 3. Zebrafish fluorescence imaging: After hatching, zebrafish were first immersed in a fluorescence imaging probe solution (0.24 g·L⁻¹). -1 The zebrafish were cultured in an iron solution for 2 hours, then washed with distilled water. Finally, they were immersed in an iron ion solution for 30 minutes. Fluorescence images were captured using a fluorescence microscope. The results are as follows: Figure 11 As shown, blue fluorescence was observed in the zebrafish, indicating that the fluorescent imaging probe prepared in Example 1 could successfully enter the zebrafish. Furthermore, the blue fluorescence of the zebrafish weakened after the introduction of iron ions, demonstrating that the fluorescent imaging probe prepared in Example 1 could successfully enter the zebrafish and dynamically monitor the iron ion concentration in the zebrafish.

[0062] Example 9: Fluorescence imaging of the fluorescent imaging probe prepared in Example 1 in mice. 1. Hemolysis rate experiment: 2% red blood cells (20 μL) were mixed with the fluorescent imaging probe solution (1 mL) prepared in Example 1 to achieve a final concentration of 0.2 g·L⁻¹. -1 0.4 g·L -1 0.6 g·L -1 0.8 g·L-1 and 1.0 g·L -1 The control group included a positive control (containing water and 2% red blood cells) and a negative control (containing 0.1M PBS and 2% red blood cells). After incubation for 1 hour, the samples were centrifuged. The optical density (OD) of the supernatant at 545 nm was measured using a microplate reader. The results are as follows: Figure 12 As shown, when the concentration of the fluorescent imaging probe prepared in Example 1 reaches 1 g·L⁻¹ -1 At that time, the hemolysis rate was only 2.68%. This indicates that the fluorescent imaging probe has good blood compatibility and can be used in animal experiments.

[0063] 2. In vivo fluorescence imaging in mice: C57 mice were anesthetized with ether, and then the fluorescence imaging probe prepared in Example 1 was injected into the C57 mice via the tail vein (200 μL), and cultured for 2 h. Subsequently, an iron ion solution was injected, and fluorescence images were captured using a small animal in vivo imaging system. The results are as follows: Figure 13 As shown, the fluorescent signal prepared in Example 1 mainly accumulated in the heart, lungs, and kidneys. The fluorescence signal weakened after the addition of iron ions, indicating that the fluorescent imaging probe prepared in Example 1 could successfully enter the mouse body, demonstrating that the fluorescent probe can visualize changes in iron ions within the mouse body. Examples 1-7 show that the present invention successfully prepared a fluorescent imaging probe, and the fluorescence intensity of the fluorescent imaging probe prepared by the present invention exhibits a good linear relationship with the iron ion concentration, enabling its use for iron ion concentration detection. Furthermore, cytotoxicity tests, zebrafish toxicity tests, and hemolysis rate tests verified that the fluorescent imaging probe provided by the present invention has excellent biocompatibility. In subsequent experiments, the fluorescent imaging probe provided by the present invention was successfully used for in vivo fluorescence imaging in HeLa cells, zebrafish, and C57 mice.

[0064] The fluorescence imaging probe provided by this invention not only enriches the means of measuring iron ion concentration, simplifies the steps of concentration measurement, and reduces the time cost in the concentration measurement process, but also enables it to be used for in vivo fluorescence imaging due to its excellent biocompatibility.

[0065] In summary, the covalent organic framework-based fluorescent imaging probe, its preparation method, and its application provided by this invention solve the technical problems of low quantum yield, poor biocompatibility, and the need to improve iron ion selectivity in related technologies.

[0066] The above technical solutions of the present invention are merely preferred embodiments of the present invention and do not limit the patent scope of the present invention. All equivalent structural transformations made under the technical concept of the present invention using the contents of the present invention specification and drawings, or direct / indirect applications in other related technical fields, are included in the patent protection scope of the present invention.

Claims

1. A fluorescent imaging probe based on a covalent organic framework, characterized in that, The structural formula of the fluorescence imaging probe is as follows: Wherein, Me represents methyl, n represents degree of polymerization, and the fluorescent imaging probe is a covalent organic framework and polyvinyldioxythiophene.

2. The fluorescence imaging probe according to claim 1, characterized in that, The degree of polymerization ranges from 1000 to 4000.

3. A method for preparing a fluorescent imaging probe as described in claim 1 or 2, comprising: 2,5-Dibromo-3,4-vinyldioxythiophene was dissolved in organic solvent A, mixed with covalent organic framework powder, stirred, and the organic solvent A was evaporated under vacuum to obtain a semi-finished product. The semi-finished product was purified to obtain a fluorescent imaging probe.

4. The preparation method according to claim 3, characterized in that, The synthesis of the covalent organic framework includes: adding 1,3,5-tris(4-aminophenyl)benzene and 2,5-dimethoxytetraphenyldialdehyde to organic solvent B, sonicating, stirring, injecting acetic acid into the mixture, stirring, centrifuging, washing, and vacuum drying to obtain covalent organic framework powder.

5. The preparation method according to claim 4, characterized in that, The organic solvent A includes one or more of acetone and diethyl ether; the organic solvent B includes one or more of acetonitrile and propionitrile.

6. The preparation method according to claim 3, characterized in that, The mass ratio of the covalent organic framework powder to 2,5-dibromo-3,4-vinyldioxythiophene is 1:(1.4-2.2).

7. The preparation method according to claim 3, characterized in that, The purification steps include: rinsing the semi-finished product; sealing under a protective gas and heating at 50-70℃ for 2.5-3 days and at 75-90℃ for 0.5-1.5 days; cooling, washing multiple times, and drying to obtain the fluorescent imaging probe.

8. The application of the fluorescent imaging probe as described in claim 1 or 2, or the fluorescent imaging probe prepared by any one of claims 3-7, for the detection of iron ions for non-diagnostic and non-therapeutic purposes.

9. The application according to claim 8, characterized in that, The process for detecting iron ions includes: Obtain the linear equation between the fluorescence intensity of the fluorescence imaging probe solution and the iron ion concentration; The fluorescent imaging probe solution was mixed with the iron ion solution to be tested, the fluorescence intensity of the fluorescent imaging probe solution was measured, and the concentration of the iron ion solution to be tested was calculated by linear equation.

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